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Published by soedito, 2018-12-29 07:06:42



Structural features Protein-coding genes Phenotype
Satellites Point
>99% X–Y Copy Expression mutation
Y–Y repeats number pattern or Deletion
Inversion polymorphism
1 gene loss
0 Ubiquitous
1 SRY Sex reversal


TSPYA array
6 IR3 None
USPgY None
7 IR1 DBY None
11 cen

Mb12 AZFa




16 P6

18 P4 CDY2, CDY2 AZFc
19 XKRY None

21 IR2 CYorf15A, CYorf15B

22 P3 EIF1AY
23 P2 RBMY1, RBMY1
25 DAZ1
26 CDY1
Yqh BPY2


The Y Chromosome and Male Fertility 133

The most significant finding from the considered evidence for the Y chromosome’s
human Y chromosome sequence project is ability to accumulate and to maintain male-
the discovery of the ampliconic sequence ness and testes genes as these gene families
blocks and the eight massive palindromes are all involved in male reproduction (see
in the MSY. These so-called “amplicons” below). Given the facts that the MSY does
exhibit intrachromosomal identities of not recombine during meiosis, the molecu-
99.9% or greater (Skaletsky et al. 2003). The lar mechanism for conserving Y gene func-
ampliconic sequences contain nine distinct tions across evolutionary time is the Y–Y
MSY-specific protein-coding gene families gene conversion, which is very common in
(∼60 transcriptions), with copy numbers the ampliconic sequences that exhibit intra-
ranging from two (VCY, XKRY, HSFY, PRY) chromosomal identities of 99.9% (Rozen
to three (BPY2) to four (CDY, DAZ) to six et al. 2003).
(RBMY) to ∼35 (TSPY) (Figure 6.2). In addi-
tion, the ampliconic sequences also contain All together, the human MSY contains
75 putative non-coding transcription units. a total of 156 transcription units, out of
Together, the ampliconic sequences contain which 78 are protein-coding genes that col-
135 of the 156 transcription units identified lectively encode 27 (18 single-copy genes
in the human MSY. All of the nine gene and 9 gene families) distinct proteins (Figure
families and the majority of the noncoding 6.2). The remaining 78 transcription units
transcription units are expressed predomi- are noncoding transcripts (Skaletsky et al.
nantly or exclusively in the testes. It is 2003).
thought that the genes in the ampliconic
region were derived through three converg- 6.3.2 Genes on the Y chromosome are
ing processes: (1) amplification of the functionally clustered
X-degenerate genes (e.g., RBMY and VCY),
(2) transposition and amplification of auto- Autosomes in mammals appear to contain
somal genes (DAZ), and (3) retroposition and randomly mixed collections of genes with
amplification of autosomal genes (CDY) extremely heterogeneous patterns of devel-
(Skaletsky et al. 2003). These processes were opmentally regulated expression in different
tissues. The mammalian sex chromosomes,

Figure 6.2 Human Y chromosome structure and gene content. From left to right: cytogenetic features of
the chromosome and their approximate locations, which are numbered from the Yp telomere. Structural
features include three satellite regions (cen, DYZ19, and Yqh), segments of X–Y identity (PAR1 and PAR2)
and high similarity, and Y–Y repeated sequences in which the regions with the greatest sequence identity
are designated “IR” for “inverted repeat” and “P” for “palindrome.” An inversion polymorphism on Yp that
distinguishes haplogroup P from most other lineages is indicated. The locations of the 27 distinct Y-specific
protein-coding genes are shown; some are present in more than one copy and their expression patterns
are summarized. Pseudoautosomal genes and Y-specific noncoding transcripts are not shown. On the right,
the phenotypes that are associated with gene inactivation or loss are indicated; some deletions produce no
detectable phenotype (black) and represent polymorphisms in the population, whereas others result in
infertility (AZFa, AZFb, and AZFc); contributions of the individual deleted genes are discussed in the text.
Reproduced with permission from the Nature Publishing Group, Jobling, M.A. and Tyler-Smith, C. 2003.
Nature Reviews Genetics 4: 598–612.

134 Quantitative Genomics of Reproduction

however, are enriched for sex-biased genes X-degenerate genes (EIF1AY, CYorf15A, and
related to sex development and reproduction 15B; DDX3Y; NLGN4Y; PCDH11Y; PRKY;
(Lahn and Page 1997; Saifi and Chandra 1999; USP9Y; RPS4Y1; RPS4Y2; JARID1D; TBL1Y;
Wang et al. 2001; Khil et al. 2004, 2005). TGIF2LY; TMSB4Y; ZFY; and UTY) that are
Analyses of the function of genes on the single copy and expressed ubiquitously. Four
human and mouse X chromosomes have genes (USP9Y, DDX3Y, UTY, and TMSB4Y)
revealed that the X has significantly higher from this group are clustered together and
number of sex- and reproduction-related play a significant role in spermatogenesis.
(SRR) genes, which are not subject to selec- The rest of the genes in this group have
tion by meiotic sex chromosome inactiva- “housekeeping” functions. Group III con-
tion (Khil et al. 2004). In contrast to the tains AMELY and a proposed growth control
chromosomal distribution of most tissue- Y (GCY) gene, which are associated with the
enriched genes which is not significantly control of embryonic growth, stature, and
different from randomness, ovary- and development of teeth (Fincham et al. 1990;
placenta-enriched genes are significantly Kirsch et al. 2004). However, the proposed
overrepresented on the X chromosome. As GCY has not been confirmed transcription-
to the testis-enriched genes, data from a ally. If the GCY gene is confirmed, it may
few studies are inconsistent. Khil et al. (2004) have a potential value for growth selection
reported underrepresented testis-enriched in animal breeding. Group IV contains the
genes on the X, whereas Wang et al. dis- nine multicopy genes (RBMY, DAZ, TSPY,
covered a roughly 15-fold enrichment on the CDY, BPY2, XKRY, PRY, HSFY, and VCY),
X chromosome for male germ cell-specific of which eight families are localized in the
spermatogonially expressed genes (Wang palindromes of the ampliconic sequences.
et al. 2001). In addition, the human X is asso- They are testis specific and are functionally
ciated with a high incidence of mental dis- coherent in spermatogenesis and fertility
ability caused by mutations in genes on the (Figure 6.2) (Lahn and Page 1997; Skaletsky
X that are required for brain development et al. 2003).
or function (Zechner et al. 2001; Delbridge
et al. 2008). This is supported by the findings 6.3.3 Genes on the Y chromosome are
of fivefold enrichment of the “intelligence” not conserved between species
genes on the X. Therefore, the X chromo-
some is “smart and sexy” (Graves 2006). In contrast to the X, which is highly con-
served between species in its size, gene
Compared with the X, the Y chromosome content and gene order (except for the order
is even more biased in its gene content in the rodent X) (O’Brien et al. 1999; Bourque
with the highest density of testis-enriched et al. 2004; Raudsepp et al. 2004a), the Y
genes. These testis genes are functionally chromosome is not conserved between
coherent in the MSY and clustered together species. It varies in size and gene content,
in the ampliconic and X-degenerate seg- and in homology relationships to the X
ments (Lahn and Page 1997). (Graves 2006). Comparative mapping among
several species, including human, mouse,
Based on gene functions, the 27 protein- cow, sheep, cat, dog, lemur, and wallaby, has
coding genes (families) in the human MSY demonstrated that the Y chromosome PAR
can be classified into four groups. Group I genes are not conserved at all (Graves et al.
contains only one gene (SRY) that is involved
in sex determination. Group II contains 15

The Y Chromosome and Male Fertility 135

1998; Graves 2006). The variation of the RPS4Y, SMCY (JARID1D), EIF2S3Y, AMELY,
PAR gene content and the origin and evolu- ZFY, UTY, DDX3Y, USP9Y, HSFY, and
tion of the PAR in mammals were explained UBE1Y, which are homologous on the X, are
by an “addition–attrition” theory, as pro- either conserved in all species or lost in
posed by Graves et al. (Graves 1995, 1998; some species. For instance, UBE1Y is absent
Graves et al. 1998). in primates, whereas AMELY is absent in
the rodent Y chromosomes. These genes
Other than human, chimpanzee, and are thought to be present on the ancestral
mouse, the gene content of MSY is poorly (or so-called proto) Y chromosome and are
known. Based on the currently available lost as a result of Y degradation during
data, a comparative map of the MSY genes the mammalian evolution in different
was constructed (Figure 6.3). From this map, lineage (Graves 2006). (3) Genes that are
we can conclude that (1) the SRY gene, the acquired from autosomes by different mech-
most important gene on the Y chromosome anisms and amplified thereafter on the Y
that triggers the male sex development, is are not conserved. For instance, the DAZ
conserved on all mammalian species studied gene family emerged on the Y chromosome
so far. (2) Genes such as RBMY, TSPY,

Human Cattle Horse Pig Cat Mouse Kangaroo

CYorf15A UBE1Y
CYorf15B SRY

Skaletsky Present Raudsepp et al. 2004b Quilter Murphy Alföldi Graves
et al. 2003 2008 2006
study Paria et al. 2008 et al. 2002 et al. 2006

Figure 6.3 A comparison of MSY genes among several mammalian species. Active genes in MSY are
marked for conserved (italic), species-specific (underlined), and not conserved on MSY (black). A few MSY
genes are pseudoautosomal in cattle and horse (#). Pseudogenes are in gray. Multiple copy genes are
labeled by an asterisk (*). Highly amplified human and bovine TSPY and the mouse Rbmy gene are indi-
cated by a vertical bar. Where available, an order and/or map position of loci is provided. PAR, pseudoau-
tosomal region (black box); PAB, pseudoautosomal boundary (dashed line); Cen, centromere; p, short arm;
q, long arm.

136 Quantitative Genomics of Reproduction

by transposition of an autosomal DAZL wasteland of repetitive DNA carrying no
gene (Saxena et al. 1996; Shan et al. 1996 genetic information apart from the sex-
Gromoll et al. 1999) and identified only in determining factor. The first association
Old World monkeys and great apes, while between Y chromosome function and sper-
DAZL is present in all vertebrates (Cooke matogenic failure was demonstrated by
et al. 1996; Saxena et al. 1996). Another Tiepolo and Zuffardi in 1976 using a Y chro-
example is the CDY gene that has been iden- mosome deletion mapping approach. Since
tified only in primates (Lahn and Page 1999; patients with de novo microscopically
Kostova et al. 2002; Rottger et al. 2002; detectable deletions of a region on Yq showed
Wimmer et al. 2002; Dorus et al. 2003). Two azoospermia and infertility, a spermatogen-
members, CDYL and CDYL2 genes, map to esis factor, designed AZoospermia Factor
autosomes and exist in most mammalian (AZF), was proposed to be located in the Yq
species (Lahn and Page 1999; Dorus et al. deleted region (Tiepolo and Zuffardi 1976).
2003; Wang et al. 2008). It is believed that However, the deleted region was not defined
the CDY gene arose by retroposition of a until the mid-1980s when Y chromosome-
processed messenger RNA derived from an specific markers (especially ∼200 sequence-
autosomal CDYL gene (Lahn and Page 1999; tagged sites [STS]) and a fine deletion interval
Dorus et al. 2003; Bhowmick et al. 2006). map were developed (Vergnaud et al. 1986;
Recent efforts in searching for novel Y chro- Vollrath et al. 1992). These markers have
mosome genes in cats, dogs (Murphy et al. permitted simple deletion analysis in infer-
2006), horses (Paria et al. 2008), and cattle tile men with azoospermia or severe oligo-
(our unpublished data) have resulted in zoospermia by polymerase chain reaction
a dozen species-specific Y-linked genes (PCR) to define AZF. By screening 76 Yq STS
(termed TETY for testis expressed transcript markers in a large group of 370 patients with
on the Y) (Figure 6.3). As we expected, these azoospermia and severe oligozoospermia,
novel genes appear to either have an autoso- Vogt et al. (1996) defined AZF to three non-
mal origin (such as ZNF280BY in bovine and overlapping regions, termed AZFa, AZFb,
TETY1 in cat) or be relics of X-degeneration and AZFc (Figure 6.2). It was later found that
(cat TETY2 and CUL4BY) as described above the AZFb and AZFc regions overlapped on
(Figure 6.3). It is clear that more species- the basis of the Y sequence (Repping et al.
specific Y-linked genes will be identified 2002; Skaletsky et al. 2003).
once a complete MSY gene content (sequence)
is available for most, if not all, mammalian The importance of Y chromosome micro-
species. deletions is underlined by the fact that they
account for 10–18% of idiopathic primary
6.4 Function of Y chromosome testiculopathies (azoospermia and severe oli-
genes in spermatogenesis and gozoospermia) (Foresta et al. 2001; Kleiman
male fertility et al. 2003; Krausz and Degl’Innocenti 2006).
Y microdeletions have been found exclu-
6.4.1 Y chromosome deletion and sively in patients with <1 million spermato-
infertility in men zoa/mL, but are very rare in patients with >5
million spermatozoa/mL. The most frequent
Initial research before the 1950s suggested deletions occurred at AZFc (∼60%), followed
that the (human) Y chromosome was a by AZFb, and AZFb+c, or AZFa+b+c (∼35%),
whereas deletions in AZFa are infrequent

