Haprin-deficient spermatozoa are incapable of in vitro fertilization.

Haprin (TRIM36) is a ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin-deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility-related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin-deficient mice having poorer sperm morphology and motility than wild-type mice. Interestingly, haprin-deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.

Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa.

Formation of spermatozoa of normal shape, number, and motility is insufficient for the male siring of pups. The spermatozoa must be accompanied by sound fertilizing ability. We found that males with disrupted testis-expressed gene 101 (Tex101) produce normal-looking but fertilization-incompetent spermatozoa, which were accompanied by a deficiency of a disintegrin and metallopeptidase domain 3 (ADAM3) on sperm plasma membrane. It was also found that the existence of TEX101 on spermatozoa was regulated by angiotensin-converting enzyme (ACE). The removal of GPI-anchored protein TEX101 by ACE was essential to produce fertile spermatozoa, and the function of ACE was not depending on its well-known peptidase activity. The finding of TEX101 as a unique specific substrate for ACE may provide a potential target for the production of an awaited contraceptive medicine for men.

Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility [corrected].

A disintegrin and metalloproteinase 3 (ADAM3) is a sperm membrane protein critical for both sperm migration from the uterus into the oviduct and sperm primary binding to the zona pellucida (ZP). Here we show that the testis-specific protein disulfide isomerase homolog (PDILT) cooperates with the testis-specific calreticulin-like chaperone, calsperin (CALR3), in the endoplasmic reticulum and plays an indispensable role in the disulfide-bond formation and folding of ADAM3. Pdilt(-/-) mice were male infertile because ADAM3 could not be folded properly and transported to the sperm surface without the PDILT/CALR3 complex. Peculiarly we find that not only Pdilt(-/-), but also Adam3(-/-), spermatozoa effectively fertilize eggs when the eggs are surrounded in cumulus oophorus. These findings reveal that ADAM3 requires testis-specific private chaperones to be folded properly and that the principle role of ADAM3 is for sperm migration into the oviduct but not for the fertilization event. Moreover, the importance of primary sperm ZP binding, which has been thought to be a critical step in mammalian fertilization, should be reconsidered.

Thirteen Ovary-Enriched Genes Are Individually Not Essential for Female Fertility in Mice.

Infertility is considered a global health issue as it currently affects one in every six couples, with female factors reckoned to contribute to partly or solely 50% of all infertility cases. Over a thousand genes are predicted to be highly expressed in the female reproductive system and around 150 genes in the ovary. However, some of their functions in fertility remain to be elucidated. In this study, 13 ovary and/or oocyte-enriched genes (Ccdc58, D930020B18Rik, Elobl, Fbxw15, Oas1h, Nlrp2, Pramel34, Pramel47, Pkd1l2, Sting1, Tspan4, Tubal3, Zar1l) were individually knocked out by the CRISPR/Cas9 system. Mating tests showed that these 13 mutant mouse lines were capable of producing offspring. In addition, we observed the histology section of ovaries and performed in vitro fertilization in five mutant mouse lines. We found no significant anomalies in terms of ovarian development and fertilization ability. In this study, 13 different mutant mouse lines generated by CRISPR/Cas9 genome editing technology revealed that these 13 genes are individually not essential for female fertility in mice.

Individual disruption of 12 testis-enriched genes via the CRISPR/Cas9 system does not affect the fertility of male mice.

More than 1200 genes have been shown in the database to be expressed predominantly in the mouse testes. Advances in genome editing technologies such as the CRISPR/Cas9 system have made it possible to create genetically engineered mice more rapidly and efficiently than with conventional methods, which can be utilized to screen genes essential for male fertility by knocking out testis-enriched genes. Finding such genes related to male fertility would not only help us understand the etiology of human infertility but also lead to the development of male contraceptives. In this study, we generated knockout mice for 12 genes (Acrv1, Adgrf3, Atp8b5, Cfap90, Cfap276, Fbxw5, Gm17266, Lrrd1, Mroh7, Nemp1, Spata45, and Trim36) that are expressed predominantly in the testis and examined the appearance and histological morphology of testes, sperm motility, and male fertility. Mating tests revealed that none of these genes is essential for male fertility at least individually. Notably, knockout mice for Gm17266 showed smaller testis size than the wild-type but did not exhibit reduced male fertility. Since 12 genes were not individually essential for male fertilization, it is unlikely that these genes could be the cause of infertility or contraceptive targets. It is better to focus on other essential genes because complementary genes to these 12 genes may exist.

