TSKS localizes to nuage in spermatids and regulates cytoplasmic elimination during spermiation.

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.

1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm-egg interaction.

Sperm acrosomal membrane proteins, such as Izumo sperm-egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)-Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.

Trim41 is required to regulate chromosome axis protein dynamics and meiosis in male mice.

Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.

FAM71F1 binds to RAB2A and RAB2B and is essential for acrosome formation and male fertility in mice.

The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation.

Multiple genes in the Pate5-13 genomic region contribute to ADAM3 processing†.

Sperm proteins undergo post-translational modifications during sperm transit through the epididymis to acquire fertilizing ability. We previously reported that the genomic region coding Pate family genes is key to the proteolytic processing of the sperm membrane protein ADAM3 and male fertility. This region contains nine Pate family genes (Pate5-13), and two protein-coding genes (Gm27235 and Gm5916), with a domain structure similar to Pate family genes. Therefore, in this study, we aimed to identify key factors by narrowing the genomic region. We generated three knockout (KO) mouse lines using CRISPR/Cas9: single KO mice of Pate10 expressed in the caput epididymis; deletion KO mice of six caput epididymis-enriched genes (Pate5-7, 13, Gm27235, and Gm5916) (Pate7-Gm5916 KO); and deletion KO mice of four genes expressed in the placenta and epididymis (Pate8, 9, 11, and 12) (Pate8-12 KO). We observed that the fertility of only Pate7-Gm5916 KO males was reduced, whereas the rest remained unaffected. Furthermore, when the caput epididymis-enriched genes, Pate8 and Pate10 remained in Pate7-Gm5916 KO mice were independently deleted, both KO males displayed more severe subfertility due to a decrease in mature ADAM3 and a defect in sperm migration to the oviduct. Thus, our data showed that multiple caput epididymis-enriched genes within the region coding Pate5-13 cooperatively function to ensure male fertility in mice.

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice.

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules and some attached projections. Radial spokes (RSs) facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 radial spoke proteins (RSP1-RSP23), although the roles of some radial spoke proteins remain unknown. Recently, RSP15 has been reported to be bound to the stalk of RS2, but its homolog in mammals has not been identified. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A and RSPH3B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was not significantly affected. These data indicate that LRRC23 is a key regulator that underpins the integrity of the RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. This article has an associated First Person interview with the first author of the paper.

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice.

Sperm-oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm-oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm-oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm-oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm-oocyte fusion.

Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice.

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.

Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice.

CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.

NAIL: an evolutionarily conserved lncRNA essential for licensing coordinated activation of p38 and NFκB in colitis.

OBJECTIVE: NFκB is the key modulator in inflammatory disorders. However, the key regulators that activate, fine-tune or shut off NFκB activity in inflammatory conditions are poorly understood. In this study, we aim to investigate the roles that NFκB-specific long non-coding RNAs (lncRNAs) play in regulating inflammatory networks. DESIGN: Using the first genetic-screen to identify NFκB-specific lncRNAs, we performed RNA-seq from the p65-/- and Ikkß-/- mouse embryonic fibroblasts and report the identification of an evolutionary conserved lncRNA designated mNAIL (mice) or hNAIL (human). hNAIL is upregulated in human inflammatory disorders, including UC. We generated mNAILΔNFκB mice, wherein deletion of two NFκB sites in the proximal promoter of mNAIL abolishes its induction, to study its function in colitis. RESULTS: NAIL regulates inflammation via sequestering and inactivating Wip1, a known negative regulator of proinflammatory p38 kinase and NFκB subunit p65. Wip1 inactivation leads to coordinated activation of p38 and covalent modifications of NFκB, essential for its genome-wide occupancy on specific targets. NAIL enables an orchestrated response for p38 and NFκB coactivation that leads to differentiation of precursor cells into immature myeloid cells in bone marrow, recruitment of macrophages to inflamed area and expression of inflammatory genes in colitis. CONCLUSION: NAIL directly regulates initiation and progression of colitis and its expression is highly correlated with NFκB activity which makes it a perfect candidate to serve as a biomarker and a therapeutic target for IBD and other inflammation-associated diseases.