The Y Chromosome and Male Fertility 137

(∼5%) (Krausz and McElreavey 1999; Krausz 1999). As a result, DAZ has been identified
and Degl’Innocenti 2006). However, reports only in Old World monkeys and great apes,
on isolated gene-specific deletions within while DAZL is present in all vertebrates
AZF regions are limited partially because (Cooke et al. 1996; Saxena et al. 1996).
most Y microdeletions involved more than
one gene. To date, gene-specific deletions The four copies of DAZ (DAZ1-4) form
have been identified for DDX3Y (Foresta two pairs within palindromic duplications
et al. 2000), HSFY (Vinci et al. 2005), and (Figure 6.2); one pair of genes (DAZ1-2) is
USP9Y (Sun et al. 1999), which are all associ- part of the P2 palindrome and the second
ated with azoospermia and/or severe oligo- pair (DAZ3-4) is part of the P1 palindrome.
zoospermia and infertility. Each gene contains a 2.4-kb repeat including
a 72-bp exon, called the DAZ repeat. The
6.4.2 Candidate genes for number of DAZ repeats is variable, and
spermatogenesis and male fertility there are several variations in the sequence
of the DAZ repeat. Alternative splicing
As discussed above (in Section 6.2), genes on produces multiple transcript variants encod-
the Y chromosome are clustered together and ing different isoforms. The DAZ family is
functionally coherent in spermatogenesis expressed exclusively in germ cells encoding
and male fertility. These clusters correspond proteins that contain a highly conserved
to the AZFa, AZFb, and AZFc regions (Figure RNA recognition motif (RRM) (Yen 2004).
6.2). To date, the following seven candidate DAZ genes are expressed only in the testis
genes have been confirmed or proposed to (Reijo et al. 1995), while DAZL is expressed
play a role in spermatogenesis and male fer- in both the testis and the ovary (Seligman
tility: DDX3Y and USP9Y in the AZFa region; and Page 1998; Dorfman et al. 1999;
RBMY, PRY, HSFY, and CDY in AZFb; and Cauffman et al. 2005). Both DAZ and DAZL
DAZ and CDY in AZFc (reviewed in Krausz are detected in the nuclei of primordial germ
and Degl’Innocenti 2006). cells (PGCs) in fetal gonads (Xu et al. 2001)
and are believed to function in the develop-
DAZ gene family ment of PGCs and in germ cell differentia-
The Deleted in Azoosermia (DAZ) gene has tion and maturation (reviewed in Yen 2004).
four copies on the human Y chromosome Mutations in these genes have been linked
and two autosomal homologs, DAZL (DAZ- to infertility in several species. In flies, loss
like) on chromosome 3 and BOLL (bol, of function of the boule gene, an ortholog of
boule-like [Drosophila]) on chromosome 2. DAZ/DAZL, leads to male sterility (Eberhart
It is believed that the ancestor member of et al. 1996). In frogs, inhibition of Xdazl
the family is BOLL, which gave rise to DAZL (Xenopus daz-like) leads to defective migra-
via duplication prior to the divergence of tion and a reduction in PGCs (Houston and
vertebrates and invertebrates (Cauffman King 2000). In mice, a disruption of the Dazl
et al. 2005). Later, during primate evolution, leads to prenatal loss of all germ cells in both
the autosomal DAZL gene gave rise to DAZ sexes during prenatal germ cell develop-
on the Y chromosome by transposition, ment, and hence infertility (Ruggiu et al.
repeat amplification, and pruning (Saxena et 1997). Further, the infertile phenotype of
al. 1996; Shan et al. 1996; Gromoll et al. Dazl null mouse (Dazl–/–) can be partially
rescued by a human DAZ transgene (Slee
et al. 1999). As the gene name suggests,

138 Quantitative Genomics of Reproduction

deletion or microdeletion in DAZ gene(s) in tin structure (Briton-Jones and Haines 2000).
the AZFc region leads to severe spermato- Biochemical analysis revealed that human
genic failure and infertility in men (Reijo et CDY/CDYL and mouse Cdyl proteins
al. 1995; Vogt et al. 1996; Ferlin et al. 1999), exhibit histone acetyltransferase activity
and DAZL transcripts in the testes are lower in vitro. Both proteins can specifically acety-
in men with spermatogenic failure com- late H4 and H2A, with H4 being the strongly
pared with fertile men (Lin et al. 2001). preferred substrate (Lahn et al. 2002).
Chromodomain proteins are localized to the
CDY gene family nucleus of late spermatids where histone
Like the DAZ family, the human chro- hyperacetylation takes place. Histone hyper-
modomain protein, Y-linked (CDY) gene acetylation is thought to facilitate the
family also consists of three members. One transition in which protamines replace his-
member locates on the Y chromosome in tones as the major DNA-packaging protein
the form of four highly related copies (CDY1 (Lahn and Page 1999; Kleiman et al. 2001,
and CDY2), which are identified only in 2003; Kostova et al. 2002; Lahn et al. 2002;
primates (Lahn and Page 1999; Kostova et al. Dorus et al. 2003). In addition to the chro-
2002; Rottger et al. 2002; Wimmer et al. modomain, the catalytic domain in the car-
2002; Dorus et al. 2003). Two members, boxy-terminal portion of the CDY protein
CDYL and CDYL2 genes, map to chromo- family can bind CoA and histone deacety-
some 6 and 16, respectively, which exist in lases, and acts as a corepressor of transcrip-
most mammalian species (Lahn and Page tion in somatic cells, as well as during the
1999; Dorus et al. 2003). It is believed that early stages of spermatogenesis (Caron et al.
in the common ancestor of mammals, the 2003).
progenitor of the gene family duplicated to
result in the two autosomal genes, CDYL Members of the CDY gene family have
and CDYL2. The CDY gene instead arose by different expression patterns. While the
retroposition and subsequent amplification human Y-linked CDY genes are specifically
of a processed messenger RNA derived from expressed in the testis (Lahn and Page 1999;
an autosomal CDYL gene (Lahn and Page Kostova et al. 2002; Dorus et al. 2003), the
1999; Dorus et al. 2003; Bhowmick et al. autosomal homologs CDYL and CDYL2 are
2006). In the simian lineage, the CDY gene expressed ubiquitously. Two protein iso-
was retained and subsequently amplified forms (540 and 554 amino acids [aa]) were
into many copies. In other mammals, this identified for CDY and three isoforms
gene has been lost (Lahn and Page 1999; (598, 544, and 309 aa) for CDYL as results
Dorus et al. 2003). of alternative spliced transcripts. In mice
and rabbits, both CDYL and CDYL2 express
The human chromosome Y has two iden- a ubiquitous long transcript and a highly
tical copies of CDY1 in P1 palindrome and abundant testis-specific short transcript
of CDY2 in P5 palindrome (Figure 6.2). (Lahn and Page 1999; Kostova et al. 2002;
Proteins encoded by genes of the CDY family Dorus et al. 2003). In cows, at least four
contain two domains: the chromodomain transcript variants for the bovine CDYL
and the enoyl-coenzyme A hydratase-isom- gene have been identified (Wang et al. 2008).
erase catalytic domain (Lahn and Page 1999; These transcripts are expressed predomi-
Dorus et al. 2003). The chromodomain has nantly or exclusively in the bovine testis
been implicated in remodeling the chroma- (Wang et al. 2008).

The Y Chromosome and Male Fertility 139

RBMY gene family within inverted repeat IR2, along with
The RNA-binding motif protein, Y-linked RBMY genes on the human Y chromosome
(RBMY) gene encodes a protein containing (Figure 6.2). PRY is expressed specifically
an RNA-binding motif in the N-terminus in the testis. It encodes a protein, which has
and four repetitions of a Ser-Arg-Gly-Tyr tet- a low degree of similarity to the protein
rapeptide motif (SRGY box) in the C-terminus tyrosine phosphatase, non-receptor type 13.
(Ma et al. 1993). Multiple copies of this gene Since PRY is located in AZFb, and is often
are found in the AZFb region of chromosome deleted in patients with severe infertility,
Y, and the encoded protein is thought to be it was proposed to play an important role
involved in spermatogenesis. Most copies in spermatogenesis (Stouffs et al. 2001).
of this locus are pseudogenes, although six However, further study indicated that the
highly similar copies have full-length open role of the PRY gene in spermatogenesis
reading frames (ORFs) and are considered could be questioned, but suggested its prob-
functional (Ma et al. 1993; Skaletsky et al. able involvement in apoptosis of defective
2003). Four functional copies of this gene are spermatozoa (Stouffs et al. 2004).
located within inverted repeat IR2, and the
remaining two in P3 palindrome, along with HSFY gene family
two copies of PRY genes (Figure 6.2). The heat shock transcription factor, Y-linked
gene (HSFY) belongs to the HSF family. Two
The mouse RBMY contains an RNA- identical HSFY copies are situated in the
binding motif with 74% similarity to the P4 palindrome in proximal AZFb on Yq,
human RBMY, followed by only one SRGY whereas there are four pseudogenes mapping
box. RBMY-deficient mice do not show the in two clusters in the P1 palindrome of AZFc
same phenotype as in humans; they have and in P3. Sequences similar to few HSFY
abnormal sperm development but are not exons are also located in Yp, X, and 22
sterile (Mahadevaiah et al. 1998). The human (Shinka et al. 2004; Tessari et al. 2004). The
RBMY gene family has an X-located member HSF family is a group of highly conserved
(RBMX), which encodes the widely expressed regulators that play a role as transcriptional
heterogeneous nuclear ribonucleoprotein G activators of heat shock protein (HSP) genes.
(hnRNPG) (Delbridge et al. 1999). There is This family consists of multiple genes in
also another member named hnRNPG-T, a mammals, and it is thought to be involved
functional retrogene on chromosome 11 in physiological pathways related to devel-
(Elliott et al. 2000). The human RBMY is opment and differentiation, other than in
expressed specifically in the nuclei of adult stress response (Wang et al. 2003; Tessari
male germ cells throughout all transcrip- et al. 2004). HSFY is characterized by an
tionally active stages of spermatogenesis, HSF-type DNA-binding domain related to
and deletion of the functional copies of the HSF2 gene on chromosome 6 (Ferlin
RBMY is associated with an arrest of meiotic et al. 2003). HSF2 is expressed at high levels
division I during spermatogenesis (Elliott and is only active in embryogenesis and
et al. 1997; Elliott 2004). spermatogenesis (Pirkkala et al. 2001). HSF2
regulates the expression of many genes, and,
PRY gene family in particular, it controls the hsp70 gene
The PTPBL-related gene on Y (PRY, also family promoter (Sistonen et al. 1992). There
known as PTPN13LY, PTPN13-like, Y- are three different transcripts and protein
linked gene) has two nearly identical copies

