Genetics of mare fertility
After REEL and gonadal dysgenesis that are caused by chromosomal abnormalities, the most common subfertile phenotype in mares is the failure to become pregnant (Coutinho da Silva, 2008). Genetic causes of pregnancy failure are unknown. Currently, there are no molecular tools for the diagnostics of mare fertility or for proper evaluation of oocytes and embryos prior their use for in vitro reproductive technologies.
Genetics of stallion fertility
Stallions are typically selected on the basis of their ancestry, athletic performance, and overall appearance, but not for their reproductive potential (Colenbrander, Gadella, & Stout, 2003). As a result, reduced fertility among breeding stallions has emerged as one of the major problems (Leeb et al., 2005; Leeb, 2007). For example, in two retrospective studies, 36–43% of prospective breeding stallions did not pass reproductive soundness test (Blanchard & Varner, 1997; Woods et al., 2000).
Genetic studies of stallion fertility started with microsatellite heterozygosity analysis (Aurich, Achman, & Aurich, 2003) and SNP search in candidate genes (Giese et al., 2002; Hamann et al., 2007; Giesecke et al., 2009a, 2010a; Giesecke et al., 2009b). The goal was to identify genetic markers associated with various reproductive phenotypes and semen parameters (Table 13.1). Not much success accompanied microsatellite analysis: genotyping of 12 markers in 110 Noriker stallions did not reveal any correlation between heterozygosity and semen parameters such as sperm count and motility (Aurich et al., 2003).
One of the first candidate gene studies dissected the molecular organization and function of AEG1 locus, also known as cystein-rich secretory protein 1 or CRISP1, which is thought to be involved in sperm-egg fusion (Giese et al., 2002). It was determined that the gene comprises of eight exons and has three possible transcription start sites. Sequencing the exons and their flanking sequences in 16 horses of 9 different breeds identified 17 SNPs of which 3 were exonic and 1 changed the amino acid sequence. However, the role of AEG1 in stallion fertility remains unclear because no attempts have been made to correlate AEG1 polymorphism with various reproductive phenotypes.
Another group of studies analyzed association of sequence polymorphism of selected candidate genes with fertility data in a population of reproductively normal Hanoverian warmblood stallions (Giesecke et al., 2009a, 2010a; Giesecke et al., 2009b; Hamann et al., 2007). Reproductive performance of the stallions was evaluated based on pregnancy rate per estrus of mares. The genes CRISP3 (Hamann et al., 2007), PRLR (Giesecke et al., 2009a), INHBA (Giesecke et al., 2010a), and SPATA1 (Giesecke et al., 2009b) were examined and the results suggested association of sequence variants of all four genes with stallion fertility (Table 13.1).
Many more gene loci potentially involved in regulating male fertility have been proposed using extensive data mining from PubMed (www.ncbi.nlm.nih.gov/pubmed/) and Web of Science (apps.isiknowledge.com/), followed by in silico analysis of the most reliable candidate genes (Ogorevc, Dovc, & Kunej, 2010). Considering data from many mammalian species, 835 candidate genes for mammalian male fertility were identified. Of these, 33 genes were proposed as the most promising candidates because they were reported in at least two different species or using two or more approaches. According to this, one of the strongest candidate genes for male fertility in multiple species, including the horse, is prolactin receptor (PRLR). A similar data search was recently conducted to identify candidate genes specific for stallion fertility (Giesecke et al., 2010b). The authors proposed 37 genes with associated SNPs and microsatellites for linkage and GWA studies. Surprisingly, only 10 candidate male fertility genes were common between the two data sets, thus further underlining the complexity of the genetic component of male reproduction.
Due to the large number of genes involved, the molecular research of fertility requires genome-wide (GW) approaches. Thus, great expectations are placed on the use of horse whole genome sequence information (Wade et al., 2009), the SNP50/SNP74 beadchips (McCue et al., 2009; Mickelson, personal communication), and GW linkage and association analysis. However, subfertile phenotypes do not facilitate collection of large numbers of samples or pedigrees for statistically sound GWA and linkage studies. Difficulties are also encountered in precise clinical characterization of subfertile phenotypes because in most cases the causes of reduced fertility are unknown or idiopathic (Blanchard, Johnson, & Roser, 2000; Turner & Casas-Dolz, 2002). This is probably why GW molecular research has been initiated only for two conditions: cryptorchidism and impaired acrosomal reaction.
