A Nonsense Mutation in Encoding a Nondescript Transmembrane Protein Causes Idiopathic Male Subfertility in Cattle

Summary

Genetic variants underlying reduced male reproductive performance have been identified in humans and model organisms, most of them compromising semen quality. Occasionally, male fertility is severely compromised although semen analysis remains without any apparent pathological findings (i.e., idiopathic subfertility). Artificial insemination (AI) in most cattle populations requires close examination of all ejaculates before insemination. Although anomalous ejaculates are rejected, insemination success varies considerably among AI bulls. In an attempt to identify genetic causes of such variation, we undertook a genome-wide association study (GWAS). Imputed genotypes of 652,856 SNPs were available for 7962 AI bulls of the Fleckvieh (FV) population. Male reproductive ability (MRA) was assessed based on 15.3 million artificial inseminations. The GWAS uncovered a strong association signal on bovine chromosome 19 (P = 4.08×10⁻⁵⁹). Subsequent autozygosity mapping revealed a common 1386 kb segment of extended homozygosity in 40 bulls with exceptionally poor reproductive performance. Only 1.7% of 35,671 inseminations with semen samples of those bulls were successful. None of the bulls with normal reproductive performance was homozygous, indicating recessive inheritance. Exploiting whole-genome re-sequencing data of 43 animals revealed a candidate causal nonsense mutation (rs378652941, c.483C>A, p.Cys161X) in the transmembrane protein 95 encoding gene TMEM95 which was subsequently validated in 1990 AI bulls. Immunohistochemical investigations evidenced that TMEM95 is located at the surface of spermatozoa of fertile animals whereas it is absent in spermatozoa of subfertile animals. These findings imply that integrity of TMEM95 is required for an undisturbed fertilisation. Our results demonstrate that deficiency of TMEM95 severely compromises male reproductive performance in cattle and reveal for the first time a phenotypic effect associated with genomic variation in TMEM95.

Introduction

Impaired reproductive performance is a prevalent condition in both sexes of many species and up to 15% of couples are affected in humans [1], [2]. The disability to reproduce is defined as infertility (i.e., sterility), whereas subfertility refers to any form of reduced fertility [3].

Low sperm concentration (i.e., oligospermia) and the absence of spermatozoa (i.e., azoospermia), respectively, are frequently diagnosed in males with impaired fertility [4]. Further aberrant semen quality traits (e.g., abnormal sperm morphology [5], reduced motility [6], [7]) account for another substantial fraction of reduced male fertility. However, semen analysis of a considerable number of males with impaired reproductive performance remains without any apparent pathological findings (i.e., unexplained/idiopathic infertility) [8], [9].

Semen quality traits have low to medium heritability in cattle populations [10]. Numerous genetic variants underlying routinely assessed semen quality traits have been identified so far in humans [11], [12], model species [13] and livestock populations [14]. However, the number of known genetic mechanisms causing idiopathic male subfertility is very small [15], [16] and identified polymorphisms explain only a small fraction of its genetic variation [17].

Artificial insemination (AI) is predominant over natural service in most cattle populations and all ejaculates are closely examined immediately after semen collection. Only semen samples without any apparent abnormalities, such as low sperm count, reduced progressive motility, low viability, abnormal morphology of spermatozoa, are used for insemination. However, the reproductive performance indicated by the proportion of successful inseminations varies considerably among AI sires [18], [19]. So far, genome-wide association studies (GWAS) for male reproductive traits were of limited success in cattle populations [20], [21] and only one putatively causative mutation has been identified [22].

Here we report a new recessively inherited variant of idiopathic male subfertility in the Fleckvieh (FV) cattle population. The mapping of the underlying genomic region was facilitated by using high-density genotypes in a large sample of artificial insemination bulls with phenotypes for reproductive performance assessed based on 15 million artificial inseminations. Exploiting whole-genome re-sequencing data revealed a causative loss-of-function mutation in the transmembrane protein 95 encoding gene TMEM95.