140 Quantitative Genomics of Reproduction

isoforms found in humans, each containing terns. A long transcript (DDX3Y-L) is ubiq-
an HSF domain typical of HSF proteins. uitously expressed, while a short transcript
These HSFY transcripts are differentially (DDX3Y-S) is testis specific (Foresta et al.
expressed, transcript 1 being present in 2000; Vong et al. 2006). DDX3Y protein was
many tissues including the testis, and tran- found only in male germ lines, while DDX3X
scripts 2 and 3 being testis specific (Tessari protein was present in all testicular and non-
et al. 2004). These observations suggest that testicular tissues, suggesting that DDX3Y is
HSFY could play an important role in sper- essential for human spermatogenesis (Ditton
matogenesis. HSFY gene-specific deletion et al. 2004). The DDX3Y and its neighbor
has been reported in an azoospermic man, genes, USP9Y and UTY, in the AZFa region,
confirming its function in spermatogenesis as well as the gene order of USP9Y-DDX3Y-
(Vinci et al. 2005). UTY, are similar in both human and mouse
Y chromosomes, indicating that they repre-
At least six different transcripts have been sent a conserved synteny block (Mazeyrat
identified from an adult testis for the bovine et al. 1998; Vong et al. 2006). In mice, partial
HSFY gene, which can be classified into two deletion in this conserved syntenic region on
groups. Group I contains three transcripts the short arm of the Y chromosome results
with a different size 3′UTR that encode in early failure of spermatogenesis and con-
a peptide of 207 aa. Group II contains the sequent sterility (Sutcliffe and Burgoyne
remaining three transcripts that encode a 1989; Simpson and Page 1991; Wood et al.
417 aa. One of the group II transcripts shows 1997; Mazeyrat et al. 1998). Although Ddx3y
a deletion of 9 bp, resulting in an isoform of was proposed to be a Y chromosome gene
414 aa. All bovine HSFY isoforms contain essential for normal spermatogonial prolif-
the conserved HSF DNA-binding domain. eration in the mouse (Mazeyrat et al. 2001),
Preliminary data indicated that multicopies a recent study indicated that the Ddx3y gene
of the bHSFY gene are present in the bovine may not be required for mouse spermatogen-
Y chromosome (W.-S. Liu, T.-C. Chang, and esis (Vong et al. 2006), signifying that there
Y. Yang, unpublished data). may be species-specific difference between
the function of DDX3Y/Ddx3y in human and
DDX3Y mice. A recent proteomics searching and
The DEAD box polypeptide 3, Y-linked functional study identified a number of sper-
gene (DDX3Y, also known as DBY, DEAD matogenesis-enriched chromatin proteins
box gene on the Y) encodes a putative with roles in fertility in Caenorhabditis
ATP-dependent RNA helicase (Abdelhaleem elegans (Chu et al. 2006). One of these pro-
2005). This gene belongs to the DEAD box teins, named glh-2, is an RNA helicase,
protein family, which is characterized by the which is the sole protein on the list that has
conserved motif Asp-Glu-Ala-Asp (DEAD) an ortholog, the DDX3Y gene, on the human
(Rosner and Rinkevich 2007). In humans, Y chromosome, suggesting a conserved func-
DDX3Y is located in the AZFa interval in tion of DDX3Y in spermatogenesis and male
MSY (Vogt et al. 1996; Lahn and Page 1997). fertility between C. elegans and humans
This gene has a homolog on the X chromo- (Chu et al. 2006).
some (DDX3X) that escapes X inactivation.
DDX3Y may have both housekeeping and Like the human DDX3Y and mouse
testis-specific functions as the gene produces Ddx3y gene, two transcripts were identified
two transcripts with different expression pat- for the bovine DDX3Y gene, which are

The Y Chromosome and Male Fertility 141

identical except for a three-base-pair inser- Other genes that may be involved in
tion and an expanded 3′UTR in the bovine spermatogenesis and male fertility
DDX3Y-L. The bovine DDX3Y is predomi- In addition to the major candidate genes/
nantly expressed in the testis. The bDDX3Y- families for spermatogenesis and fertility
-S encodes a peptide of 660 aa, while the described above, several other genes (fami-
bDDX3Y-L encodes a 661 aa as a result of lies) including BPY2, JARID1D, XKRY,
the insertion of a serine in the bDDX3Y-L RPS4Y, eIF1AY, and CYorf15A and CYorf15B
peptide. Both bDDX3Y isoforms contain the are also located within the AZFb region on
conserved motifs of DEAD-box RNA heli- the human Y chromosome (Figure 6.2). It is
cases (Liu et al. 2008). not clear if any of these genes play a role in
spermatogenesis. The basic charge, Y-linked
USP9Y 2 (BPY2-, also known as VCY2) has three
The ubiquitin-specific peptidase 9, Y-linked nearly identical copies; two of them are in
(USP9Y, previously known as DFFRY, the P1 palindrome. BPY2 is expressed spe-
Drosophila fat facets-related Y-linked) is a cifically in the testis (Lahn and Page 1997;
member of the peptidase C19 family. The Tse et al. 2003). As the PBY2-encoded protein
human USP9Y is located in AZFa clustered interacts with ubiquitin protein ligase E3A,
with DDX3Y and UTY, which have a homo- it may be involved in male germ cell devel-
log on the X chromosome (USP9X). The opment and male infertility (Wong et al.
gene contains 46 exons and has a transcript 2002; Tse et al. 2003). The jumonji, AT-rich
of 10048 bp, encoding a protein of 2555 aa interactive domain 1D (JARID1D, previ-
(Brown et al. 1998). USP9Y protein does not ously known as SMCY or HY), encodes one
contain known functional domains except human H-Y epitope. A short peptide derived
for the Cys and His domains, which are from this protein is a minor histocompati-
present in ubiquitin-specific proteases. The bility (H-Y) antigen, which can lead to graft
latter cleave the ubiquitin moiety from rejection of male donor cells in a female
ubiquitin-fused precursors and ubiquitinyl- recipient (Wang et al. 1995). The XK,
ated proteins (Lee et al. 2003). Both USP9Y Kell blood group complex subunit-related,
and USP9X genes are expressed ubiquitously Y-linked (XKRY) gene has two identical
in different human tissues (Lahn and Page copies located in the P5 palindrome in the
1997). proximal region of AZFb (Figure 6.2). XKRY
encodes a putative membrane transport
USP9Y is one of the few Yq genes protein similar to the XK precursor and is
for which isolated gene-specific deletions/ expressed specifically in the testis (Lahn
mutations have been reported: a massive and Page 1997). The ribosomal protein S4,
deletion removing the entirety of USP9Y Y-linked (RPS4Y) gene has two copies on the
(Brown et al. 1998), a 4-bp splice-donor site human Y: one copy (RPS4Y1) maps to the
deletion resulting in a severely truncated Yp near SRY, the other (RPS4Y2) maps to
protein (Sun et al. 1999), and two cases of Yq. The encoded ribosomal protein S4 is a
deletions removing part of the USP9Y gene, component of the 40S subunit. The eukary-
which were spontaneously transmitted from otic translation initiation factor 1A, Y-linked
father to son (Krausz et al. 2006). These (EIF1AY) encodes a Y isoform of an eIF1A,
mutations are associated with Sertoli cell- an essential translation initiation factor.
only (SCO) syndrome, azoospermia, and There is a homologous gene on the X
male infertility.

142 Quantitative Genomics of Reproduction

(EIF1AX). Both Y and X copies are expressed identified from the coding and noncoding
ubiquitously with a high level of expression sequences of the Y chromosome genes
in the testis (Lahn and Page 1997). described in Section 6.3.2 of this chapter.
Our recent work on the bovine Y chromo-
6.5 Polymorphisms of the Y some found that single nucleotide indels are
chromosome and male fertility popular on BTAY (W.-S. Liu, unpublished
data). Finally, the Y chromosome microdele-
6.5.1 Different types of polymorphisms tions are considered as polymorphisms
on the Y chromosome (Machev et al. 2004). Although Y chromo-
some microdeletions are the most frequent
As the MSY region is inherited as a single genetic cause of severe oligozoospermia and
haploid block in linkage from father to male azoospermia in infertile men (Krausz et al.
offspring, the Y chromosome represents an 2003; Krausz 2005), some microdeletions,
important collection of all mutations that such as gr/gr deletions in AZFc region, were
have occurred along male lineages during also observed in 3.5% of normal fertile men.
evolution. Therefore, Y chromosome DNA It was suggested that most gr/gr deletions
variations/polymorphisms are valuable for are neutral variants (Machev et al. 2004).
investigations on human evolution, forensic
analysis, paternity test, and molecular medi- 6.5.2 Polymorphisms and fertility––
cine (Krausz et al. 2004) Genotype and phenotype correlation

There are at least four types of polymor- The absence of recombination on the
phisms identified on the Y chromosome. MSY means that polymorphisms within
The first type is the variation in tandemly this region are in tight association with
repeated sequences including microsatel- potential functional variations associated
lites, short interspersed nuclear elements with Y-linked phenotypes. Thus, an indirect
(SINEs), and long interspersed nuclear way to explore whether Y chromosome
elements (LINEs). This type of Y-specific genes are involved in fertility/infertility is
markers, such as DYSl9 (a tetranucleotide the characterization of Y chromosome hap-
microsatellite) and DYS287 (Y Alu inser- logroups in infertile versus normal (fertile)
tional polymorphism or “YAP”), is applied men (Krausz et al. 2004). To date, approxi-
for human evolution and migration studies mately 600 binary markers have been
(Hammer et al. 1997). The second type of characterized on the human Y chromosome,
variation on the Y chromosome is the and the resultant haplogroups were stan-
changes in gene copy number, which is dardized (Y-chromosome-consortium 2002;
caused by gene duplication/amplification. A Vogt 2005; Karafet et al. 2008). The asso-
good example is the TSPY gene, which is ciation between Y haplogroups and sperm
estimated to be 30–60 copies on the human counts and/or spermatogenic failure has
MSY, and 50–200 copies on the bovine MSY been investigated in several populations
(Verkaar et al. 2004; Vodicka et al. 2007). (Table 6.1) (Kuroki et al. 1999; Paracchini
Another type of polymorphism is single base et al. 2000, 2002; Quintana-Murci et al.
DNA mutations including single nucleotide 2001; Carvalho et al. 2003; Ferlin et al. 2005).
polymorphisms (SNPs) and single base inser- A haplogroup (termed hg26) was found to
tions/deletions (indels). SNPs have been be associated with reduced sperm count

The Y Chromosome and Male Fertility 143

Table 6.1 A summary of association studies dealing with Y chromosome polymorphisms and spermato-
genic failure.

