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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., Thirteen testis- or epididymis-enriched genes are individually dispensable for male fertility based on phenotypic analyses of mutant mice produced by the CRISPR/Cas9 system.

Spermiation is a multiple-step process involving profound cellular changes in both spermatids and Sertoli cells. We have observed spermiation defects, including abnormalities in spermatid orientation, translocation and release, in mice deficient in the retinoic acid receptor alpha (RARA) and upon treatment with a pan-RAR antagonist. To elucidate the role of retinoid signaling in regulating spermiation, we first characterized the time course of appearance of spermiogenic defects in response to treatment with the pan-RAR antagonist. The results revealed that defects in spermiation are indeed among the earliest abnormalities in spermatogenesis observed upon inhibition of retinoid signaling. Using fluorescent dye-conjugated phalloidin to label the ectoplasmic specialization (ES), we showed for the first time that these defects involved improper formation of filamentous actin (F-actin) bundles in step 8–9 spermatids and a failure of the actin-surrounded spermatids to move apically to the lumen and to disassemble the ES. The aberrant F-actin organization is associated with diminished nectin-3 expression in both RARA-deficient and pan-RAR antagonist-treated testes. An abnormal localization of both tyrosinated and detyrosinated tubulins was also observed during spermatid translocation in the seminiferous epithelium in drug-treated testes. These results highlight a crucial role of RAR receptor-mediated retinoid signaling in regulating microtubules and actin dynamics in the cytoskeleton rearrangements, required for proper spermiation. This is critical to understand in light of ongoing efforts to inhibit retinoid signaling as a novel approach for male contraception and may reveal spermiation components that could also be considered as new targets for male contraception., A crucial role of RAR receptor-mediated retinoid signaling in regulating microtubule and actin dynamics in cytoskeleton rearrangements during spermiogenesis is demonstrated.

Retinoic acid receptor alpha (RARA), a nuclear receptor protein, has been validated as a target for male contraception by gene knockout studies and also pharmacologically using a pan-retinoic acid receptor antagonist. Retinoic acid receptor alpha activity is indispensable for the spermatogenic process, and therefore its antagonists have potential as male contraceptive agents. This review discusses the effects of systematic dosing regimen modifications of the orally bioavailable and reversible pan-antagonist BMS-189453 as well as studies with the alpha-selective antagonists BMS-189532 and BMS-189614 in a murine model. We also provide an overview of structure–activity studies of retinoic acid receptor alpha antagonists that provide insight for the design of novel alpha-selective ligands., Fertility studies with retinoic acid receptor antagonists in male mice and the current state of RARA-selective antagonist development are reviewed.

One of the mechanisms that cells have developed to fulfil their specialized tasks is to express different molecular variants of a particular protein that has unique functional properties. Na,K-ATPase (NKA), the ion transport mechanism that maintains the transmembrane Na+ and K+ concentrations across the plasma membrane of cells, is one of such protein systems that shows high molecular and functional heterogeneity. Four different isoforms of the NKA catalytic subunit are expressed in mammalian cells (NKAα1, NKAα2, NKAα3, and NKAα4). NKAα4 (ATP1A4) is the isoform with the most restricted pattern of expression, being solely produced in male germ cells of the testis. NKAα4 is abundant in spermatozoa, where it is required for sperm motility and hyperactivation. This review discusses the expression, functional properties, mechanism of action of NKAα4 in sperm physiology, and its role in male fertility. In addition, we describe the use of NKAα4 as a target for male contraception and a potential approach to pharmacologically block its ion transport function to interfere with male fertility., This paper reviews the role and mechanisms of action of Na,K-ATPase α4 and its potential use of as a target for male contraception.

There is concern that using depot-medroxyprogesterone acetate (DMPA) for pregnancy prevention heightens HIV susceptibility. While no clinical data establishes causal link between HIV acquisition and use of this injectable progestin, prior work from our laboratory showed that DMPA comparably lowers genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and weakens genital epithelial barrier function in female mice and women. We likewise saw DMPA increase mouse susceptibility to multiple genital pathogens including HIV. Herein, we sought to confirm and extend these findings by comparing genital epithelial barrier function in untreated rhesus macaques (RM) vs. RM treated with DMPA or DMPA and estrogen (E). Compared to controls, genital tissue from RM with pharmacologically relevant serum levels of medroxyprogesterone acetate displayed significantly lower DSG1 levels and greater permeability to low molecular mass molecules. Conversely, DMPA-mediated effects on genital epithelial integrity and function were obviated in RM administered DMPA and E. These data corroborate the diminished genital epithelial barrier function observed in women initiating DMPA and identify RM as a useful preclinical model for defining effects of exogenous sex steroids on genital pathogen susceptibility. As treatment with E averted DMPA-mediated loss of genital epithelial barrier function, our results also imply that contraceptives releasing progestin and E may be less likely to promote transmission of HIV and other sexually transmitted pathogens than progestin-only compounds.