Results

Male subfertility in the Fleckvieh cattle population

Phenotypes for male reproductive ability (MRA) were obtained for 7962 bulls of the FV population based on 15.3 Mio artificial inseminations (AI). The values for MRA range from −40 to +13 and reflect the bulls' reproductive performance as percentage deviation from the population mean. Male reproductive ability is highly correlated (r = 0.59) with the 56-day non-return rate (NRR56) in cows. The NRR56 is the proportion of cows that are not re-inseminated within a 56-day interval after the first insemination. After visual inspection of the distribution of MRA, forty-nine bulls with exceptionally poor reproductive performance (MRA<−20) were considered as subfertile (Figure S1 and Table 1). Animals with values for MRA below −20 ( = five standard deviations below the population mean) were used as case group in a case-control design.

Bovine male subfertility maps to chromosome 19

Using MRA as quantitative trait in a genome-wide association study (GWAS) yielded a strong association signal on bovine chromosome (BTA) 19 (P = 4.38×10⁻²⁰, Figure S2). However, the association signal was more pronounced using 49 subfertile animals (MRA<−20) as case group and the remaining 7913 animals as controls (Table 1 and Figure 1A). The most significantly associated SNP is located at 30,220,186 bp (ARS-BFGL-NGS-11488; P = 4.08×10⁻⁵⁹).

Autozygosity mapping revealed a common 1386 kb segment (26,580,096 bp–27,956,634 bp) of extended homozygosity in 40 subfertile bulls containing 80 genes (Figure 1B and Table S1). None of 7913 bulls with normal reproductive performance was homozygous for the 1386 kb segment, indicating recessive inheritance. Semen samples of 40 homozygous bulls had been used for 35,671 artificial inseminations with an average of 892 inseminations per bull. This is a typical number for test inseminations performed with semen samples of young bulls in progeny testing based breeding programmes. However, only 619 (1.74%) of those inseminations were successful (Table S2).

There was no evidence for the presence of large structural variants (i.e., copy number variations) within the segment of extended homozygosity (Figure S3). The proportion of missing genotypes did not significantly differ between cases and controls (P>0.09) for all SNPs located within the associated region.

The frequency of the subfertility-associated haplotype amounts to 7.2%. Of 7962 genotyped bulls with phenotypes for MRA, 1068 (13.41%) carry the deleterious haplotype in heterozygous state. The carrier frequency increased considerably within the last years (P = 0.0002, Figure S4). The reproductive performance of heterozygous bulls is normal, indicating recessive inheritance (Figure 2). Of 1952 primiparous cows, 291 are heterozygous and 17 are homozygous for the subfertility-associated haplotype. The haplotype neither affects reproductive performance nor milk production traits in females (Table S3). The haplotype distribution does not deviate from the Hardy-Weinberg equilibrium, neither in females (P = 0.303) nor in males (P = 0.817).

Both, haplotype and pedigree analysis allowed to trace the mutation back to the bull HAXL (*1966) (Figure S5). HAXL appears in the pedigrees of 7779 out of 7962 bulls (97.70%) and can be considered as the most important ancestor of the current FV population [23].

Exploiting whole-genome re-sequencing data for mutation detection

Whole genome re-sequencing of 43 animals and subsequent multi-sample variant calling yielded genotypes at 17.17 million sites [23]. Among them, 5965 (5287 SNPs and 678 INDELs) are located within the subfertility-associated region on BTA19 (26,580,096 bp to 27,956,634 bp). Six of the 43 re-sequenced animals were identified as carriers of the associated haplotype via high-density genotypes. The sequence data were filtered for variants compatible with the supposed recessive inheritance, i.e., heterozygous in carriers and homozygous for the reference allele in non-carriers (see Material & Methods, Figure S6). After filtering, 26 SNPs and six INDELs were retained as candidate causal mutations (Table S4 and S5). The functional effects of those variants were predicted based on the gene annotation of the UMD3.1 assembly of the bovine genome [24]. Four of the 32 compatible variants were located in coding regions (Table 2). Among them, we considered a nonsense mutation in TMEM95 (rs378652941, c.483C>A, p.Cys161X, Chr19: 27,689,622 bp) as the prime candidate causal mutation (Figure 3A and 3B). The nonsense mutation was subsequently confirmed in the re-sequenced animals by classical Sanger sequencing (Figure S7 and S8).