Phenotype Population No. of No. of No. of Y Association References
patients controls
markers found

AZF deletion European 50 50 9 No Quintana-Murci et al. (2001)
AZF deletion European 73 299 11 No Paracchini et al. (2000)
AZF deletion Japanese 15 No Carvalho et al. (2003)
Oligo/azoospermia Japanese 6 84 No Carvalho et al. (2003)
Azoospermia Japanese 51 57 3 No Kuroki et al. (1999)
Oligo/azoospermia Italian 106 156 8 Yes
Teratozoospermia 74 216
Oligo/azoospermia Danish 3 Yes Krausz et al. (2001)
Sperm count Italian 43 128 11 No Paracchini et al. (2002)
Spermatogenic Italian 41 Not recorded Yes Ferlin et al. (2005)
337 263 3
impairment Brazilian No Carvalho et al. (2006)
Azoospermia Chinese 117 122 22 Yes Lu et al. (2007)
Oligo/azoospermia Chinese 285 515 12 Yes Yang et al. (2008)
Oligo/azoospermia 414 262 11

(<20 × 106 spermatozoa/mL) in a Danish compared with AMELY/X genes in 84 infer-
population (Krausz et al. 2001; McElreavey tile men and 40 controls. Surprisingly, the
and Quintana-Murci 2003), while several number of TSPY copies was significantly
“at-risk” Y haplogroups including hgK, hgC, higher in infertile men compared with the
and hgO3* were associated with spermato- controls (P = 0.002). The potential of using
genic failure in Chinese Han populations (Lu the relative number of TSPY copies in clini-
et al. 2007; Yang et al. 2008). However, cal diagnostics was also evaluated, and the
similar findings have not been observed in result indicated that the ratio TSPY/AMELY
other Japanese, Brazilian, and European pop- was a good marker to separate the infertile
ulations (Table 6.1) (Paracchini et al. 2000, from the control samples (Vodicka et al.
2002; Quintana-Murci et al. 2001; Carvalho 2007). It remains to be seen whether evalu-
et al. 2003, 2006), indicating that an associa- ation of the TSPY copy number offers a new
tion between Y chromosome polymorphism diagnostic approach in relation to the genetic
and a phenotype in one population may not cause of male infertility.
be relevant for another. This can be explained
by the fact that some haplotypes are largely It is rare to find that a point mutation in
confined to particular populations. the Y-linked genes causes spermatogenic
failure and infertility. The only report was
It has been confirmed that decrease (dele- the identification of a 4-bp deletion in a
tion) in the number of DAZ and CDY copies splice-donor site of the human USP9Y. This
in the AZFc region is associated with infer- deletion leads to an exon being skipped and
tility (Moro et al. 2000). However, it was not protein truncation. The man with this muta-
clear until recently that increase in gene tion showed non-obstructive azoospermia,
copy number has any effects on Y-linked but his fertile brother has a normal Y without
phenotypes. By a quantitative fluorescence the deletion, suggesting that the USP9Y
PCR approach, Vodicka et al. (2007) evalu- mutation caused spermatogenic failure (Sun
ated the relative number of TSPY copies et al. 1999).

144 Quantitative Genomics of Reproduction

An SNP (A/G transition) occurring in the available for the porcine (∼20) (McGraw
RNA-binding domain of human DAZL gene, et al. 1988; Mileham et al. 1988; Quilter
leading to Thr54→Ala change (T54A) of et al. 2002) and ovine (<10) (Meadows et al.
DAZL protein, conferred susceptibility to 2004) Y chromosomes. We have tested 38
severe spermatogenic failure in men (Teng bovine Y-linked MSs on male and female
et al. 2002). Interestingly, there is no report, genomic DNAs of sheep and goat by PCR.
to date, on whether a single point mutation The results indicated that 11 MSs were
(SNP or indel) in any Y-linked genes could amplified in male sheep DNA, and 10 MSs
directly lead to spermatogenic failure. in male goat DNA only, while 21 and 18
MSs amplified products from both male and
6.5.3 The potential to use Y-linked female DNA in sheep and goat, respectively.
markers for male fertility selection in All but one MS amplified multiple bands,
farm animals indicating the existence of multiple copies
of these markers in ovine and caprine Y
In contrast to sequence data on humans, chromosomes (W.-S. Liu and A.F. Ponce de
chimpanzees, and mice, Y chromosome León, unpublished data).
sequence information is very limited for
other mammals. Although considerable As far as Y-linked marker variation is con-
efforts have been made in the past few years cerned, 14 of the BTAY MSs were found to
by animal geneticists to characterize the Y be polymorphic in a small population of 17
chromosome of farm animals, progress in bulls. These polymorphic markers were
Y-specific marker development and associa- combined to produce unique haplotypes that
tion study is scarce. To date, over 300 DNA could be used to identify an individual or a
markers have been reported on the bovine Y group of bulls (Liu et al. 2003). It is expected
chromosome (reviewed by Liu and Ponce de that more BTAY polymorphic MSs could be
León 2007), which includes ∼80 microsatel- discovered in a large population or in animals
lites (MSs), ∼30 SNPs, and ∼200 STSs. The with more diversified genetic backgrounds,
majority of these markers have multicopies as data from an earlier investigation of four
or are part of the repetitive sequences on BTAY-specific MSs showed that they were
BTAY as demonstrated by radiation hybrid all highly polymorphic in different bovid
(RH) mapping (Liu et al. 2002; Liu and de species including domestic cattle, bison,
Leon 2004; Liu and Ponce de León 2007). mithan, buffalo, and yak (Edwards et al.
Approximately 1800 Y-linked BACs were 2000). Furthermore, Y halotypes on the basis
isolated and fingerprinted. A high-resolution of an intronic SNP (A/C) in bUTY and an
BTAY physical map is under construction intronic 2-bp indel in bZFY have been suc-
(W.S. Liu, unpublished data). The horse Y cessfully applied to the study of the origin
chromosome project has generated ∼300 of domestic cattle in Europe (Gotherstrom
Y-unique STSs including 32 gene-specific et al. 2005). In contrast to BTAY, the horse
markers (Raudsepp et al. 2004b; Wallner et Y chromosome is very low in variability.
al. 2004; Chowdhary et al. 2008). A BAC- One investigation found a single haplotype
based physical map of the equine Y chromo- in the domestic horse after analyzing three
some is available (Raudsepp et al. 2004b). Y-linked MSs in 49 male horses of 32 differ-
Compared with the bovine and equine Y ent breeds, whereas notable variation has
chromosomes, there are fewer markers been observed in the other members of the
genus Equus (Wallner et al. 2004). A separate

The Y Chromosome and Male Fertility 145

research obtained a similar result (Lindgren chromosome haplotypes in farm animals for
et al. 2004). Lindgren et al. investigated 34 marker-assisted selection (MAS) of fertility-
equine Y-linked STSs in 52 male horses related traits to select sires at an early age
from 15 breeds and found that all stallions (as early as newborn) in a breeding program.
carried the same Y chromosome haplotype. DNA-based MAS would allow us not only
Based on their sequence data, the authors to eliminate those young male animals that
further predicted that the Y chromosome may have genetic defects on the Y chromo-
polymorphism level of the domestic horse some, but also to select those that have a
was at least 10–30 times lower than that of potential to become a higher fertility male
humans (Lindgren et al. 2004). early on during the progeny test. This is par-
ticularly important for large animals such as
At present, there is no report on the asso- cattle and horses as subfertile or infertile
ciation of Y-linked polymorphic marker(s) bulls/stallions are usually not identified
with male fertility in farm animals except until the age of 18–24 months when they are
for one report on the bovine DAZL gene (Liu expected to breed. Most importantly, early
et al. 2008). There are two main reasons for decision on selecting/eliminating a young
this. One is that the development of farm male in progeny test will significantly reduce
animal Y chromosome markers, especially breeding costs.
in searching for testis gene polymorphic
markers, is insufficient to carry out the 6.6 Future research directions
association study with male fertility. The
other reason is that, unlike humans, within The mammalian Y chromosome is unique
which male patients of infertility or subfer- in many aspects. It is present in the males
tility are recorded and analyzed in details, only and is full of repeated sequences. It is
male animals of infertility or subfertility are responsible for vital biologic roles in sex
usually eliminated from the breeding program determination and male fertility. Since very
without genetic evaluation. Therefore, the limited numbers of autosomal genes associ-
lack of adequate DNA samples further slows ated with measures of male germ cell defects
down the molecular study of male infertility have been identified in mammals (Tung
(or subfertility) in farm animals. et al. 2006; Matzuk and Lamb 2008), the Y
chromosome “testis genes” have become the
Infertility or subfertility is a common most important source for molecular study
problem in farm animals (Steffen 1997). of male fertility/infertility. The sequencing
For example, it was estimated in the beef of the human Y chromosome has provided
industry that as high as 18–30% of beef us with a big (but not complete) picture of
bulls used in natural service were reproduc- the mammalian Y chromosome in terms of
tive deficient (Coulter 1980; Coulter and its structure and gene content. Functional
Kozub 1980). Given the facts that the Y studies of the human and mouse Y chromo-
chromosome genes in humans and mice some genes have identified several candi-
play an essential role in spermatogenesis dates for azoospermia/oligozoospermia and
and fertility, and the recent findings that infertility. The association of Y chromo-
species-specific genes are present on the Y some polymorphisms (haplogroups) with
chromosome of cattle, horses, cats, and dogs azoospermia/oligozoospermia in humans
(Murphy et al. 2006; Paria et al. 2008), I
believe that there is a huge potential to
use Y-linked gene polymorphisms and Y

146 Quantitative Genomics of Reproduction

has opened the door for a possibility of using Briton-Jones, C. and Haines, C.J. 2000.
Y chromosome haplotypes/haplogroups for Microdeletions on the long arm of the
male fertility selection in farm animals. Y chromosome and their association
But significant improvements in animal Y with male-factor infertility. Hong Kong
chromosome sequence information (such Medical Journal 6: 184–189.
as gene content, polymorphisms, and hap-
lotypes) and association analysis with fertil- Brown, G.M., Furlong, R.A., Sargent, C.A.,
ity traits are desperately necessary before Erickson, R.P., Longepied, G., Mitchell,
designing any Y chromosome gene-based M., Jones, M.H., Hargreave, T.B., Cooke,
MAS strategy for male fertility selection. H.J., and Affara, N.A. 1998. Characterisa-
The recent discovery of novel Y chromo- tion of the coding sequence and fine
some genes by “direct cDNA selection” mapping of the human DFFRY gene and
in cattle, horses, and cats holds promise comparative expression analysis and
for the further identification and analysis mapping to the Sxrb interval of the mouse
of genetic factors that are involved in regu- Y chromosome of the Dffry gene. Human
lating spermatogenesis and fertility. This Molecular Genetics 7: 97–107.
approach will eventually lead to the comple-
tion of a “mammalian Y gene catalog” even Caron, C., Pivot-Pajot, C., van Grunsven,
without sequencing the Y chromosome in L.A., Col, E., Lestrat, C., Rousseaux, S.,
all species. In conclusion, the Y chromo- and Khochbin, S. 2003. Cdyl: A new tran-
some is a “gold mine” in the field of molecu- scriptional co-repressor. EMBO Reports 4:
lar evolution and male fertility; the more we 877–882.
study the Y chromosome, the more knowl-
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Mitochondriomics of Reproduction and Fertility

Zhihua Jiang, Galen A. Williams, Jie Chen, and Jennifer J. Michal

7.1 Introduction smaller than those of free-living bacteria and
thus containing significantly fewer genes
Mitochondria are membrane-enclosed organ- (Selosse et al. 2001). For example, in humans,
elles found in most eukaryotic cells but only 37 genes are encoded by mitochondrial
located in the cytoplasm outside their nuclei. DNA (mtDNA) and transcribed within the
In animals, a typical cell contains 1000– mitochondrion. Most of the original mito-
2000 mitochondria. Primarily, mitochondria chondrial genes have been transferred to the
convert nutrients into chemical energy, thus nucleus, leading to a marked loss of coding
supporting a cell’s activities and functions. capacity compared with that of their closest
In addition, mitochondria also play an impor- bacterial relatives (Smith et al. 2005).
tant role in many other metabolic tasks, Approximately 1000–1500 mitochondrial
such as apoptosis-programmed cell death, genes were functionally transferred to the
glutamate-mediated excitotoxic neuronal nucleus, leading to the birth of a set of so-
injury, cellular proliferation, regulation of called nucleus-encoded mitochondrial genes
the cellular redox state, heme synthesis, and (Chinnery 2003).
steroid synthesis (McBride et al. 2006; Nisoli
et al. 2007; Sas et al., 2007; Schwarz et al. Therefore, in a broad sense, the mitochon-
2007). drial genome encompasses genes located
in both the cytoplasm and the nucleus.
It has been widely believed that the mito- Recently, the scientific and medical com-
chondrial genomes in eukaryotic cells are munity has found that the functional
of endosymbiotic origin; that is, they are status of mitochondria contributes to the
derived from a once-free living bacterium, quality of oocytes and spermatozoa, and
almost certainly an α-proteobacterium plays a part in the process of fertilization
(Gray 1999). Interestingly, the mitochon- and embryo development. This chapter
drial genome size has gradually receded over reviews recent information about involve-
a long period of symbiosis, being ∼100-fold ments of both cytoplasm and nuclear