Cryptorchidism, or failure of testes to descend, affects about 8% of live male births in horses, and is more frequent in ponies, draft horses, and breeds like Percheron, American Saddlebred, and Quarter Horse (Hayes, 1986). Cryptorchidism can occur as a single defect or as a part of complex congenital abnormalities accompanying intersex conditions or chromosomal aneuploidies (Constant et al., 1994; Chowdhary & Raudsepp, 2000). In all cases, failure of testes descent affects normal testicular functions and can result in reduced fertility or infertility. Etiology of cryptorchidism is complex, involves interaction of environmental, genetic, and epigenetic factors, and is, as yet, not well understood. Studies in mouse and human suggest complex genetic regulation of the condition involving genes for gubernaculum development and androgen signaling pathway (Ferlin et al., 2008; Foresta et al., 2008). Indeed, a missense mutation in relaxin/insulin-like family peptide receptor 2 (RXFP2) was recently associated with cryptorchidism in mouse (Harris et al., 2010). Genetic studies of the condition in horses are limited to a linkage analysis using a Thoroughbred pedigree of 23 cryptorchids and 24 unaffected horses (Diribarne et al., 2009). Gentoyping of 16 microsatellite markers for seven candidate genes, namely AR, INSL3, RXFP2, CALCA, HOXA10, NR5A1, and ESR1, did not reveal any association with the cryptorchid phenotype. In contrast, recent gene expression profiling showed significant (P < 0.001) up-regulation of RXFP2, FGF10, HOXA10, and down-regulation of INSL3, HOXA11, and HSD3B1 in cryptorchid compared to normal testes (our unpublished data). It is likely that the observed differences in gene expression reflect the consequences rather than the primary genetic causes of abnormal testicular descent. Research is currently under way to explore the epigenetic landscape of cryptorchidism by comparing global DNA methylation levels of selected candidate genes between normal and cryptorchid testes, as well as to study the possible involvement of genomic copy number variation in the etiology of cryptorchidism (our ongoing research).
Impaired acrosomal reaction
Impaired acrosomal reaction (IAR) is a reproductive disorder characterized by the inability of sperm to perform acrosomal reaction, resulting in reduced fertility. The condition has been found in Thoroughbreds, and currently efforts are made to identify the genomic region(s) associated with the condition using the equine SNP50 (McCue et al., 2009) and the 21,000-element expression oligoarray (Bright et al., 2009). GW association analysis reveals statistically significant association of the subfertile phenotype to ECA13p around 8–9 Mb position (Raudsepp et al., 2011). Notably, some of the differentially expressed genes, as detected by microarray analysis, originate from the same region. Re-sequencing of two most significant candidate genes has identified a few statistically significant (p<0.05) amino acid changing SNPs in FKBP6, suggesting this as a susceptibility locus for IAR. With the exception of the Y chromosome (see Chapter 5), this is the first report about a direct association of a genomic region with a reproductive disorder in horses.
Functional Genomics of Reproduction
Functional genomics of reproduction studies gene expression profiles in male and female reproductive tissues and the zygote, to identify protein coding genes and other functional elements (i.e., small and long non-coding regulatory RNAs) that are critical for male and female fertility. The studies seek for understanding the functions and interactions of genes during different stages of sexual development, sexual maturation, and reproduction, both in normal and subfertile individuals. One of the main goals is to identify functional signatures that can lead to the development of molecular tests for fertility evaluation. Gene expression studies typically do not require the procurement of hundreds of samples, as is necessary for linkage and GWA analyses, and are therefore more feasible for reproduction-related research. Functional analysis can be carried out on individual genes of interest or across the whole genome. The former is typically based on reverse transcriptase (RT) and quantitative real-time (qRT) PCR (Das et al., 2010a; Das et al., 2010b) or differential display (Turner & Casas-Dolz, 2002). GW analysis, on the other hand, uses whole genome expression arrays (Ing et al., 2004; Das et al., 2010b) or direct mRNA sequencing, also known as RNA-Seq (Coleman et al., 2010; Das et al., 2011c).
Gene expression studies in the mare
Similarly to molecular genetics, functional genetics of mare reproduction lags far behind that of the stallion. A few studies have analyzed the expression of selected genes in ovaries and the uterus. It has been shown that high expression level of steroidogenic acute regulatory (StAR) gene in ovaries is required during the early stages of pregnancy (Watson, Thomson, & Howie, 2000; Watson et al., 2004). Ovarian expression of dopamine receptors 1 and 2 (D1R and D2R) suggests that dopamine can act directly on ovarian tissues through its interaction with dopamine receptors during fertilization (King et al., 2005). Additionally, there are preliminary studies on differential gene expression in blastocysts (Paris et al., 2010) and the endometrium during pregnancy recognition (Bruemmer et al., 2010), and the early stages of pregnancy (Merkl et al., 2010). Despite the important role of ovarian genes in follicular maturation and early embryogenesis in human, mouse, and chicken (Juengel et al., 2004; Elis et al., 2009), no gene expression studies have, as yet, been initiated in equine oocytes.