A loss-of-function mutation in TMEM95 causes male subfertility in cattle

Genotypes for two non-synonymous substitutions in ACDVL and KIF1C and for the nonsense mutation in TMEM95 were obtained for cases and controls using TaqMan genotyping assays (Table 3). Only c.483C>A, introducing the premature stop-codon in TMEM95 (p.Cys161X), was perfectly associated. All animals, which are homozygous for the subfertility-associated haplotype, are homozygous for the non-reference allele, whereas none of 1396 FV bulls with normal reproductive performance are homozygous. The polymorphism is present in the FV breed only; 277 Holstein-Friesian and 278 Braunvieh animals are homozygous for the reference allele. The c.483C>A-mutation is not segregating among 15 Jersey, 47 Angus and 129 Holstein-Friesian animals which were sequenced in the context of the 1000 bull genomes project [25].

TMEM95 encodes a highly conserved single-pass type I transmembrane protein consisting of 183 amino acids with a predicted extracellular N-terminal signal peptide, a 23-amino acid transmembrane domain (amino acid position 153 to 175) and a 8-amino acid intracellular C-terminal domain (Figure 3C and Figure S9 and S10). The premature stop codon introduced by the c.483C>A-mutation is located within the predicted transmembrane domain and truncates the protein by 22 amino acids.

The c.483C>A-mutation does not affect semen quality

Semen quality (morphology, vitality, total motility) was analysed using cryopreserved semen samples of 30 bulls (10 wt/wt, 10 wt/mt, 10 mt/mt). In all ejaculates, spermatozoa showed less than 20% morphological alterations and less than 5% morphological alterations of the head. Total motility after thawing ranged from 50 to 65%. Statistical analysis showed no significant differences in the proportion of motile spermatozoa from wt/wt, wt/mt and mt/mt bulls (Table 4). As shown by eosin staining, 40 to 70% of the spermatozoa were viable after thawing. There were no significant differences in the percentages of viable spermatozoa between wt/wt, wt/mt and mt/mt bulls. Additionally, ejaculate volume, sperm concentration and progressive motility were assessed in fresh semen samples of 203 AI bulls (177 wt/wt, 21wt/mt, 5 mt/mt). Ejaculate volume was above 5 ml, sperm count was above 1.42 Mio/µl and the proportion of spermatozoa with progressive motility was above 70% for all animals (Table 5).

Localization of TMEM95 in spermatozoa

A mouse-derived polyclonal antibody generated against human transmembrane protein 95 was used to locate its position in spermatozoa of 33 bulls (10 wt/wt, 10 wt/mt and 13 mt/mt). In spermatozoa of wt/wt bulls, TMEM95 was distinctly located on the plasma membrane of the acrosome (Figure 4A). Staining was also visible on the equatorial segment of the head. The sperm neck was regularly labelled. Spermatozoa of wt/mt and wt/wt bulls showed an identical staining pattern (Figure 4B), whereas spermatozoa of mt/mt bulls did not show any staining on the head (Figure 4C). Weak fluorescence was detected in the midpiece of the tail in spermatozoa of all animals due to the autofluorescence of the mitochondria. In the negative controls, there was no signal detectable on the sperm head whereas the midpiece of the tail showed weak autofluorescence (Figure S11).