158 Quantitative Genomics of Reproduction

mitochondrial genomes in fertility and size of the mitochondrial genome varies
reproduction. from 16,319 bp (NC_012346) to 17,245 bp
(NC_001913) among these 12 agriculturally
7.2 Cytoplasm mitochondrial related species.
genomes in fertility
and reproduction In mammals, each circular mtDNA mol-
ecule harbors genetic material that encodes
7.2.1 Mammalian mitochondrial the same 37 genes: 13 for proteins (polypep-
genomes in the cytoplasm tides), 22 for transfer RNA (tRNA), and one
each for the small and large subunits of ribo-
The mitochondrial genome is present in somal RNA (rRNA). Among these 12 agri-
mitochondria as a chromosome or a circular culturally related species, all 37 genes plus
DNA molecule. Unlike nuclear chromo- D-loop and replication of origin regions are
somes that are paired in mammals (except X placed on the circular DNA in the same ori-
and Y sex chromosomes), there are many entation (Table 7.1). Of 13 coding mitochon-
copies of the mitochondrial chromosome in drial genes, four genes––COX1, ND4L, ND4,
every cell. However, the copy number can and ND6 remain evolutionally consistent in
be extremely variable in different cells. For terms of their sizes of coding sequence. The
example, an egg contains 100,000–1,000,000 D-loop region seems very variable, ranging
mtDNA molecules, while a sperm contains from 888 bp in B. bison to 1800 bp in O.
only 100–1000. During fertilization, the cuniculus. Interestingly, one-third of these
sperm mtDNA is degraded. Therefore, no genes/regions (13/39) in the mitochondrial
matter whether we are male or female, we genome have the potential to overlap with
inherit our mitochondrial chromosome from one of the adjacent neighbors (Table 7.1).
our mother. In other words, the mitochon- In addition, the genetic code in mitochon-
drial chromosome is transmitted in a matri- dria is different from the genetic code in
linear manner. the nucleus. For example, UGA is read as
tryptophan rather than “stop,” AGA and
As the mitochondrial genomes are rela- AGG as “stop” rather than arginine, AUA
tively small in size, it is relatively easy to as methionine rather than isoleucine, and
sequence them. Up to date, mtDNA has AUA or AUU is sometimes used as an initia-
been completely sequenced in species related tion codon instead of AUG (Anderson et al.
to farming and agriculture, such as Bison 1982).
bison (American bison, NC_012346), Bos
grunniens (domestic yak, NC_006380), 7.2.2 Special features of
Bos indicus (zebu, AF492350), Bos taurus mitochondrial genetics
(domestic cattle, NC_006853), Bubalus
bubalis (water buffalo, NC_006295), Capra The cytoplasmic location of mtDNA and
hircus (goat, NC_005044), Equus asinus the high copy number contribute to certain
(donkey, NC_001788), Equus caballus unique features of mitochondrial genetics.
(horse, NC_001640), Lama glama (llama, First, as mentioned above, mtDNA is mater-
NC_012102), Oryctolagus cuniculus (rabbit, nally inherited. Three theories have been
NC_001913), Ovis aries (sheep, NC_001941), developed to explain the maternal control
and Sus scrofa (swine, NC_000845). The of mitochondrial inheritance in mammals.
The mtDNA dilution theory is based on the

Mitochondriomics of Reproduction and Fertility 159

Table 7.1 Characterization of mitochondrial genomes in 12 agriculturally related animals.1

Gene or region Orientation Gene size (in bp) Gene distance (in bp)

D-loop + 888–1800 1
tRNA-Phe + 67–71 1
s-rRNA + 955–975 0–3
tRNA-Val + 66–68 1
l-rRNA + 1569–1581 1
tRNA-Leu + 74–75 3–4
ND1 + 955–957 (−1)–2
tRNA-Ile + 69–70 (−2)
tRNA-Gln − 72–73 2–3
tRNA-Met + 69–70 1
ND2 + 1041–1044 (−1)–1
tRNA-Trp + 67–70 2–7
tRNA-Ala − 67–69 1–2
tRNA-Asn − 73–75 1
Rep-origin + 23–35 (−5)–10
tRNA-Cys − 66–68 0–1
tRNA-Tyr − 66–68 2–8
COX1 + 1545 (−2)–4
tRNA-Ser − 69–71 4–7
tRNA-Asp + 67–70 1–2
COX2 + 684–688 1–4
tRNA-Lys + 67–69 1–2
ATP8 + 201–204 (−42)–(−39)
ATP6 + 679–681 0–2
COX3 + 781–784 1–4
tRNA-Gly + 68–70 (−1)–1
ND3 + 340–350 1–2
tRNA-Arg + 67–69 1–2
ND4L + 297 (−6)
ND4 + 1378 1
tRNA-His + 69–71 1
tRNA-Ser + 59–60 1–2
tRNA-Leu + 70–71 1
ND5 + 1812–1821 (−4)–(−16)
ND6 − 528 1
tRNA-Glu − 68–69 4–7
CYTB + 1139–1140 1–5
tRNA-Thr + 66–74 0–2
tRNA-Pro − 64–70 1

1 Gene distance ≤0 means that there is (are) ≥1 nucleotide(s) overlapped, while gene distance ≥2 means that there is (are) ≥1 intergenic
nucleotide(s) between two adjacent neighbor genes. When gene distance is equal to 1, it means that no overlap or intergenic sequences
exist between two adjacent neighbor genes.

fact that the mammalian egg contains dria cannot enter into the oocyte cytoplasm
about 100,000 copies of mtDNA, whereas at fertilization. Using the polymerase chain
the sperm harbors 100–1500 molecules of reaction (PCR), Gyllensten and colleagues
mtDNA (Manfredi et al. 1997). This means (1991) detected paternally inherited mtDNA
that the theoretic ratio of paternal versus molecules in mice at a frequency of 10(−4),
maternal mitochondria is ∼1 : 1000. However, relative to the maternal contributions.
this does not mean that paternal mitochon- Therefore, the dilution theory cannot explain

160 Quantitative Genomics of Reproduction

why mtDNA is only maternally inherited. Third, mitochondria undergo replicative
The oxidative damage theory (Allen 1996) segregation at cell division. Each cell
suggests that sperm mitochondria are recog- contains hundreds of mitochondria, each
nized by oocyte cytoplasm because of the having 2–10 copies of mtDNA molecules.
oxidative damage they suffer during passage This polyploid nature of the mitochondrial
through the female genital tract. However, genome––up to several thousand copies per
in vitro fertilization experiments do not cell––gives rise to an important feature of
support this hypothesis because oxidative mitochondrial genetics, homoplasmy, and
damage is minimal during the process heteroplasmy. In simple terms, homoplasmy
(Aitken 1995). Sutovsky and colleagues is when all copies of the mitochondrial
(2000) proposed a feasible explanation for genome are identical; heteroplasmy is when
maternal mtDNA inheritance in mammals. there is a mixture of two or more mitochon-
Using bull semen and cow eggs, the authors drial genotypes. At cell division, the mito-
observed that sperm mitochondria are ubiq- chondria and their genomes are randomly
uitinated inside the oocyte cytoplasm and distributed to the daughter cells, a process
later subjected to proteolysis during preim- known as replicative segregation. Therefore,
plantation development. Therefore, paternal when mutant and wild-type mtDNA coexist
mitochondria are degraded during early in affected individuals (the condition of
embryogenesis, leaving only maternal mito- heteroplasmy), the proportion of mutant
chondria in the cytoplasm. mtDNA transmitted from mother to off-
spring is variable because of a genetic bottle-
Second, mtDNA genes have a much higher neck, and the “dose” of mutant mtDNA
mutation rate than nuclear DNA genes. received influences the severity of the phe-
Various surveys of mutation accumulation notype (Marchington et al. 1998).
in mitochondrial and nuclear genomes of
animals and plants have consistently found Fourth, clinical expression of mitochon-
that the former genomes accumulate delete- drial disease or defect depends on the thresh-
rious mutations at a higher rate than the old effect. In the presence of heteroplasmy,
latter genomes. In a recent review, Neiman there is a threshold level of mutation that is
and Taylor (2009) concluded that asexuality important for the clinical expression of
might not be the primary determinant of disease and for biochemical defects. This
the high mutation load in mtDNA. Instead, means that the clinical expression of a
the authors proposed that a high rate of pathogenic mtDNA mutation is largely
accumulation of mildly deleterious muta- determined by the relative proportion of
tions in mtDNA may result from the small normal and mutant mtDNA genomes in
effective population size associated with different tissues. A minimum critical
effectively haploid inheritance, because this number of mutant mtDNAs is required to
type of transmission is nearly ubiquitous cause mitochondrial dysfunction in a par-
among mitochondrial genomes. In addition, ticular organ or tissue, resulting in a mito-
polymorphisms accumulate approximately chondrial disease. This is the so-called
10–17 times faster in mtDNA compared threshold effect. For example, the threshold
with nuclear DNA. This may be explained varies for different mtDNA mutation types
by the lack of an efficient DNA repair system and is about 60% for deleted mtDNA
in mitochondria and histones associated (Hayashi et al. 1991). For the mutation
with mtDNA (Wallace et al. 1997). 8344A>G that causes the syndrome of

Mitochondriomics of Reproduction and Fertility 161

myoclonic epilepsy and ragged-red fibers, evidence suggests that defects in mitochon-
the threshold level is about 85% mutated drial respiration result in an overall reduc-
DNA (Chomyn 1998). tion in fertility and reduced sperm motility
(Nakada et al. 2006). This may be a clue to
Fifth, somatic mtDNA mutations accu- the molecular basis for the relationship
mulate in post-mitotic tissues with age, between mtDNA content and fertility status
reducing the ATP-generating capacity. At in the reports above. Despite what is known
cell division, the proportion of mutant about mtDNA and its role in male fertility,
mtDNAs in daughter cells may shift and the this field is still in its infancy.
phenotype may change accordingly. This
phenomenon, called mitotic segregation, 7.2.4 Mitochondria and female fertility
explains how certain patients with mtDNA-
related disorders may actually manifest dif- In many mammalian species, mitochondria
ferent mitochondrial diseases at different are the most abundant organelle in fully
stages of their lives. grown oocytes detected by electron micros-
copy (Van Blerkom 2004). However, deter-
7.2.3 Mitochondria and male fertility mining if mtDNA mutations cause pre- or
postimplantation embryo demise can be dif-
Faulty mitochondrial function and reduced ficult because blastocysts used for in vitro
copy numbers are linked to male subfertility fertilization (IVF) may be unavailable and
and sperm dysfunction (Folgero et al. 1993; even when they are available, they may be
Kao et al. 1995). In addition, new assays for an unsuitable model to study mtDNA
evaluating the functional status of mito- defects (Van Blerkom 2004). It is unclear
chondria in sperm have been developed. whether mtDNA plays a role in apoptosis of
Using mitochondria-specific stains and fluo- oocytes in adult life (Jansen and de Boer
rochromes, it has been shown that higher 1998). Overall, there are little data available
mitochondrial function is correlated to relating to mitochondriomics of oogenesis.
increased fertilization rates in vitro. These Using 422 beef cattle of two different breeds
stains also enable the assessment of sperm (purebred Hereford and composite multi-
viability when using different semen extend- breed), Sutarno and colleagues (2002) found
ers (Huo and Scarpulla 2001). Interestingly, a significant association between calving
increased mtDNA amplification was found rate and mitochondrial polymorphisms in
in abnormal sperm from infertile patients both breeds. Calving rate is defined as the
(May-Panloup et al. 2003). Further, signifi- mean number of live calves born per year
cantly higher mtDNA deletions have been over 4 years. As pointed out by the authors,
detected in diabetic men when compared the association may have implications for
with normal controls (Agbaje et al. 2007). genetically improving cow fertility.
An analysis of human mtDNA polymerase
revealed an association between male infer- 7.2.5 Mitochondria and
tility and the absence of a common CAG reproductive aging
microsatellite allele (Rovio et al. 2001).
While differences in mtDNA are associated It has long been known that aging in mammals
with male infertility, these measures are results in reduced fertility and increased
currently considered “proxys” for male fer- frequency of abnormal development. The
tility status (St. John et al. 1997). However,