Gene-deficient mouse model established by CRISPR/Cas9 system reveals 15 reproductive organ-enriched genes dispensable for male fertility.

Since the advent of gene-targeting technology in embryonic stem cells, mice have become a primary model organism for investigating human gene function due to the striking genomic similarities between the two species. With the introduction of the CRISPR/Cas9 system for genome editing in mice, the pace of loss-of-function analysis has accelerated significantly. This has led to the identification of numerous genes that play crucial roles in male reproductive processes, including meiosis, chromatin condensation, flagellum formation in the testis, sperm maturation in the epididymis, and fertilization in the oviduct. Despite the advancements, the functions of many genes, particularly those enriched in male reproductive tissues, remain largely unknown. In our study, we focused on 15 genes and generated 13 gene-deficient mice [4933411K16Rik, Adam triple (Adam20, Adam25, and Adam39), BC048671, Cfap68, Gm4846, Gm4984, Gm13570, Nt5c1b, Ppp1r42, Saxo4, Sh3d21, Spz1, and Tektl1] to elucidate their roles in male fertility. Surprisingly, all 13 gene-deficient mice exhibited normal fertility in natural breeding experiments, indicating that these genes are not essential for male fertility. These findings have important implications as they may help prevent other research laboratories from duplicating efforts to generate knockout mice for genes that do not demonstrate an apparent phenotype related to male fertility. By shedding light on the dispensability of these genes, our study contributes to a more efficient allocation of research resources in the exploration of male reproductive biology.

Ingestion of Soybean Sprouts Containing a HASPIN Inhibitor Improves Condition in a Mouse Model of Alzheimer's Disease.

The MATP/tau protein is hyperphosphorylated in Alzheimer's patients. Therefore, research into the regulation of tau protein phosphorylation is important for understanding Alzheimer's disease. HASPIN is a serine/threonine kinase that is expressed in various cells. To examine whether HASPIN is involved in the onset of Alzheimer's disease through tau protein phosphorylation, we investigated the effects of a diet including soybean sprouts rich in the HASPIN inhibitor coumestrol in a mouse model of Alzheimer's disease (5xFAD). The results showed that HASPIN was expressed in the hippocampus and phosphorylated tau protein, while the ingestion of soybean sprouts containing coumestrol suppressed the development of spatial cognitive dysfunction in 5xFAD. These results indicate that HASPIN may be one of the target molecules for the repression of tau phosphorylation in the treatment of Alzheimer's disease.

Distinct bidirectional regulation of LFA1 and α4ß7 by Rap1 and integrin adaptors in T cells under shear flow.

Bidirectional control of integrin activation plays crucial roles in cell adhesive behaviors, but how integrins are specifically regulated by inside-out and outside-in signaling has not been fully understood. Here, we report distinct bidirectional regulation of major lymphocyte homing receptors LFA1 and α4ß7 in primary T cells. A small increase of Rap1 activation in L-selectin-mediated tether/rolling was boosted by the outside-in signaling from ICAM1-interacting LFA1 through subsecond, simultaneous activation of Rap1 GTPase and talin1, but not kindlin-3, resulting in increased capture and slowing. In contrast, none of them were required for tether/rolling by α4ß7 on MAdCAM1. High Rap1 activation with chemokines or the loss of Rap1-inactivating proteins Rasa3 and Sipa1 increased talin1/kindlin-3-dependent arrest with high-affinity binding of LFA1 to membrane-anchored ICAM1. However, despite increased affinity of α4ß7, activated Rap1 severely suppressed adhesion on MAdCAM1 under shear flow, indicating the critical importance of a sequential outside-in/inside-out signaling for α4ß7.