Identification of transmembrane protein 168 mutation in familial Brugada syndrome.

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.

Tmprss12 is required for sperm motility and uterotubal junction migration in mice†.

Spermatozoa are produced in the testis but gain their fertilizing ability during epididymal migration. This necessary step in sperm maturation includes posttranslational modification of sperm membrane proteins that includes protein processing by proteases. However, the molecular mechanism underpinning this epididymal sperm maturation remains unknown. In this study, we focused on transmembrane serine protease 12 (Tmprss12). Based on multi-tissue expression analysis by PCR, Tmprss12 was specifically expressed in the testis, and its expression started on day 10 postpartum, corresponding to the stage of zygotene spermatocytes. TMPRSS12 was detected in the acrosomal region of spermatozoa by immunostaining. To reveal the physiological function of TMPRSS12, we generated two knockout (KO) mouse lines using the CRISPR/Cas9 system. Both indel and large deletion lines were male sterile showing that TMPRSS12 is essential for male fertility. Although KO males exhibited normal spermatogenesis and sperm morphology, ejaculated spermatozoa failed to migrate from the uterus to the oviduct. Further analysis revealed that a disintegrin and metalloprotease 3 (ADAM3), an essential protein on the sperm membrane surface that is required for sperm migration, was disrupted in KO spermatozoa. Moreover, we found that KO spermatozoa showed reduced sperm motility via computer-assisted sperm analysis, resulting in a low fertilization rate in vitro. Taken together, these data indicate that TMPRSS12 has dual functions in regulating sperm motility and ADAM3-related sperm migration to the oviduct. Because Tmprss12 is conserved among mammals, including humans, our results may explain some genetic cases of idiopathic male infertility, and TMPRSS12 and its downstream cascade may be novel targets for contraception.

CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproduction†.

Developing a safe and effective male contraceptive remains a challenge in the field of medical science. Molecules that selectively target the male reproductive tract and whose targets are indispensable for male reproductive function serve among the best candidates for a novel non-hormonal male contraceptive method. To determine the function of these genes in vivo, mutant mice carrying disrupted testis- or epididymis-enriched genes were generated by zygote microinjection or electroporation of the CRISPR/Cas9 components. Male fecundity was determined by consecutively pairing knockout males with wild-type females and comparing the fecundity of wild-type controls. Phenotypic analyses of testis appearance and weight, testis and epididymis histology, and sperm movement were further carried out to examine any potential spermatogenic or sperm maturation defect in mutant males. In this study, we uncovered 13 testis- or epididymis-enriched evolutionarily conserved genes that are individually dispensable for male fertility in mice. Owing to their dispensable nature, it is not feasible to use these targets for the development of a male contraceptive.

Seminal vesicle secretory protein 7, PATE4, is not required for sperm function but for copulatory plug formation to ensure fecundity†.

Seminal vesicle secretions (SVSs), together with spermatozoa, are ejaculated into the female reproductive tract. SVS7, also known as PATE4, is one of the major SVS proteins found in the seminal vesicle, copulatory plug, and uterine fluid after copulation. Here, we generated Pate4 knockout (-/-) mice and examined the detailed function of PATE4 on male fecundity. The morphology and weight of Pate4-/- seminal vesicles were comparable to the control. Although Pate4-/- cauda epididymal spermatozoa have no overt defects during in vitro fertilization, Pate4-/- males were subfertile. We found that the copulatory plugs were smaller in the vagina of females mated with Pate4-/- males, leading to semen leakage and a decreased sperm count in the uterus. When the females mated with Pate4-/- males were immediately re-caged with Pate4+/+ males, the females had subsequent productive matings. When the cauda epididymal spermatozoa were injected into the uterus and plugged artificially [artificial insemination (AI)], Pate4-/- spermatozoa could efficiently fertilize eggs as compared to wild-type spermatozoa. We finally examined the effect of SVSs on AI, and observed no difference in fertilization rates between Pate4+/+ and Pate4-/- SVSs. In conclusion, PATE4 is a novel factor in forming the copulatory plug that inhibits sequential matings and maintains spermatozoa in the uterus to ensure male fecundity.

CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.

Lvrn expression is not critical for mouse placentation.

Preeclampsia is a systemic disease caused by abnormal placentation that affects both mother and fetus. It was reported that Laeverin (LVRN, also known as Aminopeptidase Q) was up-regulated in the placenta of preeclamptic patients. However, physiological and pathological functions of LVRN remained to be unknown. Here we characterized Lvrn function during placentation in mice. RT-PCR showed that Lvrn is expressed in both fetus and placenta during embryogenesis, and several adult tissues. When we overexpressed Lvrn in a placenta-specific manner using lentiviral vectors, we did not see any defects in both placentae and fetuses. The mice carrying Lvrn overexpressing placentas did not show any preeclampsia-like symptoms such as maternal high blood pressure and fetal growth restriction. We next ablated Lvrn by CRISPR/Cas9-mediated genome editing to see physiological function. In Lvrn ablated mice, maternal blood pressure during pregnancy was not affected, and both placentas and fetuses grew normally. Collectively, these results suggest that, LVRN is irrelevant to preeclampsia and dispensable for normal placentation and embryonic development in mice.

Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice.

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

Physiological function of seminal vesicle secretions on male fecundity.

BACKGROUND: A mixture of spermatozoa and accessory gland secretions (from seminal vesicles, prostates, and coagulating glands) is ejaculated into the female reproductive tract at copulation. However, the physiological function of accessory glands on male fecundity remains unclear. METHODS: Publications regarding the physiological functions of male accessory glands were summarized. MAIN FINDINGS RESULTS: The functions of accessory glands have been studied using male rodents surgically removed coagulating glands (CG), prostates (PR), or seminal vesicles (SV). CG-removed males are fertile or subfertile, while the fecundity of PR-removed males is controversial. SV-removed males show copulatory plug defects, leading to fewer sperm in the uterus and severe subfertility. TGM4, SVS2, and PATE4 were identified as essential factors for copulatory plug formation. When the sufficient number of epididymal spermatozoa was artificially injected into a uterus (AI method), they could efficiently fertilize oocytes, implicating that accessory gland secretions are not essential. Seminal vesicle secretions (SVSs) improved fertilization rates only when low numbers of spermatozoa were used for AI. The changes of uterine environment by SVSs could not improve the pregnancy rate. CONCLUSION: Accessory gland factors are critical for copulatory plug formation and support sperm fertilizing ability.

Protein biomarkers for male artificial insemination subfertility in bovine spermatozoa.

Background: Although artificial insemination (AI) technique is an established biotechnology for bovine reproduction, the results of AI (conception rates) have a tendency to decline gradually. To our annoyance, moreover, AI-subfertile bulls have been occasionally found in the AI centers. To resolve these serious problems, it is necessary to control the sperm quality more strictly by the examinations of sperm molecules. Methods: We reviewed a number of recent articles regarding potentials of bovine sperm proteins as the biomarkers for bull AI-subfertility and also showed our unpublished supplemental data on the bull AI-subfertility associated proteins. Main findings: Bull AI-subfertility is caused by the deficiency or dysfunctions of various molecules including regulatory proteins of ATP synthesis, acrosomal proteins, nuclear proteins, capacitation-related proteins and seminal plasma proteins. Conclusion: In order to control the bovine sperm quality more strictly by the molecular examinations, it is necessary to select suitable sperm protein biomarkers for the male reproductive problems which happen in the AI centers.