Sexually transmitted infections are highly prevalent, and over 40% of pregnancies are unplanned. We are producing new antibody-based multipurpose prevention technology products to address these problems and fill an unmet need in female reproductive health. We used a Nicotiana platform to manufacture monoclonal antibodies against two prevalent sexually transmitted pathogens, HIV-1 and HSV-2, and incorporated them into a vaginal film (MB66) for preclinical and Phase 1 clinical testing. These tests are now complete and indicate that MB66 is effective and safe in women. We are now developing an antisperm monoclonal antibody to add contraceptive efficacy to this product. The antisperm antibody, H6-3C4, originally isolated by Shinzo Isojima from the blood of an infertile woman, recognizes a carbohydrate epitope on CD52g, a glycosylphosphatidylinositol-anchored glycoprotein found in abundance on the surface of human sperm. We engineered the antibody for production in Nicotiana; the new antibody which we call “human contraception antibody,” effectively agglutinates sperm at concentrations >10 μg/ml and maintains activity under a variety of physiological conditions. We are currently seeking regulatory approval for a Phase 1 clinical trial, which will include safety and “proof of principle” efficacy endpoints. Concurrently, we are working with new antibody production platforms to bring the costs down, innovative antibody designs that may produce more effective second-generation antibodies, and delivery systems to provide extended protection., We are engineering monoclonal antibodies and delivery systems for multipurpose prevention technology products that will concurrently prevent sexually transmitted infections and unplanned pregnancies, addressing an important unmet need in reproductive health.

The development of non-hormonal contraceptives is critical to increase options for women. In combination with prevention against sexually transmitted infections, they can become an important component of multipurpose prevention technologies (MPTs) which address multiple reproductive health needs with a single product. Resulting from multiple rounds of expert consultations, this framework aims to guide the development of non-hormonal contraceptive MPTs. Key informant interviews with experts in family planning and HIV and STI prevention and MPT product developers and funders from around the globe were conducted, reviewed, and coded. Identified key themes were discussed by experts at the November 2019 Eunice Kennedy Shriver National Institute of Child Health and Human Development Contraceptive Development Meeting in Houston, Texas. Seven action strategies were identified to address key research gaps and priorities for advancing the field. They highlight the importance of identifying target populations, a systematic approach to collaborative research, and leveraging knowledge from other fields, including regulatory and patenting, manufacturing, and commercialization expertise. Employing expanded target product profiles and setting go/no-go decisions for non-hormonal MPTs will help to prioritize the most promising candidates in the drug development pipeline. Further, they call for optimizing investments and engagement of stakeholders from public and private sectors. These action strategies aim to facilitate collaboration and innovation amongst multidisciplinary MPT stakeholders. Paramount to success will be enhancing strategic alliances and reconciling the essential social–behavioral context and market forces that drive product use with the complexities of research and development, regulatory approval, and commercialization., The objective of this paper is to present an actionable framework that guides development of non-hormonal contraceptive MPTs.

Cyclin-dependent kinase 2 (CDK2) is a member of the larger cell cycle regulating CDK family of kinases, activated by binding partner cyclins as its name suggests. Despite its canonical role in mitosis, CDK2 knockout mice are viable but sterile, suggesting compensatory mechanisms for loss of CDK2 in mitosis but not meiosis. Here, we review the literature surrounding the role of CDK2 in meiosis, particularly a cyclin-independent role in complex with another activator, Speedy 1 (SPY1). From this evidence, we suggest that CDK2 could be a viable nonhormonal male contraceptive target. Finally, we review the literature of pertinent CDK2 inhibitors from the preclinical to clinical stages, mostly developed to treat various cancers. To date, there is no potent yet selective CDK2 inhibitor that could be repurposed as a contraceptive without appreciable off-target toxicity. To achieve selectivity for CDK2 over closely related kinases, developing compounds that bind outside the conserved adenosine triphosphate-binding site may be necessary., Cyclin-dependent kinase 2 (CDK2) is a validated and underexploited target for male contraception.