162 Quantitative Genomics of Reproduction

mitochondrial theory of aging is by no means the transferred genes physically make that
universally accepted, but it is likely that intracellular journey––as RNA, as cDNA, as
mitochondria do play a role in male and pieces of organelle DNA, or as whole organ-
female reproductive aging. An analysis elle chromosomes? Two opinions exist:
of mtDNA samples from females widely direct DNA transfer and cDNA-mediated
varying in age found a considerably large transfer. But Henze and Martin (2001) pro-
(∼5 kb) deletion in tissues from abnormal posed that direct DNA transfer, rather than
tissues in the reproductive tract as well as cDNA-mediated transfer, probably prevailed
nonreproductive muscle. This deletion was during the early phases of organelle evolu-
predominately found in menopausal and tion. Mathematical model analysis revealed
postmenopausal women and suggested that that the rate of gene transfer from mitochon-
aging may be closely related to ovarian dria to the nucleus could be affected by three
dysfunction (Kitagawa et al. 1993). In addi- factors: the intensity of intracellular compe-
tion to this common 5-kb mtDNA deletion, tition, the probability of paternal organelle
additional deletions in mtDNA are more transmission, and the effective population
common in oocytes from older women when size (Yamauchi 2005). Gene transfer rate
compared with those from younger women tends to increase with decreasing intracel-
(Keefe et al. 1995). Others reported a correla- lular competition, increasing paternal organ-
tion between increased oocyte volume and elle transmission, and decreasing effective
maternal age, but to date this has not been population size. Intense intracellular compe-
associated with mtDNA mutations or func- tition tends to suppress gene transfer because
tional defects (Müller-Höcker et al. 1996). it is likely to exclude mutant mitochondria
Interestingly, mtDNA deletions are more that lose the essential gene due to the produc-
frequent in oocytes than embryos, partially tion of lethal individuals. In fact, most
supporting a “bottleneck” theory that filters researchers believe that functional transfer
out mutated mtDNA in conjunction with of mitochondrial genes has ceased in animals
perhaps another nuclear mechanism (Brenner (Boore 1999).
et al. 1998). Similarly, rearrangements in
mtDNA are more common in oocytes than There are several databases publicly avail-
in embryos (Barritt et al. 1999). able that provide information on nucleus-
encoded mitochondrial genes/proteins. The
7.3 Nuclear mitochondrial Human Mitochondrial Protein Database
genomes in fertility (HMPDb) ( provides com-
and reproduction prehensive data on mitochondrial and
human nuclear encoded proteins involved
7.3.1 Mammalian mitochondrial in mitochondrial biogenesis and function.
genomes in the nucleus This database consolidates information
from SwissProt, LocusLink, Protein Data
A central component of the endosymbiotic Bank (PDB), GenBank, Genome Database
theory is that mitochondria originated as (GDB), Online Mendelian Inheritance
bacterial intracellular symbionts. However, in Man (OMIM), Human Mitochondrial
many of the original genes from bacteria were Genome Database (mtDB), MITOMAP,
transferred to the nucleus. In what form did Neuromuscular Disease Center, and Human
2-D PAGE Databases. The database also
provides tools for database search, mtDNA

Mitochondriomics of Reproduction and Fertility 163

sequence visualization, mtDNA polymor- mitochondrial genes are not yet annotated
phism, mitochondrial protein-related dis- in domestic animals. Here, we propose to
eases, and 3D structures of mitochondrial use a comparative annotation approach to
proteins. The MitoP2 database (http://www. retrieve both cDNA and genomic DNA integrates informa- sequences for each nucleus-encoded mito-
tion on mitochondrial proteins, their molec- chondrial gene in livestock species in three
ular functions, and associated diseases steps. In step 1, we use the cDNA sequences
(Prokisch et al. 2006). As stated by the devel- of the human orthologs as references for
opers, the MitoP2 enables (1) the identi- BLAST (basic local alignment search tool)
fication of putative orthologous proteins searches to retrieve the orthologous cDNA
between these species to study evolution- sequences against the GenBank database
arily conserved functions and pathways, “nr” or the orthologous expressed sequence
(2) the integration of data from systematic tags (ESTs) against the GenBank database
genome-wide studies such as proteomics and “est_others” with a species option limited
deletion phenotype screening, (3) the pre- to livestock species, for example, B. taurus.
diction of novel mitochondrial proteins The cDNA sequences in the “nr” database
using data integration and the assignment represents three categories: cDNA sequences
of evidence scores, and (4) systematic derived from a full-length cDNA library,
searches that aim to find the genes that known gene cDNA sequences, or annotated
underlie common and rare mitochondrial cDNA sequences compiled by the GenBank
diseases. Other databases include the Human staff. In step 2, we choose the longest cDNA
Genome Resources at the National Center sequence retrieved from the “nr” database
for Biotechnology Information (NCBI), the or one assembled from several ESTs retrieved
MitoProteome—an object-relational mito- from the “est_others” database to form a
chondrial protein sequence database at the primary cDNA sequence for the cattle gene.
University of California, San Diego, CA This sequence will then be used to perform
(Cotter et al. 2004), and the MitoRes—a a species-specific BLAST search against the
bio-sequences resource for mitochondria- “est_others” database in order to expand the
related genes, transcripts, and proteins at the primary sequence to a full-length cDNA
Institute of Biomedical Technologies, CNR, sequence. For step 3, we use the full-length
Italy (Catalano et al. 2006). cDNA sequence to search for genomic DNA
contigs. Such a process will retrieve both
Approximately 1300 nucleus-encoded cDNA and genomic DNA sequences of each
mitochondrial protein-coding genes have nucleus-encoded mitochondrial gene from
been well annotated in the human genome, the public database of different livestock
including 113 on HSA1, 75 on HSA2, 64 species.
on HSA3, 46 on HSA4, 54 on HSA5, 53 on
HSA6, 59 on HSA7, 36 on HSA8, 55 For example, the annotation of the
on HSA9, 61 on HSA10, 79 on HSA11, 68 on bovine mitochondrial transcription factor
HSA12, 17 on HSA13, 51 on HSA14, 37 B1 (TFB1M) is shown in Figure 7.1 and dem-
on HSA15, 57 on HSA16, 76 on HSA17, onstrates how to utilize the EST database
16 on HSA18, 70 on HSA19, 21 on HSA20, for annotation as outlined above. First, a
12 on HSA21, 44 on HSA22, and 50 cDNA sequence of the human ortholog
on human × chromosome, respectively. (NM_016020) was used as a reference, and a
However, most of these nucleus-encoded BLAST search retrieved more than 50 bovine

164 Quantitative Genomics of Reproduction

Figure 7.1 Compilation and annotation of both cDNA and genomic DNA sequences for the bovine TFB1M
gene. Sequence length is not in the same scale.

ortholgous ESTs against the GenBank tures. First, these mitochondrial genes are
database “est_others.” Second, three ESTs not evenly distributed in the nuclear genome.
(DT811342, DT815256, and CB447193) were Based on the locations of 1298 human
chosen and assembled to form a primary nucleus-encoded mitochondrial genes, the
cDNA sequence for the bovine TFB1M gene, mitochondrial gene density is calculated as
which was then used to perform a species- Mitochondrial Gene Density = (N(mt)/N)/
specific EST search for expansion. Three (T(mt)/T), where N(mt) is the number of
additional ESTs (DT840260, CK943822, and mitochondrial genes within a window of
CB430084) were added to form a full-length 5 Mbp on a chromosome, N is the number
cDNA sequence of 1626 bp. Finally, the full- of genes within a window of 5 Mbp on a
length cDNA sequence retrieved a genomic chromosome, T(mt) is the number of total
DNA contig (AAFC03094296) from the mitochondrial genes on all chromosome
7.15X bovine genome sequence database and (1298), and T is the number of total genes on
thus determined its genomic organization all chromosomes (28,644) (Build 33.0). The
(Figure 7.1). mitochondrial gene density plots are illus-
trated along each chromosome with an
7.3.2 Special features of mitochondrial interval of 1 Mb (Figure 7.2). Overall, the
genome in the nucleus mitochondrial genes tend to be clustered but
are not evenly distributed along each human
The transfer of mitochondrial genes to the chromosome. Some chromosomes (e.g.,
nucleus contributes to certain unique fea- human chromosome 1, HSA1) are very rich

165 Figure 7.2 Distribution of nucleus-encoded mitochondrial genes in the human nuclear genome.

166 Quantitative Genomics of Reproduction

30,000 Chi-squares
25,000 Pearson’s = 1056.55 (P = 0.000000000)



5000 Total Overlapping Overlapping
1363 468 2204

0 Nuclear genes
Mito-nuclear genes

Figure 7.3 Overlapping genes associated with mitochondrial genes and nuclear genes.

in mitochondrial genes, while some (e.g., with neighbors (Figure 7.3). In contrast,
HSA13) contain few mitochondrial genes. among the rest of the nuclear genes in the
human genome, only 8% (2204/27,254) con-
Second, nucleus-encoded mitochondrial tribute to the overlapping cases. Therefore,
genes tend to overlap adjacent neighbors or transfer of mitochondrial genes into the
cause overlapping of adjacent genes with nucleus might play an important role in the
their neighbors. In mammalian genomes, evolutionary formation of overlapping genes
hundreds of pairs of protein-coding overlap- in the nucleus.
ping genes have been reported so far.
Overlapping genes might play an important Third, translation efficiency remains in
role in various levels of gene expression nucleus-encoded mitochondrial genes. The
control, such as transcription, mRNA pro- Kozak consensus sequence is referred to as
cessing, splicing, stability, transport, and a sequence that occurs on eukaryotic mRNA
translation. However, the evolutionary and has the consensus (gcc)gccRccAUGG,
origin of such genes is not known, existing where R is a purine (adenine or guanine)
hypotheses can explain only selected cases three bases upstream of the start codon
of mammalian gene overlaps, which could (AUG), which is followed by another “G”
originate as a result of rearrangements, over- (Kozak 1987). The Kozak consensus sequence
printing and/or adoption of signals in the plays a major role in the initiation of transla-
neighboring gene locus (Makalowska et al. tion. Figure 7.4 shows frequencies of the
2005). Based on a total of 1363 mitochon- Kozak consensus nucleotides between mito-
drial coding genes and pseudogenes in the chondrial and nuclear genes in the human
human nuclear genome, we found that 468 nuclear genome. The mitochondrial genes
(34%) genes overlap adjacent neighbors or adapted the translation systems after trans-
cause overlapping of their adjacent genes fer into the nucleus and even contain a

Mitochondriomics of Reproduction and Fertility 167



–9 –8 –7 –6 –5 –4 –3 –2 –1 Start 12345

Nuclear mitochondria genes Nuclear genes

Figure 7.4 The Kozak sequence between mitochondrial genes in nucleus and other nuclear genes.

stronger consensus sequence within the lar development, and Sertoli cell function,
translation binding site. as well as appropriate maturation, motility,
and penetration of the sperm. The following
Fourth, transfer of mitochondrial genes nucleus-encoded mitochondrial genes affect
into the nucleus might contribute to genome spermatogenesis in males based on knock-
evolution. For example, the TFAM gene out or deficiency mice models, including
neighborhood is rearranged in both human cannabinoid receptor 1 (CNR1), guani-
and bovine genomes, causing a singleton dinoacetate N-methyltransferase (GAMT),
(one gene) synteny between two species. For Huntingtin interacting protein 1 (HIP1),
the TFB1M gene, intron 5 harbors a func- inner mitochondrial membrane peptidase
tional gene CLDN20, while its 3′UTR over- 2-like (IMMP2L), pantothenate kinase 2
laps with the 3′UTR of TIAM2 in human, (PANK2), protein phosphatase 1, catalytic
while CLDN20 might no longer be expressed subunit, gamma isoform (PPP1CC), sirtuin
in cattle. In humans, a gene (LOC100132379) (silent mating type information regulation
similar to TBC1 domain family member 3 is 2 homolog) 1 (Saccharomyces cerevisiae)
inserted into the adjacent region of TFB2M, (SIRT1), sperm mitochondria-associated cys-
which does not occur in the bovine genome. teine-rich protein (SMCP), TAF7-like RNA
polymerase II, TATA box binding protein
7.3.3 Nucleus-encoded mitochondrial (TBP)-associated factor (TAF7L), and voltage-
genes and male fertility dependent anion channel 3 (VDAC3).