Testis-enriched ferlin, FER1L5, is required for Ca2+-activated acrosome reaction and male fertility.

Spermatozoa need to undergo an exocytotic event called the acrosome reaction before fusing with eggs. Although calcium ion (Ca2+) is essential for the acrosome reaction, its molecular mechanism remains unknown. Ferlin is a single transmembrane protein with multiple Ca2+-binding C2 domains, and there are six ferlins, dysferlin (DYSF), otoferlin (OTOF), myoferlin (MYOF), fer-1-like 4 (FER1L4), FER1L5, and FER1L6, in mammals. Dysf, Otof, and Myof knockout mice have been generated, and each knockout mouse line exhibited membrane fusion disorders such as muscular dystrophy in Dysf, deafness in Otof, and abnormal myogenesis in Myof. Here, by generating mutant mice of Fer1l4, Fer1l5, and Fer1l6, we found that only Fer1l5 is required for male fertility. Fer1l5 mutant spermatozoa could migrate in the female reproductive tract and reach eggs, but no acrosome reaction took place. Even a Ca2+ ionophore cannot induce the acrosome reaction in Fer1l5 mutant spermatozoa. These results suggest that FER1L5 is the missing link between Ca2+ and the acrosome reaction.

Calsperin is a testis-specific chaperone required for sperm fertility.

Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3(-/-) males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.

OAZ-t/OAZ3 is essential for rigid connection of sperm tails to heads in mouse.

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t-disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines.

A Haspin promoter element induces tissue-specific methylation of a transcription region and the regulation of gene expression in mouse ova.

HASPIN is a nuclear serine-threonine kinase originally identified in the mouse testis. Its 193 bp DNA promoter element (hereafter, 193PE) regulates bidirectional, synchronous gene expression in the germ cells of male mice. Recent studies have shown that Haspin is also expressed in trace amounts in somatic cells; HASPIN also functions in oocytes. Haspin expression is regulated by the tissue-specific methylation of Haspin genomic DNA regions, including somatic cells. This study investigated relationship between 193PE and DNA methylation by examining methylation status of transgenic mice carrying 193PE and a reporter gene. In somatic (liver) cells carrying the reporter gene, 193PE induced methylation as well as trace expression of the reporter gene. In the testis, 193PE induced hypomethylation and intense reporter gene expression. Expression of HASPIN in an egg was assessed using human chorionic gonadotrophin to induce ovulation in female transgenic mice. The results showed that 193PE induced tissue-specific methylation, which resulted in reporter gene expression in a mouse egg.

Inhibitory Effect of the HASPIN Inhibitor CHR-6494 on BxPC-3-Luc, A Luciferase-Expressing Pancreatic Cancer Cell Line.

HASPIN acts in chromosome segregation via histone phosphorylation. Recently, HASPIN inhibitors have been shown to suppress growth of various cancer cells. Pancreatic cancer has no symptom in the early stages and may progress before detection. So, the 5-year survival rate is low. Here, we reported that administration of the HASPIN inhibitor, CHR-6494, to mice bearing pancreatic BxPC-3-Luc cancer cells significantly suppressed growth of BxPC-3-Luc cells. CHR-6494 might be a useful agent for treating pancreatic cancer.

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice.

Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.

Evaluation of the antiproliferative effects of the HASPIN inhibitor CHR-6494 in breast cancer cell lines.

HASPIN is a serine/threonine kinase that regulates mitosis by phosphorylating histone H3 at threonine 3. The expression levels of HASPIN in various cancers are associated with tumor malignancy and poor survival, suggesting that HASPIN inhibition may suppress cancer growth. As HASPIN mRNA levels are elevated in human breast cancer tissues compared with adjacent normal tissues, we examined the growth-suppressive effects of CHR-6494, a potent HASPIN inhibitor, in breast cancer cell lines in vitro and in vivo. We found that HASPIN was expressed in breast cancer cells of all molecular subtypes, as well as in immortalized mammary epithelial cells. HASPIN expression levels appeared to be correlated with the cell growth rate but not the molecular subtype of breast cancer. CHR-6494 exhibited potent antiproliferative effects against breast cancer cell lines and immortalized mammary epithelial cells in vitro, but failed to inhibit the growth of MDA-MB-231 xenografted tumors under conditions that have significant effects in a colorectal cancer model. These results imply that CHR-6494 does have antiproliferative effects in some situations, and further drug screening efforts are anticipated to identify more potent and selective HASPIN inhibition for use as an anticancer agent in breast cancer patients.