Current modern contraceptives rely heavily on the use of hormones. These birth control drug products, including pills, patches, injections, and IUDS, have been extremely beneficial to millions of women and their families over the past 50 years. But a surprisingly high number of women abandon such modern methods, many because they cannot tolerate the side effects and others because they have medical issues for which hormonal methods are contraindicated. In addition, modern hormonal methods are simply not available to many women. The extent of this problem is steadily becoming more apparent. We present the case for developing simple nonhormonal vaginal products that women can use when needed, ideal products that are multipurpose and offer both contraception and sexually transmitted disease protection. Gel-based vaginal products are particularly well suited for this purpose. Gels are easy to use, highly acceptable to many women, and can be safely formulated to enhance natural vaginal defenses against infection. However, the development of a new chemical entity for this application faces significant technical and regulatory hurdles. These challenges and our solutions are described for polyphenylene carboxymethylene (PPCM), a novel topical drug in a vaginal gel nearing human clinical trials. We have advanced PPCM from benchtop to IND-enabling studies and provide a brief description of the complex development process. We also describe a simple lab assay which can be used as a biomarker for contraceptive activity to enable pharmacodynamic studies in vaginal contraceptive development, both preclinically and in early human clinical trials., Nonhormonal contraceptive and microbicidal multipurpose drug products are needed to fill a gap in available products: development of polyphenylene carboxymethylene, a candidate vaginal gel is described.

Efforts to develop a male contraceptive method beyond condoms and vasectomy have been on-going for nearly 70 years. During this time, there have been ebbs and modest flows of resources available to support product development, but not at a level sufficient to carry research efforts through to market. The small community of researchers that have continued to pursue the development of male contraceptives is comprised of dedicated scientists who have a great deal of knowledge and experience to offer. While collaboration has been an organic outcome of limited resources, competing research objectives and geographically diverse locations have made consistent and sustained progress challenging, particularly for those working in the earliest stages of developing nonhormonal, reversible male contraceptive methods. While the past decade has seen an increase in funding to the field, the levels are still modest when placed in the context of actual costs to bring products to market. In addition, there are challenges still to be identified given that there is no regulatory precedent for these products. These challenges present an excellent use case for the application of design-thinking or human centered design, as a means of generating novel solutions. By engaging those with deep technical expertise in the field of male contraception as well as thought leaders from other fields of practice, design-thinking offers an opportunity to identify potential strategies, including nontraditional approaches, capable of driving the product development process forward, in a faster and more efficient manner., An organizational framework that engages and supports researchers and demonstrates appreciable collaborative outputs will result in a faster path to market for nonhormonal, reversible male contraceptive products.

Families with sequence similarity 170 members A and B (FAM170A and FAM170B) are testis-specific, paralogous proteins that share 31% amino acid identity and are conserved throughout mammals. While previous in vitro experiments suggested that FAM170B, an acrosome-localized protein, plays a role in the mouse sperm acrosome reaction and fertilization, the role of FAM170A in the testis has not been explored. In this study, we used CRISPR/Cas9 to generate null alleles for each gene, and homozygous null (−/−) male mice were mated to wild-type females for 6 months to assess fertility. Fam170b−/− males were found to produce normal litter sizes and had normal sperm counts, motility, and sperm morphology. In contrast, mating experiments revealed significantly reduced litter sizes and a reduced pregnancy rate from Fam170a−/− males compared with controls. Fam170a−/−;Fam170b−/− double knockout males also produced markedly reduced litter sizes, although not significantly different from Fam170a−/− alone, suggesting that Fam170b does not compensate for the absence of Fam170a. Fam170a−/− males exhibited abnormal spermiation, abnormal head morphology, and reduced progressive sperm motility. Thus, FAM170A has an important role in male fertility, as the loss of the protein leads to subfertility, while FAM170B is expendable. The molecular functions of FAM170A in spermatogenesis are as yet unknown; however, the protein localizes to the nucleus of elongating spermatids and may mediate its effects on spermatid head shaping and spermiation by regulating the expression of other genes. This work provides the first described role of FAM170A in reproduction and has implications for improving human male infertility diagnoses., The testis-specific protein FAM170A plays a role in sperm head morphology and motility that is critical for fertility, while its paralog, FAM170B, is not essential.