Discussion

The genome-wide association study (GWAS) with imputed genotypes for 7962 artificial insemination bulls identified a genomic region on BTA19 for male reproductive ability (MRA) in the FV population. Autozygosity mapping revealed a common 1386 kb segment of extended homozygosity in 40 bulls with unexplained exceptionally poor reproductive performance. None of the bulls with normal reproductive performance was homozygous indicating recessive inheritance. Only 1.74% of inseminations performed with semen samples of affected bulls were successful, although semen quality parameters were within a normal range, reflecting idiopathic subfertility [26]. The newly identified congenital defect is denominated as “Bovine Male Subfertility” and accounts for 82% of FV bulls with exceptionally poor reproductive performance. However, we cannot exclude the possibility that homozygous males are infertile and that the very low proportion of successful inseminations reflects errors in parentage recording which might be as high as 10% in dairy cattle breeding programmes [27]. In progeny testing based breeding programmes, semen doses of young bulls are used for approximately 1000 test inseminations [28]. These artificial inseminations are performed within very short time, precluding the early identification of subfertile/infertile bulls. Identifying and culling bulls with poor fertility prognosis (i.e., homozygous bulls) before they are used for artificial insemination is now possible. There was no evidence for any additional genomic region underlying idiopathic male subfertility in the FV population, although the reproductive ability of nine bulls which are not homozygous for the c.483C>A-mutation, is very low. However, the number (n = 9) of subfertile bulls not attributable to the BTA19 locus might not be sufficient for detecting additional loci (Figure S12 and Table S6).

The potential of targeted or whole genome re-sequencing for the identification of causal trait variants has been demonstrated in several species (e.g., [29]–[31]) including cattle [32]–[34]. Causal trait variants for monogenic disorders are traditionally identified by sequencing case/control-panels and by subsequently comparing allele counts in affected and unaffected individuals. However, the concept of the present study is different: the identification of the underlying mutation was based on whole genome re-sequencing data of 43 unaffected FV animals explaining a vast majority of the population's genomic variation [23]. As the frequency of the mutation was reasonably high (7.2%), the affected haplotype was present in heterozygous state in six of the re-sequenced animals. Filtering the re-sequencing data for variants compatible with the supposed recessive inheritance pattern revealed a plausible candidate causative loss-of-function mutation in TMEM95 encoding the transmembrane protein 95.

The nonsense mutation was perfectly associated in 1990 animals representing three different breeds. To our knowledge, this is the first report of a phenotypic effect associated with variation in TMEM95 in any organism. So far, there are no clues about the precise function of TMEM95. However, it seems likely that TMEM95 is involved in sperm-egg interactions, which has been shown to be the main function of sperm-specific transmembrane proteins (e.g., [35], [36]). The phenotype in the present study resembles phenotypic patterns of Caenorhabditis elegans resulting from an impaired function of sperm-specific transmembrane proteins [37], [38]. Taken together, our findings evidence genomic variation within TMEM95 to severely compromise the reproductive performance in cattle.

The causative polymorphism (c.483C>A, rs378652941) introduces a premature stop codon in TMEM95 (p.Cys161X). The affected codon resides within the predicted transmembrane domain of TMEM95 most likely resulting in a disturbed anchorage of the truncated protein in the sperm plasma membrane bilayer. It is also likely that the resulting truncated protein is absent due to nonsense-mediated mRNA decay [39]. Our data show no evidence that the mutation affects any of the routinely assessed semen quality parameters in vitro. However, we cannot exclude the possibility that the mutation affects semen quality parameters, e.g., vitality and motility, in vivo [40], [41].

Transmembrane protein 95 is primarily located on the acrosomal membrane of the sperm head indicating that it may be involved in the acrosome reaction. Spermatozoa of mt/mt animals showed no fluorescence at the acrosomal membrane implying deficiency of TMEM95. Thus, successful fertilization by spermatozoa of mt/mt animals might be compromised. This is supported by the fact that the equatorial segment of the acrosome, which provides the first contact of the spermatozoon with the cell membrane of the oocyte [42], is also labelled in spermatozoa of fertile animals. The sperm neck contains the centriole and is essential for cell division and development of the early embryo [43]. Labelling of the neck indicates an additional potential role of TMEM95 after fertilization during the first cell divisions of the early embryo.