Functional genomics of the stallion
The majority of functional studies in the stallion have focused on a small number of selected candidate genes comparing their transcription and/or protein expression profiles at different time points in reproductive and somatic tissues. For example, it was determined that AEG1 is highly expressed in epididymis but not in testis (Giese et al., 2002). Interesting findings were reported for the expression of anti-Müllerian hormone (AMH) which induces regression of Müllerian ducts during male fetal development (Ball et al., 2008). Equine AMH is highly expressed in Sertoli cells of fetal, neonatal, and prepubertal testes, but not in normal adult testes. Notably, AMH is also expressed in cryptorchid testes (in animals up to 3–4 years of age), in Sertoli cell tumors, and male intersex gonads, making it a useful biomarker for the detection of these conditions in the horse (Ball et al., 2008). A novel testis-specific equine serine/threonine kinase, STK31 was recently discovered in stallion testes (Sabeur et al., 2008). Detailed characterization of the RNA transcript (3.1 kb), the functional domains of the protein (protein kinase and putative RNA-binding domains), and the protein localization in the equatorial segment of testicular spermatozoa suggest that STK31 may have a role in reorganization of sperm chromatin during spermiogenesis. Systematic studies have also been carried out on horse Y chromosome genes (Paria, 2009) by determining their expression in testis and somatic tissues, and generating full-length cDNA sequences for selected genes of interest (see Chapter 5 for more details).
Promising solutions for functional genomics of reproduction are offered by global gene expression microarrays (Farber & Lusis, 2008) and RNA-Seq (Wang, Gerstein, & Snyder, 2009). The well-annotated 21,000-element oligoarray for the horse (Bright et al., 2009) is currently the most cost-effective tool for functional analysis of tens of thousands of genes from a small number of normal and affected individuals, providing a critical link between the genes and the phenotype. RNA-Seq technology, on the other hand, is the most powerful method for transcriptional profiling of any cell or tissue (Shendure, 2008; Wang et al., 2009; Montgomery et al., 2010). This includes the identification of novel genes, alternative transcripts, and small and long non-coding regulatory RNAs.
Application of microarray technology for transcriptional profiling of stallion testis (Laughlin et al., 2002; Ing et al., 2004) can be considered the formal beginning of functional genomics in horses. In these studies, gene expression profiles in dark and light testicular tissues of three prepubertal colts were analyzed using a human WG cDNA array. Assessment of transcriptional profiles of 9,132 human genes showed that only 93 genes (1.3%) are differentially expressed between the light and dark testicular parenchyma.
Recently, testis transcriptome of reproductively normal stallions was studied using the 21,000-element equine whole genome expression oligoarray (Das et al., 2010b) and direct mRNA sequencing (Coleman et al., 2010). The two studies closely agree about the overall number of testis mRNA transcripts, which is between 11,000 and 15,000 according to microarray analyses and 12,013 according to RNA-Seq. Efforts are currently made to generate a comprehensive catalog of testis transcripts characteristic to a normal stallion. This includes annotations, expression profiles, and putative functions of all detected genes. The catalog will serve as an important foundation for the study of functional changes in the testis of subfertile animals, leading to the identification of genes that are critically involved in stallion fertility. Furthermore, recently optimized protocol for RNA isolation from stallion testis biopsies using a 14 gauge 22 mm deep needle and yielding about 12 micrograms of sequencing quality total RNA (Das et al., 2010a) offers an alternative to excision surgery for sample procurement.
Despite the achievements of testis transcriptome analysis, difficulties remain regarding the procurement of samples, because conducting excision surgery or biopsy on the testis of prospective breeding animals is potentially harmful. A promising solution is to use sperm instead of testis as the source of RNA. This builds on recent discoveries in humans showing that ejaculated sperm contain a rich repertoire of RNAs, including mRNAs, and small and long non-coding regulatory RNAs (Ostermeier et al., 2002; Krawetz, 2005; Ostermeier et al., 2005a; Ostermeier et al., 2005b; Boerke et al., 2007). Thus, the sperm’s contribution to the oocyte is not only the paternal DNA but also a complex host of RNAs. Some sperm mRNAs might be needed for early embryonic development (Boerke et al., 2007) and for protein synthesis during capacitation using mitochondrial-type ribosomes (Gur & Breitbart, 2006, 2007, 2008). Sperm microRNAs might serve as transgenerational signals for heritable epigenetic modifications (Cuzin & Rassoulzadegan, 2010). While the exact functions of sperm RNAs are still a matter of debate, most researchers agree that the majority of sperm mRNAs are historical records of spermatogenesis, and that sperm RNA profiles can be used as a measure of male fertility (Krewetz, 2005; Boerke et al., 2007; Platts et al., 2007). Most importantly, the use of sperm instead of testis as the source of RNA will grant noninvasive and feasible procurement of samples.