Contributions from a diverse set of scientific disciplines will be needed to help individuals make fully informed decisions regarding contraceptive choices least likely to promote HIV susceptibility. This commentary recaps contrasting interpretations of results from the Evidence for Contraceptive Options and HIV Outcomes (ECHO) Trial, a study that compared HIV risk in women using the progestin-only injectable contraceptive depot medroxyprogesterone acetate (DMPA) vs. two other contraceptive choices. It also summarizes results from basic and translational research that establish biological plausibility for earlier clinical studies that identified enhanced HIV susceptibility in women using DMPA., Basic and translational research studies provide strong indication that the contraceptive depot medroxyprogesterone acetate weakens genital mucosal barrier function, a vital first-line defense against HIV and other sexually transmitted pathogens.

A significant global unmet need for new contraceptive options for both women and men remains due to side effect profiles, medical concerns, and inconvenience of many currently available products. The pharmaceutical industry has largely abandoned early research and development for contraception and will not likely engage to bring new products to the market unless they have been significantly de-risked by showing promise in early phase clinical trials. This lack of interest by big pharma comes at a time when scientific and technological advances in biology and medicine are creating more opportunities than ever for the development of new and innovative drug products. Novel partnerships between the academic sector, small biotechnology companies, foundations, non-government organizations (NGOs), and the federal government could accelerate the development of new contraceptive products. We discuss the challenges and opportunities that we have encountered as an NGO with a mission to develop novel contraceptive products for low- and middle-income countries and how it differs from developing products for higher-income markets. We hope that our experiences and “lessons learned” will be of value to others as they proceed down the product development path, be it for female or male or for hormonal or nonhormonal contraceptives., Based on our experience and lessons learned as an NGO engaged in contraceptive research, we describe opportunities and challenges facing contraceptive developers outside the pharmaceutical industry across the stages of product development.

Semen liquefaction is a proteolytic process where a gel-like ejaculated semen becomes watery due to the enzymatic activity of prostate-derived serine proteases in the female reproductive tract. The liquefaction process is crucial for the sperm to gain their motility and successful transport to the fertilization site in Fallopian tubes (or oviducts in animals). Hyperviscous semen or failure in liquefaction is one of the causes of male infertility. Therefore, the biochemical inhibition of serine proteases in the female reproductive tract after ejaculation is a prime target for novel contraceptive development. Herein, we will discuss protein components in the ejaculates responsible for semen liquefaction and any developments of contraceptive methods in the past that involve the liquefaction process., We propose inhibition of semen liquefaction has the potential to be developed as a non-hormonal contraceptive method.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes., Ten testis-enriched genes are dispensable for male fertility, as determined by phenotypic analyses of knockout mice.

Mammalian spermatozoa are produced in the testis through spermatogenesis and matured in the epididymis to acquire fertilizing ability. Spermatozoa are ejaculated and migrate from the uterus to the oviducts to fuse with oocytes. Although over 2000 genes are expressed abundantly in mouse testes, the genes responsible for male fertility are not yet fully clarified. Here, we focused on two testis-enriched serine protease genes, Serine protease (Prss) 51 and Prss55, which overlap their gene loci partially in both mice and humans. To characterize their functions in male fertility, we first generated Prss51 and Prss55 double knockout (DKO) mice by CRISPR/Cas9 system and found that the DKO mice were sterile. DKO spermatozoa exhibit impaired migration from the uterus to the oviduct and impaired ability to bind the zona pellucida (ZP) of oocytes. Moreover, a sperm membrane protein, ADAM3 (a disintegrin and metalloprotease 3), which plays a role in sperm migration through uterotubal junction (UTJ) and sperm–ZP binding, disappeared in the DKO spermatozoa from the epididymis. We next generated single knockout (KO) mice lacking Prss51 and found that Prss51 KO mice are fertile. We also generated single KO mice lacking Prss55 and found that Prss55 KO mice phenocopy the DKO mice, demonstrating impaired sperm migration and sperm–ZP binding and a severe defect in fertility. We conclude that Prss55, but not Prss51, is required for male fertility in mice, by stabilizing ADAM3 protein for efficient sperm–UTJ migration and sperm–ZP binding. Our findings have implications for understanding additional genetic causes of the idiopathic male infertility and for the development of male or female contraceptives., The testis-specific serine protease gene Prss55, but not Prss51, is required for sperm ADAM3 maturation and male fertility in mice; Prss55 KO sperm fail both to migrate from the uterus into the oviduct and to bind the ZP of oocytes.