Although spermatozoa of mt/mt animals showed fluorescence, neither on the acrosomal membrane and the equatorial segment nor at the centriole, weak unspecific fluorescence was observed at the midpiece of the tail. This fluorescence pattern is also present in spermatozoa of wt/wt and wt/mt animals. The midpiece is the only region of spermatozoa that contains mitochondria [44]. The weak fluorescence of the midpiece is due to unspecific autofluorescence of the mitochondria, which has been described in several organs and species [45]–[47].

Male subfertility is also present in other species besides cattle [9], [48]–[50], and compromised sperm surface proteins account for a substantial number of males with distinctly reduced reproductive ability in humans [15], [40]. Our results demonstrate that TMEM95 is another sperm surface protein, which is likely to be involved in sperm-egg plasma membrane interactions. Its protein sequence is highly conserved among species (Figure S10) and genetic variants disrupting TMEM95 are likely to induce male subfertility also in other species than cattle. Numerous polymorphic sites have been identified in human TMEM95, among them several potential loss-of-function variants (Figure S13). Based on our findings it is highly recommended to systematically survey variants in TMEM95 as potentially causal for idiopathic male in- or subfertility in any species.

Frequencies of variants that disrupt protein-coding genes are usually low in human populations [51], [52]. However, in livestock populations, the frequency of deleterious alleles might increase rapidly because individual sires can generate tens of thousands of progeny by artificial insemination (e.g., [53], [54], [32]). The loss-of-function mutation in TMEM95 can be traced back to HAXL (*1966), the most important ancestor of the current FV population. Within eight generations, the frequency of the deleterious allele increased to 8.9% and 1443 (13.92%) animals of the present study are heterozygous. This increase of the allele frequency had been possible because phenotypic effects become apparent in homozygous males only. There are no phenotypic effects detectable neither in heterozygous nor in homozygous females (Table S3).

In agreement with previous findings in livestock [55] and humans [15], our results evidence that standard assessment of spermatozoa (i.e., morphology, motility and vitality) is not sufficient to reliably anticipate male reproductive performance. All routinely assessed semen parameters of bulls homozygous for the nonsense mutation in TMEM95 comply with current requirements for artificial insemination in cattle [56]. It might be advisable to develop functional assays, e.g., for the integrity of sperm surface proteins, for an efficient prospective monitoring of male fertility.

Materials and Methods

Ethics statement

Semen samples were collected by approved commercial artificial insemination stations as part of their regular breeding and reproduction measures in cattle industry. No ethical approval was required for this study.

Animals and phenotypes

Male reproductive ability (MRA) was evaluated in 7962 AI bulls of the German FV population. Semen samples of those bulls were used for 15,321,171 artificial inseminations with an average of 1924 artificial inseminations per bull. The phenotypes for MRA were obtained from routine breeding value estimation for reproductive traits, which are jointly estimated for males and females [57]. The resulting phenotypes for MRA represent the bulls' reproductive performance adjusted for environmental and genetic effects (i.e., year, season, flock, female mating partner). The lower the value for MRA, the worse is the bull's reproductive performance (i.e., the smaller the proportion of successful inseminations).