Sperm transcriptome studies were recently initiated in stallions. As a first step, total RNA isolation methodology from sperm was optimized for the horse (Das et al., 2010a; Sudderth et al., 2010). This was necessary because sperm typically contain only 10–20 fg of RNA; the ejaculates contain somatic cells and immature sperm that must be removed before RNA extraction; sperm are depleted of ribosomal RNAs, thereby negating the use of rRNA integrity as a marker of quality; and differences between species in sperm attributes and packaging require adjustments in RNA isolation protocols. The optimized protocol allows isolation of about 2 micrograms of hybridization- and sequencing-quality total RNA from 100 million sperm (Das et al., 2010a). Comparison of the RNA quality and quantity with different sperm-processing and storage conditions showed that fresh semen remains the gold standard for sample submission. Processing methods utilizing an extender provide a useful alternative for shipping samples from a distance for evaluation, and flash-frozen semen is the least desirable submission type, but may be utilized if necessary (Sudderth et al., 2010).
Prior to the use of sperm RNA as a diagnostic tool for stallion fertility, the sperm RNA profiles that define the normal fertile male must be determined. Preliminary data about the number, identity, biological functions, and transcriptional profiles of mRNAs and regulatory non-coding RNAs in stallion sperm were recently obtained using microarray hybridization (Das et al., 2010b) and RNA-Seq (Das et al., 2011c).
Microarray analysis of stallion sperm identified more than 6,000 mRNA transcripts, which is roughly half of the number of mRNAs in stallion testis (Coleman et al., 2010; Das et al., 2010b). Notably, about 2.4% of sperm transcripts are sperm-specific and not detected in testis (Table 13.2), while about 200 sperm mRNAs show differential expression between the sperm and the testis (Table 13.3). Many sperm transcripts are associated with membranes and are involved in molecular functions related to G-protein coupled receptor (GPCR) activities. This might be important for fertilization because GPCRs are implicated in sperm chemotaxis, capacitation, and acrosome reaction.
In another study, total RNA from the sperm of one reproductively normal stallion was sequenced using SOLiD (Applied Biosystems) platform (Das et al., 2011c). Altogether, 65 million 35-base-pair reads were generated and aligned with the horse draft assembly EcuCab2 (Wade et al., 2009). Sequence map locations were determined for 19,257 reads, of which 31% were identified as dbESTs, mRNAs and 33 known microRNAs, while 69% had no annotation in EcuCab2. Like in microarray analysis, many of the transcripts detected by RNA-Seq show direct relevance to a variety of known sperm functions (Table 13.4).
It is anticipated that transcriptional profiling of stallion sperm will lead to the identification of functional signatures for high- and low-fertility stallions, so that noninvasive molecular tests for stallion fertility can be developed. Global transcriptional profiling of stallion sperm will also serve as a valuable non-rodent model for dissecting the genetics of male fertility in humans and other mammals.
Summary and Future Prospects
Genomics of equine reproduction is a challenging area of research that encompasses a wide range, from chromosomes to fine-tuning of gene expression. The stability and structural integrity of chromosomes is certainly a key component of normal sexual development and fertility in horses, and cytogenetic analysis will continue to be an essential part of clinical tests for subfertile animals. While the role of chromosomal abnormalities in equine fertility is well established, the knowledge about the fertility genes, their structure, function, and associated phenotypes is still limited. Furthermore, almost nothing is known about the role of structural genomic rearrangements, copy number variants (CNVs), gene expression regulation, and epigenetic mechanisms in male and female fertility. This is why great expectations are placed on the use of new genomics tools such as SNP beadchips (McCue et al., 2009; J. Mickelson, personal communication), the recently constructed WG tiling array (Qu et al., 2011), the expression microarrays (Bright et al., 2009), various next-generation sequencing platforms, and improved bioinformatics methods. It is anticipated that these tools will take equine genome analysis to levels that allow the detection of submicroscopic genomic rearrangements underlying the currently unknown causes of subfertility, will lead to the discovery of reproduction-related mutations in nuclear and mitochondrial genes, and will identify genetic and epigenetic regulatory mechanisms critical for stallion and mare reproduction. However, all the advanced tools will be of little value without the establishment of collections of phenotypically well-characterized samples. Thus, increased interaction between the geneticists and clinicians remains as one of the most critical factors for any success.
The research on the genomics of stallion fertility has been supported by the LINK Endowment and by the American Quarter Horse Research Foundation.
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