Unique alternative translation from two open reading frames on Acpin1 mRNA yields an acrosomal protein and a salivary-gland-specific protein.

OBJECTIVE: To examine the expression profiles of the proteins translated from Acpin1 mRNA in germ cells. METHODS: Northern and western blotting of various tissues and immunohistochemical analysis of germ cells were carried out in a mouse model. RESULTS: ACPIN1 protein was transcribed from the longer, 3' open reading frame (ORF) of Acpin1. An alternative-splicing variant, Acpin1vs, contained only the smaller, 5' ORF of the full-length Acpin1 gene. Its gene product, SAGSIN1, was expressed specifically in salivary glands. Retrotransposed regions of Acpin1 homology were also detected in various chromosomes, and intronless paralogous genes on the X chromosome were expressed in the testis and other tissues. The genomic structure of Acpin1 is highly conserved in mammals. CONCLUSION: The two ORFs on the Acpin1 mRNA are independently translated in differentiated cells. Analysis of gene Acpin1 might clarify the molecular mechanism of spermatogenesis.

Author Correction: Anchoring cortical granules in the cortex ensures trafficking to the plasma membrane for post-fertilization exocytosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anchoring cortical granules in the cortex ensures trafficking to the plasma membrane for post-fertilization exocytosis.

Following fertilization, cortical granules exocytose ovastacin, a metalloendopeptidase that cleaves ZP2 in the zona pellucida surrounding mouse eggs to prevent additional sperm binding. Using high- and super-resolution imaging with ovastacinmCherry as a fluorescent marker, we characterize cortical granule dynamics at single granule resolution in transgenic mouse eggs. Newly-developed imaging protocols provide an unprecedented view of vesicular dynamics near the plasma membrane in mouse eggs. We discover that cortical granule anchoring in the cortex is dependent on maternal MATER and document that myosin IIA is required for biphasic trafficking to the plasma membrane. We observe local clearance of cortical actin during exocytosis and determine that pharmacologic or genetic disruption of trafficking to the plasma membrane impairs secretion of cortical granules and results in polyspermy. Thus, the regulation of cortical granule dynamics at the cortex-plasma membrane interface is critical for exocytosis and the post-fertilization block to sperm binding that ensures monospermic fertilization.

Isolation and characterization of the spermatid-specific Smrp1 gene encoding a novel manchette protein.

The manchette, which is the structure that appears around the nuclei of elongated spermatids, is assumed to be involved in nuclear shaping during spermiogenesis and the transport of various proteins between the nucleus and sperm tail. In this report, we describe the molecular cloning and characterization of a mouse spermatid-specific manchette-related protein 1 (Smrp1) from a spermatid-specific subtracted mouse testis cDNA library. The isolated Smrp1 cDNA clones could be divided into three variants based on sequence analysis. Computer-assisted analysis showed that these variants were splice variants from a single locus of the mouse genome. The three putative proteins consisted of 296, 260, and 175 amino acids, respectively. Although 155 amino acids of the N terminus were common to the three proteins, they were distinguished by their C-terminal regions. Western blot analyses using specific antisera showed that SMRP1 expression was specific to the testes and that only the 261-amino-acid form was translated into protein. Immunohistochemistry revealed that SMRP1 was localized to the cytoplasm of step 9-12 elongated spermatids. The protein appeared in a cap formation that covered the caudal sides of the elongated nuclei. This localization pattern coincided with that of the manchette. SMRP1 may play an important role as a functional protein that co-operates with manchette proteins.