JQ1 is a small-molecule inhibitor of the bromodomain and extra terminal (BET) protein family that potently inhibits the bromodomain testis-specific protein (BRDT), which is essential for spermatogenesis. JQ1 treatment produces a reversible contraceptive effect by targeting the activity of BRDT in mouse male germ cells, validating BRDT as a male contraceptive target. Although JQ1 possesses favourable physical properties, it exhibits a short half-life. Because the details of xenobiotic metabolism play important roles in the optimization of drug candidates and in determining the role of metabolism in drug efficacy, we investigated the metabolism of JQ1 in human and mouse liver microsomes. We present the first comprehensive view of JQ1 metabolism in liver microsomes, distinguishing nine JQ1 metabolites, including three monohydroxylated, one de-tert-butylated, two dihydroxylated, one monohydroxylated/dehydrogenated, one monohydroxylated-de-tert-butylated and one dihydroxylated/dehydrogenated variant of JQ1. The dominant metabolite (M1) in both human and mouse liver microsomes is monohydroxylated on the fused three-ring core. Using recombinant cytochrome P450 (CYP) enzymes, chemical inhibitors and the liver S9 fraction of Cyp3a-null mice, we identify enzymes that contribute to the formation of these metabolites. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is the main contributor to the production of JQ1 metabolites in vitro, and the CYP3A4/5 inhibitor ketoconazole strongly inhibits JQ1 metabolism in both human and mouse liver microsomes. Our findings suggest that JQ1 half-life and efficacy might be improved in vivo by co-administration of a selective CYP inhibitor, thereby impacting the use of JQ1 as a probe for BRDT activity in spermatogenesis and as a probe or therapeutic in other systems., Nine JQ1 metabolites were identified and characterized in liver microsomes, and the enzyme CYP3A4 mediates the formation of most JQ1 metabolites.

Male contraception is a very active area of research. Several hormonal agents have entered clinical trials, while potential non-hormonal targets have been brought to light more recently and are at earlier stages of development. The general strategy is to target genes along the molecular pathways of sperm production, maturation, or function, and it is predicted that these novel approaches will hopefully lead to more selective male contraceptive compounds with a decreased side effect burden. Protein kinases are known to play a major role in signaling events associated with sperm differentiation and function. In this review, we focus our analysis on the testis-specific serine kinase (TSSK) protein family. We have previously shown that members of the family of TSSKs are postmeiotically expressed in male germ cells and in mature mammalian sperm. The restricted postmeiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggests that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the Tssk6 knockout (KO) mice and of the double Tssk1 and Tssk2 KO mice and by the male subfertile phenotype observed in a Tssk4 KO mouse model., The established role of TSSKs in spermiogenesis and in male fertility, together with their unique kinase features, strongly supports this family of protein kinases as a very promising drug discovery target for development of a non-hormonal male contraceptive.

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., TMPRSS12 has dual functions for uterotubal junction migration ability by affecting ADAM3 processing and sperm motility.

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 KO (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying potential targets for nonhormonal contraception., Absence of serine-rich single-pass membrane protein 1 (SSMEM1) leads to male infertility because of globozoospermia in mice.

Efforts to develop new male or female nonhormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, nonhormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institute., An acute-acting inhibitor of soluble adenylyl cyclase (sAC: ADCY10) may provide orally available, on-demand, non-hormonal contraception for men without adverse, mechanism-based effects.

This manuscript endeavors to present research considerations for the preclinical development of non-hormonal contraceptives. Topics include (1) how advances in genomics and bioinformatics impact the identification of novel targets for non-hormonal contraception, (2) the importance of target validation prior to investment in a contraceptive development campaign, (3) considerations on targeting gametogenesis vs gamete maturation/function, (4) how targets from the male reproductive system are expanding women’s options for ‘on demand’ contraception, and (5) some emerging non-hormonal methods that are not based on a specific molecular target. Also presented are ideas for developing a pipeline of non-hypothalamic-pituitary-gonadal-acting contraceptives for men and women while balancing risk and innovation, and our perspective on the pros and cons of industry and academic environments on contraceptive development. Three product development programs are highlighted that are biologically interesting, innovative, and likely to influence the field of contraceptive development in years to come.

