A population of bulls with 1.7% 56-day non-return rate were found to share a 1386 kb segment of homozygosity (Pausch et al. 2014 PLoS Genet. 10, e1004044). That study identified a nonsense mutation in the transmembrane protein 95 (TMEM95)-encoding gene as the most likely candidate for the subfertility phenotype. Sperm from homozygous bulls (mt/mt) exhibit normal motility and morphology. However, they are not as successful as sperm from wildtype (wt/wt) bulls in binding to the zona pellucida (ZP) or penetrating the plasma membrane of oocytes, which results in lower in vitro fertility (Fernandez-Fuertes et al. 2017 Biol. Reprod. 97, 50-60, 10.1093/biolre/iox065). Sperm from mice deficient for another transmembrane protein, TMEM16E, have normal total motility but cannot penetrate the ZP because they have sluggish motility and are not able to swim in a straight line (Gyobu et al. 2015 Mol. Cell. Biol. 36, 645-659). To determine whether this is one reason behind mt/mt bull subfertility, we assessed the ability of sperm from these bulls to migrate through cervical mucus of oestrus cows. In addition, because mt/mt bulls are homozygous for such an extensive chromosomal segment, this study aimed to determine whether TMEM95 alone is responsible for the loss of sperm function. To test this, sperm from a wt/wt bull was incubated in the presence of anti-TMEM95 antibody (Ab; 1:20 dilution) 1 h before fertilization. In all experiments, data were assessed for normality of distribution and analysed using a one-way ANOVA. In experiment 1, bull genotype had no effect on the number of sperm present in mucus-filled capillaries or on their distribution along them, following 30-min incubation. In experiment 2, incubation of sperm from wt/wt bulls with Ab decreased oocyte cleavage rates compared with the untreated control (59 ± 0.3 v. 76 ± 0.9%, respectively; P < 0.05). However, this percentage was still higher than in the mt/mt group (59 ± 0.3 v. 31 ± 2.9% respectively; P < 0.05). Surprisingly, the Ab did not affect the percentage of blastocysts that developed at Day 8 after fertilization. Three hours after fertilization, a lower number of Ab-treated sperm bound to the ZP compared with untreated sperm (4 ± 1.2 v. 9 ± 2.1, respectively; P < 0.05). This number did not differ between the Ab and mt/mt groups. Consistent with the previous results, Ab-treated sperm were less able to penetrate ZP-free oocytes than untreated sperm (47 ± 1.5 and 58 ± 4.2%, respectively; P < 0.05). However, lower penetration rates were observed in the mt/mt than in the Ab-treated group (24 ± 3.9 and 47 ± 1.5%, respectively; P < 0.05). We concluded that sperm from mt/mt bulls could traverse cervical mucus from oestrus cows. Transmembrane protein 95 seems to be directly responsible for the lower ZP binding and, partially, for the lower plasma membrane penetration observed. However, how this protein exerts these functions and what other factors might be involved remain to be determined. Research was supported by the Irish Department of Agriculture, Food and The Marine under the Research Stimulus Programme (Grant No. 11S104).
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