Genotypes, quality control and genotype imputation

A total of 3545 animals (1475 AI bulls, 2070 primiparous cows) of the FV population were genotyped with the Illumina BovineHD Bead chip comprising 777,962 SNPs. Another 7073 AI bulls were genotyped with the BovineSNP50 Bead chip comprising ∼54,000 SNPs. The chromosomal position of the SNPs was determined according to the UMD3.1 assembly of the bovine genome [58]. Mitochondrial, Y-chromosomal and those SNPs with unknown chromosomal position were not considered for further analyses. Stringent quality control was carried out for each dataset separately using PLINK v1.07 [59]. Animals and SNPs with call-rate <0.95 were excluded, as well as SNPs with minor allele frequency <0.5% and those SNPs deviating significantly from the Hardy-Weinberg equilibrium (P<10⁻⁶). The pairwise genomic relationship [60] was compared with the pedigree relationship tracing pedigree records back to 1920 [61]. Animals showing major discrepancies of the pedigree and the genomic relationship were removed from the dataset, as such patterns indicate sample swaps. After quality control, the high-density dataset contained 3332 animals and 652,856 SNPs with an average per-individual call-rate of 99.17%. The medium-density dataset contained 7031 animals and 42,907 SNPs with an average per-individual call-rate of 99.75%. Genotype imputation was performed to extrapolate medium-density genotypes to higher density using a pre-phasing based approach. Haplotypes were inferred for the two datasets separately using Beagle [62] and subsequent haplotype-based imputation was performed with Minimac [63]. This approach yields high imputation accuracy in cattle [64]. The imputed dataset comprised 10,363 animals (8411 AI bulls/1952 primiparous cows) and genotypes for 652,856 SNPs. Phenotypic records for MRA were available for 7962 bulls.

Genome-wide association study

Genome-wide association studies were performed applying a variance component based approach to account for population stratification. We used EMMAX [65] to fit the mixed model , where Y is a vector of phenotypes, b is the SNP effect, X is a design matrix of imputed SNP genotypes, u is a vector of additive genetic effects assumed to be normally distributed with mean 0 and (co)variance , with being the additive genetic variance and G being the realized genomic relationship matrix (GRM) of the 7962 bulls with phenotype information built based on 635,224 autosomal SNPs (see above) and where e is a vector of random normal deviates .

Exploiting whole genome re-sequencing data for mutation screening

The genomes of 42 key and contemporary animals of the FV population were sequenced at low- to medium coverage (ø 7.4-fold) and one animal was sequenced at high coverage (25-fold) using Illumina GA IIx and HiSeq 2000 instruments [66], [23]. Paired-end reads were obtained and mapped to the bovine reference sequence (UMD3.1 [58]) using the Burrows-Wheeler Aligner (BWA) [67]. PICARD (http://picard.sourceforge.net) was used to mark PCR-duplicates. Subsequent multi-sample variant calling with mpileup [68] yielded genotypes at 17.17 million sites. The re-sequencing data were contributed to the 1000 bull genomes project [25] and all variants were submitted to dbSNP [23]. Beagle phasing and imputation within the 43 sequenced animals improved the primary genotype calls (a detailed overview of the entire variant calling pipeline and all obtained variants is presented in Jansen et al. [23]). Of 17.17 million sites, 5287 SNPs and 678 INDELs were located within the 1386 kb segment (26,580,096 bp to 27,956,634 bp) of extended homozygosity on BTA19. Of the 43 sequenced animals, six were identified as carriers of the subfertility-associated haplotype via high-density genotypes, among them the animal sequenced at high coverage. Assuming perfect correlation between the subfertility-associated haplotype and the causal mutation, the allele frequency of the causal mutation should be 7% (6 of 86 affected alleles) in the sequence-derived genotypes. To account for inaccurately genotyped variants due to the low-coverage sequence data (e.g., mis-calling of heterozygous genotypes for rare variants [69]) [70] and for possible phasing errors, a conservative mutation scan was performed to identify variants compatible with recessive inheritance (Figure S6). The 5965 polymorphic sites were filtered for variants that met three conditions: (i) the frequency of the non-reference allele is below 10%, (ii) the variant is heterozygous in the animal sequenced at high coverage and (iii) the variant is present in heterozygous state in at least three of five carrier sequenced at low coverage.