Spermatogenesis is a complex process that CNR1 encodes one of two cannabinoid
requires normal germ cell migration, testicu- receptors. The cannabinoids, basically

168 Quantitative Genomics of Reproduction

delta-9-tetrahydrocannabinol and synthetic observed that velocities, amplitude of lateral
analogs, are psychoactive ingredients of head displacements, and numbers and per-
marijuana. The cannabinoid receptors are centages of sperm in the motile, rapid, and
members of the guanine-nucleotide-binding progressive categories were all significantly
protein (G-protein) coupled receptor family, reduced in Hip1−/−mice. The authors con-
which inhibit adenylate cyclase activity in cluded that HIP1 plays an important role in
a dose-dependent, stereoselective and per- stabilizing actin and microtubules, which
tussis toxin-sensitive manner. It has been are important cytoskeletal elements enabling
reported that Cnr1 is expressed in mature normal spermatid and Sertoli cell mor-
sperm, and the sperm from Cnr1-knockout phology and function (Khatchadourian et al.
mice show a dramatic increase in motility 2007).
in the caput epididymis (Rossato et al. 2008).
The mitochondrial inner membrane pep-
GAMT encodes a protein called methyl- tidase (IMP) complex generates mature,
transferase, which catalyses the synthesis active proteins in the mitochondrial inter-
of creatine from guanidinoacetate and membrane space by proteolytically remov-
S-adensylmethionine. Schmidt et al. (2004) ing the mitochondrial targeting presequence
generated a knockout mouse model for Gamt of nuclear-encoded proteins. IMMP2L is
deficiency by gene targeting in embryonic one of the catalytic subunits of the IMP
stem cells. Although some Gamt-deficient complex (Burri et al. 2005). Using a trans-
males are—at least temporarily—fertile, the genic insertional mutagenesis strategy, Lu
authors observed severely attenuated sper- et al. (2008) generated a mouse mutant,
matogenesis in knockout mice at the level Immp2lTg(Tyr)979Ove, with a mutation in
of spermatid development; that is, elongated the Immp2l gene. The mutation affected the
spermatids were hardly present and mature signal peptide sequence processing of mito-
spermatozoa were almost absent in the chondrial proteins cytochrome c1 and glyc-
lumen of seminiferous tubules. Furthermore, erol phosphate dehydrogenase 2. The authors
the authors found that the structure of the observed that the homozygous mutant males
seminiferous tubules was highly unordered were severely subfertile due to erectile
with a large number of resorption holes dysfunction.
and a larger lumen. In addition, breeding
between male Gamt−/− mice and wild-type PANK2 encodes a protein belonging to the
or heterozygous mutant females produced pantothenate kinase family and is the only
no litters, indicating impaired fertility in member of that family that is expressed in
Gamt-deficient males (Schmidt et al. 2004). mitochondria. Pantothenate kinase cata-
lyzes the first committed step in the univer-
HIP1 is an endocytic adaptor protein sal biosynthetic pathway leading to CoA and
with clathrin assembly activity that binds is itself subject to regulation through feed-
to cytoplasmic proteins, such as F-actin, back inhibition by acyl CoA species. Kuo
tubulin, and huntingtin. Khatchadourian and colleagues found that homozygous
and colleagues (2007) performed a quantita- Pank2 knockout male mice are infertile
tive analysis of sperm counts from the caudal due to azoospermia. At 6 weeks, these
epididymis of Hip1−/−mice and found a sig- Pank2−/−males had smaller testes and lacked
nificant decrease compared with wild-type sperm in the epididymis. Histological analy-
littermates. In addition, using computer- ses showed a complete absence of elongated
assisted sperm analyses, the authors also spermatids in the seminiferous tubules of

Mitochondriomics of Reproduction and Fertility 169

these males. In particular, the spermato- Nayernia and coworkers (2002) reported that
cytes and spermatids are somewhat disor- all Smcp−/− males with a 129/Sv background
dered in the Pank2 testes tubule (Kuo et al. are infertile despite normal sexual behavior
2005). toward female mice and production of copu-
lation plugs. In vitro fertilization revealed
Serine/threonine protein phosphatase 1 that only 24.5% of oocytes with intact zona
(PP1) consists of four ubiquitously expressed pellucida were fertilized by Smcp-deficient
major isoforms, two of which, PP1gamma1 sperm and 14% of all oocytes developed into
and PP1gamma2, are derived by alternative normal blastocysts, while 82.6% of eggs
splicing of a single gene, protein phospha- were fertilized with sperm from the wild-
tase 1, catalytic subunit, gamma isoform type counterparts and 54% of them devel-
(PPP1CC). Targeted disruption of the Ppp1cc oped into blastocysts. These results indicate
gene causes male infertility in mice due to that the infertility of the male Smcp−/− mice
impaired spermiogenesis, as reported by with a 129/Sv background is due to reduced
Chakrabarti et al. (2007). Seminiferous motility of the spermatozoa and decreased
tubules from Ppp1cc-null testes had multi- capability of the spermatozoa to penetrate
ple vacuoles, sloughing of germ cells into oocytes (Nayernia et al. 2002).
the lumen, and mislocated germ cells.
In comparison with wild-type animals, the TAF7L is an X-linked germ cell-specific
number of elongating spermatids in each paralogue of TAF7, which is a generally
seminiferous tubule was reduced in Ppp1cc- expressed component of TFIID. Cheng et al.
null testes. Furthermore, epididymides from (2007) generated Taf7l mutant mice by
Ppp1cc-null males contained immature homologous recombination in embryonic
germ cells. stem cells. The authors observed that the
weight of Taf7l−/Y testis was lower, and the
In mammals, SIRT1 is a member of the amount of sperm in the epididymides was
sirtuin family of proteins and plays an sharply reduced, although spermatogenesis
important physiological role in the regula- is completed in mutant mice. Mutant epi-
tion of glucose metabolism, cell survival, didymal sperm exhibited abnormal mor-
and mitochondrial respiration. Coussens phology, including folded tails. Overall,
et al. (2008) produced Sirt1−/−mice and found Taf7l−/Y males are fertile, but with reduced
that Sirt1 deficiency markedly attenuates sperm motility and litter size (Cheng et al.
spermatogenesis. The authors observed that 2007). Evidence has shown that there is a
numbers of mature sperm and spermato- possible association of point mutation in
genic precursors were significantly reduced exon 13 of the human TAF7L with infertility
(∼2- to 10-fold less; P ≤ 0.004 in Sirt1−/−mice. (Akinloye et al. 2007).
However, the proportion of mature sperm
with elevated DNA damage (∼7.5% of total Voltage-dependent anion channels
epididymal sperm; P = 0.02) was signifi- (VDACs), also known as mitochondrial
cantly increased in adult Sirt1−/− males porins, are small channel proteins involved
(Coussens et al. 2008). in the translocation of metabolites across the
mitochondrial outer membrane. Sampson
SMCP is a cysteine- and proline-rich et al. (2001) reported that mice lacking Vdac3
structural protein that is closely associated are healthy, but males are infertile. When
with the keratinous capsules of sperm mito- Vdac3-deficient male mice were mated with
chondria in the mitochondrial sheath sur- female mice, they demonstrated normal
rounding the outer dense fibers and axoneme.

170 Quantitative Genomics of Reproduction

copulatory behavior, as evidenced by the uteri appeared threadlike in appearance with
presence of vaginal plugs in their mates, but noticeable thinning of the endometrium,
no pregnancies were observed in over 100 and the ovaries contained no corpora lutea.
matings. Although there are normal sperm In addition, circulating concentrations of
numbers, the sperm exhibit markedly pituitary luteinizing hormone were also
reduced motility. In particular, 68% of reduced in Crtc1 mutants compared with
Vdac3−/− epididymal sperm axonemes (247/ the controls.
362) in cross section had some structural
aberration, most commonly loss of one outer DEAD (Asp-Glu-Ala-Asp) box polypeptide
doublet from the normal 9 + 2 microtubule 20 (DDX20) is one of the DEAD box proteins
doublet arrangement. This compared with that are implicated in a number of cellular
structural abnormalities in 9% of wild-type processes involving alteration of RNA sec-
axonemes (37/423) (Sampson et al. 2001). ondary structure such as translation initia-
tion, nuclear and mitochondrial splicing,
7.3.4 Nucleus-encoded mitochondrial and ribosome and spliceosome assembly.
genes and female fertility Some members of this family are involved
in embryogenesis, spermatogenesis, and cel-
Female fertility and reproduction is a com- lular growth and division. Mouillet and col-
plicated process. As described in Chapter 2, leagues (2008) disrupted Ddx20 gene in mice
in order to produce offspring, females must and found that Dp103+/− females have heavier
efficiently reach puberty; display estrus; ovaries than wild-type mice. The weights of
shed one or more competent ova; create the the testes or adrenal glands were similar
appropriate oviductal environment for fertil- between the two genotypes. Histological
ization to take place; undergo the necessary examination of ovarian sections showed
systemic, ovarian, and uterine modifications that both wild-type and Dp103 heterozygous
to support pregnancy; deliver the offspring; mice have a similar number of follicles and
lactate; and successfully return to estrus corpora lutea, but the number of follicles
after offspring are weaned. Gene knockout that were devoid of oocytes was significantly
or deficiency mice revealed nucleus-encoded higher in the latter mice compared with the
mitochondrial genes affecting female fertil- former mice. In addition, the estrous cycle
ity as described below. was altered and the basal secretion of adre-
nocorticotrophic hormone was reduced in
It has been widely recognized that CREB the Dp103+/− females (Mouillet et al. 2008).
(the cyclic AMP responsive element-binding
protein-1) regulated transcription coactiva- Nuclear receptor coactivator 3 (NCOA3)
tor 1 (CRTC1) is one the regulatory circuits interacts with nuclear hormone receptors
that control some of the most fundamental to enhance their transcriptional activator
aspects of metabolism, cell growth, prolif- functions. Xu and colleagues (2000) reported
eration, survival, and differentiation at that mouse Ncoa3 is expressed in a tissue-
both the cellular and organismal levels specific fashion and distributed mainly in
(Bhaskar and Hay 2007). Altarejos et al. oocytes, mammary glands, hippocampus,
(2008) reported that adult Crtc1−/−mice are olfactory bulb, smooth muscle, hepatocytes,
infertile; no offspring were obtained from and vaginal epithelium. The authors also
either Crtc1−/− males or Crtc1−/− females found that genetic disruption of Ncoa3
mated with wild-type mice. Crtc1−/− female in mice results in a series of changes,
such as dwarfism, delayed puberty, reduced

