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Actin-related proteins (Arps) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp, and is highly conserved in rodents and primates. ACTL7B is specifically expressed in round and elongating spermatids during spermiogenesis. Here, we have generated an Actl7b-null allele in mice to unravel the role of ACTL7B in sperm formation. Male mice homozygous for the Actl7bnull allele (Actl7b−/−) were infertile, whereas heterozygous males (Actl7b+/−) were fertile. Severe spermatid defects, such as detached acrosomes, disrupted membranes and flagella malformations start to appear after spermiogenesis step 9 in Actl7b−/− mice, finally resulting in spermatogenic arrest. Abnormal spermatids were degraded and levels of autophagy markers were increased. Co-immunoprecipitation with mass spectrometry experiments identified an interaction between ACTL7B and the LC8 dynein light chains DYNLL1 and DYNLL2, which are first detected in step 9 spermatids and mislocalized when ACTL7B is absent. Our data unequivocally establish that mutations in ACTL7B are directly related to male infertility, pressing for additional research in humans.

Post-copulatory sexual selection is thought to influence the evolution of genes involved in reproduction. However, the detection of straightforward effects has been proven difficult due to the complexity and diversity of reproductive landscapes found in different taxa. Here, we compare the possible effect of relative testes mass as a sperm competition proxy on protamine genotype (protamine 1/protamine 2 ratio) and the link to sperm head phenotype in two rodent groups, mice, and voles. In mice, protamine expression ratios were found to increase from low values toward a 1:1 ratio in a positive association with testes mass, and relative sperm head area. In contrast, in voles, decreasing protamine expression ratios were found in species with larger testes but, surprisingly, they range from high values, again toward a 1:1 ratio, and showing a negative correlation with relative sperm head area. Altogether, we found differences in the way protamines seem to be selected and involved in adaptations of the sperm head in voles and mice. However, sexual selection driven by sperm competition seems to exhibit a common evolutionary pattern in both groups toward an equilibrium in the expression of the two protamines.

[Background]: Cysteine-RIch Secretory Proteins (CRISP) are expressed in the reproductive tract of mammalian males and are involved in fertilization and related processes. Due to their important role in sperm performance and sperm-egg interaction, these genes are likely to be exposed to strong selective pressures, including postcopulatory sexual selection and/or male-female coevolution. We here perform a comparative evolutionary analysis of Crisp genes in mammals. Currently, the nomenclature of CRISP genes is confusing, as a consequence of discrepancies between assignments of orthologs, particularly due to numbering of CRISP genes. This may generate problems when performing comparative evolutionary analyses of mammalian clades and species. To avoid such problems, we first carried out a study of possible orthologous relationships and putative origins of the known CRISP gene sequences. Furthermore, and with the aim to facilitate analyses, we here propose a different nomenclature for CRISP genes (EVAC1–4, “EVolutionarily-analyzed CRISP”) to be used in an evolutionary context., [Results]: We found differing selective pressures among Crisp genes. CRISP1/4 (EVAC1) and CRISP2 (EVAC2) orthologs are found across eutherian mammals and seem to be conserved in general, but show signs of positive selection in primate CRISP1/4 (EVAC1). Rodent Crisp1 (Evac3a) seems to evolve under a comparatively more relaxed constraint with positive selection on codon sites. Finally, murine Crisp3 (Evac4), which appears to be specific to the genus Mus, shows signs of possible positive selection. We further provide evidence for sexual selection on the sequence of one of these genes (Crisp1/4) that, unlike others, is thought to be exclusively expressed in male reproductive tissues., [Conclusions]: We found differing selective pressures among CRISP genes and sexual selection as a contributing factor in CRISP1/4 gene sequence evolution. Our evolutionary analysis of this unique set of genes contributes to a better understanding of Crisp function in particular and the influence of sexual selection on reproductive mechanisms in general.