Validation of three identified polymorphisms

PCR primers (5′-CACCCTGCCTTGTCTTTCAT-3′ and 5′-AGGCTCTGTCCTCGTTTTCA-3′) were designed for exon 6 of TMEM95 to scrutinize the rs378652941-polymorphism by classical Sanger sequencing in the re-sequenced animals as recommended by Jansen et al. [23]. Genomic PCR products were sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies) on the ABI 3130x1 Genetic Analyzer (Life Technologies). Genotypes for rs378652941 (TMEM95:c.483C>A, p.Cys161X, Chr19:27689622), rs381722524 (KIF1C:c.197A>G, p.Gln66Arg, Chr19:27042848) and rs385135118 (ACADVL:c.706C>A, p.Pro236Thr, Chr19:27570146) were obtained by TaqMan genotyping assays (Life Technologies) in 1990, 1222 and 1206 animals, respectively, representing three different breeds (BV, FV, HF). The primer and probe sequences are listed in Table S7.

Topology prediction for TMEM95

The topology of bovine transmembrane protein 95 (NCBI reference sequence: XP_002695846.1) was predicted with SPOCTOPUS [71] and PHOBIUS [72]. Both methods predict the transmembrane protein topology while accounting for the existence of a N-terminal signal peptide sequence. The protein topology was visualized with SOSUI [73]. ClustalW [74] was used for multi-species alignment of the protein sequence and for the prediction of conserved regions.

Assessment of sperm morphology, motility and viability

Cryopreserved (−196°C) sperm specimens of 30 bulls (10 wt/wt, 10 wt/mt, 10 mt/mt) were obtained from artificial insemination companies. Two different ejaculates of each bull were evaluated with two straws pooled per ejaculate. After thawing (37°C, 30 s), sperm morphology was assessed by staining with Diff-Quik (Siemens Healthcare, Germany). Sperm total motility was investigated immediately after thawing by phase-contrast microscopy using the Leica DM 1000 microscope (Leica, Germany). Viability of spermatozoa was investigated after thawing by mixing 5 µl of the thawed ejaculate with aqueous Eosin Y solution (Sigma Aldrich, Germany) in a volume ratio of 1∶1. Intact and viable spermatozoa stay colourless whereas spermatozoa with disturbed membrane integrity stain red. Counting of viable sperm was done within 10 seconds after mixing. Two hundred spermatozoa in at least two different fields of view were investigated at a magnification of 400× to analyse morphology, viability and motility.

Assessment of ejaculate volume, sperm count and progressive motility

Fresh semen traits (ejaculate volume, sperm count and progressive motility) of 203 AI bulls (177 wt/wt, 21wt/mt, 5 mt/mt) were provided by an artificial insemination company. Semen quality was analysed based on 10,682 ejaculates with an average of 52.6 ejaculates per bull. At semen collection, the age of the bulls ranged from 1.1 to 2.5 years.

Immunohistochemical localization of TMEM95

Immunohistochemistry on cryopreserved sperm specimens of 33 bulls (10 wt/wt, 10 wt/mt,13 mt/mt) was repeated for 3 times. After thawing (37°C, 30 s), spermatozoa were washed in phosphate buffered saline (PBS) twice and were diluted in PBS to a concentration of 500,000 spermatozoa/ml. Thereafter, drops of 7 µl were placed on 3-aminopropyl-ethoxysilane-coated slides and dried on a heating-plate at a temperature of 38°C. Subsequently, the slides were fixed in Bouin's solution for 7 min and washed in PBS twice. Non-specific binding was blocked by incubation in blocking buffer (0.1% bovine serum Albumin in PBS, Sigma-Aldrich, Germany) for 5 minutes and in normal goat serum (dilution 1∶5 in PBS, Invitrogen, Germany). Next, the spermatozoa were incubated with the first antibody Yomics Ab989 (mouse-derived against human TMEM95, Primm, USA) in a dilution of 1∶200 in blocking buffer at 4°C overnight. The secondary antibody was the Fluorescein (FITC)-conjugated AffiniPure Goat anti Mouse IgG (H+L) (Dianova, Germany, dilution 1∶200). Negative controls were done by a) replacing the first antibody with PBS and b) using a non-relevant anti-mouse antibody directed against Villin (1∶75, Beckman Coulter). Specimens were evaluated by using a confocal laser scanning microscope (Leica DM IRBE) in magnifications from 400 to 800.

Supporting Information

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