Mitochondriomics of Reproduction and Fertility 171

female reproductive function, and blunted homeostasis. Deficiency in genes involved
mammary gland development. Hormonal in mitochondrial processes and biogenesis
analysis further revealed that Ncoa3 plays a often results in a severe embryonic lethality.
role in both the growth hormone regulatory The nucleus-encoded mitochondrial genes
pathway and the production of estrogen (Xu that are essential for embryonic develop-
et al. 2000). ment, include coenzyme Q7 homolog, ubi-
quinone (yeast) (COQ7), cullin 7 (CUL7),
Nitric oxide (NO) synthase 3 (NOS3) endonuclease G (ENDOG), ligase III, DNA,
(endothelial cell) is one of the NO synthases ATP dependent (LIG3), nuclear respiratory
that produce NO in almost all tissues factor 1 (NRF1), optic atrophy 1 (autosomal
and organs. NO is a reactive free radical dominant) (OPA1), v-raf-leukemia viral
that exerts a variety of biologic actions oncogene 1 (RAF1), succinate dehydrogenase
under both physiological and pathological complex, subunit D, integral membrane
conditions. Pallares and coworkers (2008) protein (SDHD), solute carrier family
generated Nos3-knockout mice and found 25 (mitochondrial thiamine pyrophosphate
significant differences in mean number carrier), member 19 (SLC25A19), and surfeit
of corpora lutea (9.7 ± 1.2 in Nos3−/− vs. 1 (SURF1). In addition to infertility problems
14.2 ± 1.2 in Nos3+/+; P < 0.01), rate of anovu- in females, both DDX20 and NOS3 are
lation (48.3 ± 7.3% in Nos3−/− vs. 29.7 ± 6.3 also important to embryonic development.
in Nos3+/+; P < 0.05), total mean number Homozygous Ddx20 null mice die early in
of recovered oocytes/zygotes (4.0 ± 1.1 in embryonic development prior to the four-cell
Nos3−/− vs. 10.4 ± 1.6 in Nos3+/+; P < 0.01), stage during early embryonic development
and non-fertilization rate (50.7 in Nos3−/− vs. (Mouillet et al. 2008). As for the Nos3 gene,
3.3% in Nos3+/+; P < 0.001) between the embryo losses were detected between days
knockout mice (groups Nos3−/− and the wild- 8.5 and 13.5, in 62.5% of Nos3-knockout
type mice (groups Nos3+/+). dams and, at days 10.5 and 11.5, in 16.7% of
the control females (P < 0.005) (Pallares et al.
In addition to affecting male fertility, both 2008).
IMMP2L and PANK2 affect female fertility.
Lu et al. (2008) found that homozygous COQ7 encodes a protein similar to a
Immp2lTg(Tyr)979Ove females are infertile mitochondrial di-iron-containing hydroxy-
due to defects in folliculogenesis and ovula- lase in S. cerevisiae that is involved with
tion. Kuo et al. (2005) observed that female ubiquinone biosynthesis. Nakai et al. (2001)
Pank2 knockout mice produced viable off- generated Coq7-deficient mice to investi-
spring when they mated with wild-type gate the biologic role of COQ7 in mammals.
males. However, the Pank2−/− females gave The authors observed the death of Coq7-
a much fewer number of offspring when deficient mouse embryos at days 10.5 with
compared with wild-type or heterozygote the neuroepithelial cells failing to show the
females probably due to an in utero effect of radial arrangement in the developing cere-
maternal Pank2 deficiency. bral wall, aborting neurogenesis. Electron
microscopic analysis further indicated the
7.3.5 Nucleus-encoded mitochondrial enlarged mitochondria with vesicular cristae
genes and embryonic development and enlarged lysosomes filled with disrupted
membranes. Biochemical analysis demon-
The mitochondrion is involved in energy strated that Coq7-deficient embryos do not
generation, apoptosis regulation, and calcium

172 Quantitative Genomics of Reproduction

synthesize CoQ9 but produce demethoxyu- duplex DNA and play essential roles in
biquinone 9 (DMQ9) instead. In addition, the DNA replication, recombination, repair, and
Coq7-deficient embryos were smaller than maintenance of genomic integrity. LIG3
normal (Nakai et al. 2001). gene is a member of the DNA ligase family.
Puebla-Osorio et al. (2006) made a targeted
CUL7 is a member of the cullin family. interruption of Lig3 gene in mice. Mice het-
Evidence has shown that CUL7 is a new erozygous for the Lig3 mutation were fertile
oncogene, which cooperates with Myc in and did not display phenotypic abnormali-
transformation by blocking Myc-induced ties. The authors then bred heterozygous
apoptosis in a p53-dependent manner, thus mice to generate homozygous Lig3 mutant
playing an essential role in numerous cellu- mice, which yielded no viable Lig3−/− pups
lar and biologic activities (Kim et al. 2007). in nearly 800 young mice genotyped by
To further understand the physiological role Southern blotting or PCR. These results
of CUL7, Arai and colleagues (2003) gener- clearly indicate that Lig3 is essential for
ated mice lacking the gene. The authors mouse embryonic development and sur-
observed that Cul7−/−embryos are runted and vival. To further determine the effects of
die immediately after birth because of respi- Lig3 gene inactivation on embryonic devel-
ratory distress. Dermal and hypodermal opment, the group collected embryos or yolk
hemorrhage was also detected in mutant sacs from heterozygous mice crossbred at
embryos in late gestation. In addition, Cul7−/− different days of gestation and genotyped by
placentas had defects in the differentiation of PCR. The authors found that the mutant
the trophoblast lineage with an abnormal embryonic developmental process stops at
vascular structure (Arai et al. 2003). 8.5 days postcoitum (dpc), and excessive cell
death occurs at 9.5 dpc.
ENDOG encodes a nuclear-encoded endo-
nuclease that is localized in the mitochon- NRF1 encodes a protein that homodimer-
drion. This gene plays an important role in izes and functions as a transcription factor,
both nuclear DNA fragmentation during which activates the expression of some key
apoptosis by cleaving DNA at GC tracts metabolic genes regulating cellular growth
and mtDNA replication by generating the and nuclear genes required for respiration,
RNA primers required by DNA polymerase heme biosynthesis, and mtDNA transcrip-
gamma to initiate the replication of mtDNA. tion and replication. Huo and Scarpulla
Zhang et al. (2003) found that Endog homo- (2001) found that embryos homozygous for
zygous mutant embryos die between embry- Nrf1 disruption die between embryonic days
onic days 2.5 and 3.5. DNA fragmentation is 3.5 and 6.5. The authors detected the beta-
also reduced in Endog+/− thymocytes and galactosidase staining in growing oocytes
splenocytes compared with wild-type cells, as well as in 2.5- and 3.5-day-old embryos,
as well as in Endog+/− thymus in vivo com- indicating that the Nrf1 gene is expressed
pared with that of the wild-type mice, on during oogenesis and during early stages of
activation of apoptosis. These findings embryogenesis.
further confirmed that EndoG is essential
during early embryogenesis and plays a criti- OPA1 encodes a nuclear-encoded mito-
cal role in normal apoptosis and nuclear chondrial protein, which is similar to dyna-
DNA fragmentation. min-related GTPases. Mutations in this
gene have been associated with optic atrophy
DNA ligases catalyze the joining of strand type 1, which is a dominantly inherited
breaks in the phosphodiester backbone of

Mitochondriomics of Reproduction and Fertility 173

optic neuropathy resulting in progressive ers (2004) constructed a null allele of the
loss of visual acuity, leading in many cases Sdhd gene by target replacement of the
to legal blindness (Gränse et al. 2003). Alavi wild-type allele with a nonfunctional allele
and coworkers (2007) generated the first lacking exons 2, 3, and 4. Among 152 animals
mouse model carrying a splice site mutation born from mating between heterozygous
(c.1065 + 5G→A) in the Opa1 gene. The parents, only 50 (33%) Sdhd+/+ and 102 (66%)
mutation induces a skipping of exon 10 Sdhd+/− mice were found, indicating lethal-
during transcript processing and leads to an ity for all homozygous mutant embryos. The
in-frame deletion of 27 amino acid residues authors then examined the embryos from
in the GTPase domain. Using magnetic reso- heterozygous pregnant females mated with
nance imaging, the authors reported that the heterozygous males and found that at 9.5 dpc,
homozygous mutant mice die in utero approximately one-fourth of embryos are
during embryogenesis with the first notable stalled. The use of specific primers for both
developmental delay at day 8.5 (Alavi et al. the wild-type and mutant alleles confirmed
2007). that the stalled embryos are homozygous
Sdhd−/− mice. These observations indicate
RAF1 protein kinase has been identified that Sdhd−/− mutants die at early embryonic
as an integral component of the Ras/Raf/ stages (Piruat et al. 2004).
MEK/ERK signaling pathway in mammals.
In the pathway, RAF1 is activated by its SURF1 encodes a protein localized to the
binding to the Ras family of membrane- inner mitochondrial membrane and is
associated GTPases and then the protein thought to be involved in the biogenesis of
can phosphorylate to activate the dual the cytochrome c oxidase complex. Agostino
specificity protein kinases MEK1 and et al. (2003) created a constitutive knockout
MEK2. In turn, MEK1 and MEK2 phosphory- mouse for Surf1, which is characterized
late to activate the serine/threonine-specific by the high postimplantation embryonic
protein kinases, ERK1 and ERK2. Activated lethality, affecting approximately 90% of
ERKs have pleiotropic effects on cell physi- the Surf1−/− individuals. The authors
ology and play an important role in the obtained homozygous Surf1−/− mice from
control of gene expression related to the cell Surf1+/− intercrosses and observed 10-fold
division cycle, apoptosis, cell differentia- lower (2.7%) Surf1−/− pups than expected by
tion, and cell migration. Hüser et al. (2001) Mendelian transmission of a recessive trait
found that Raf1−/− mice die in embryogenesis (25%). PCR-genotyping revealed that the −/−
and show vascular defects in the yolk sac genotype in blastocysts occurred 26% of the
and placenta as well as increased apoptosis time, but dropped to 14% at E6.5–E7.5 dpc,
of embryonic tissues. to 10% at E8.5–E12, and to ∼2% at E13–E18.
These results show that the loss of Surf1−/−
Complex II of the respiratory chain embryos started at a stage as early as gastru-
includes four nuclear-encoded subunits lation (E4–E7 dpc) and continued during
and is localized in the mitochondrial inner organogenesis (E8.5–E12 dpc), body-mass
membrane, which is specifically involved growth, and organ maturation (E13–E18 dpc).
in the oxidation of succinate. The SDHD Interestingly, Surf1−/− embryos at different
gene encodes one of the two membrane- developmental stages did not show gross
anchoring proteins of succinate dehydro- morphological abnormalities, but were con-
genase (complex II) of the mitochondrial sistently smaller in size in comparison with
electron transport chain. Piruat and cowork-

174 Quantitative Genomics of Reproduction

their +/+ or −/+ littermates (Agostino et al. exists between the nucleus and mitochon-
2003). dria, ultimately resulting in neuronal cell
death (Dawson and Dawson 2004). On the
SLC25A19 encodes a mitochondrial other hand, regulation of mitochondrial
protein that belongs to the solute carrier biogenesis and proliferation is influenced
family. Recent studies have shown that by external factors, such as nutrients,
this protein functions as the mitochondrial hormones, temperature, and aging. Com-
thiamine pyrophosphate carrier, which munications are also required for eliciting
transports thiamine pyrophosphates into mitochondrial responses to specific stress
mitochondria. Lindhurst et al. (2006) gener- pathways (Ryan and Hoogenraad 2007).
ated a knockout mouse model of Slc25a19. Therefore, there will be a need in the future
The authors observed that these mutant to study the mechanisms of mitochondrial
animals have 100% prenatal lethality by biogenesis and the way cells respond to
embryonic day 12. At embryonic day 10.5, external signals to maintain mitochondrial
these Slc25a19−/− embryos have a neural- function and thus maintain high reproduc-
tube closure defect with ruffling of the tive efficiency in livestock species.
neural fold ridges, a yolk sac erythropoietic
failure, and elevated α-ketoglutarate in the References
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