WEE2 oocyte meiosis inhibiting kinase is a well-conserved oocyte specific kinase with a dual regulatory role during meiosis. Active WEE2 maintains immature, germinal vesicle stage oocytes in prophase I arrest prior to the luteinizing hormone surge and facilitates exit from metaphase II arrest at fertilization. Spontaneous mutations at the WEE2 gene locus in women have been linked to total fertilization failure indicating that selective inhibitors to this kinase could function as non-hormonal contraceptives. Employing co-crystallization with WEE1 G2 checkpoint kinase inhibitors, we revealed the structural basis of action across WEE kinases and determined type I inhibitors were not selective to WEE2 over WEE1. In response, we performed in silico screening by FTMap/FTSite and Schrodinger SiteMap analysis to identify potential allosteric sites, then used an allosterically biased activity assay to conduct high-throughput screening of a 26 000 compound library containing scaffolds of known allosteric inhibitors. Resulting hits were validated and a selective inhibitor that binds full-length WEE2 was identified, designated GPHR-00336382, along with a fragment-like inhibitor that binds the kinase domain, GPHR-00355672. Additionally, we present an in vitro testing workflow to evaluate biological activity of candidate WEE2 inhibitors including; (1) enzyme-linked immunosorbent assays measuring WEE2 phosphorylation activity of cyclin dependent kinase 1 (CDK1; also known as cell division cycle 2 kinase, CDC2), (2) in vitro fertilization of bovine ova to determine inhibition of metaphase II exit, and (3) cell-proliferation assays to look for off-target effects against WEE1 in somatic (mitotic) cells., Oocyte specific WEE2 is critical for exit from metaphase II arrest in mammalian oocytes and selective inhibitors to this kinase can be developed into non-hormonal contraceptives for women that prevent fertilization.

High unintended pregnancy rates are partially due to lack of effective nonhormonal contraceptives; development of safe, effective topical vaginal methods will address this need. Preclinical product safety and efficacy assessment requires in vivo testing in appropriate models. The sheep is a good model for the evaluation of vaginally delivered products due to its close similarities to humans. The study objective was to develop an ovine model for efficacy testing of female nonhormonal contraceptives that target human sperm. Fresh human semen was pooled from male volunteers. Nonpregnant female Merino sheep were treated with control or vaginal contraceptive product (IgG antibody with action against sperm or nonoxynol-9 [N9]). Pooled semen was added to the sheep vagina and mixed with product and vaginal secretions. Microscopic assessment of samples was performed immediately and progressive motility (PM) of sperm was compared between treatments. Cytokines CXCL8 and IL1B were assessed in vaginal fluid after instillation of human semen. No adverse reactions or elevations in proinflammatory cytokines occurred in response to human semen. N9 produced signs of acute cellular toxicity while there were no cellular changes after IgG treatment. N9 and IgG had dose-related effects with the highest dose achieving complete sperm immobilization (no sperm with PM). Surrogate post-coital testing of vaginally administered contraceptives that target human semen was developed in an ovine model established for vaginal product preclinical testing. This expanded model can aid the development of much needed nonhormonal topical vaginal contraceptives, providing opportunities for rapid iterative drug development prior to costly, time-intensive human testing.

MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB–SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB–SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein–protein interactions or promote degradation of meiosis-specific proteins., A cell-based bimolecular fluorescence complementation screening assay identifies isocotoin that promotes degradation of MEIOB and SPATA22.

Spermatogenesis is a complex developmental process that involves the proliferation of diploid cells, meiotic division, and haploid differentiation. Many genes are shown to be essential for male fertility using knockout (KO) mice; however, there still remain genes to be analyzed to elucidate their molecular mechanism and their roles in spermatogenesis. Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitously expressed protein that possesses three paralogs: CIB2, CIB3, and CIB4. It is reported that Cib1 KO male mice are sterile due to impaired haploid differentiation. In this study, we discovered that Cib4 is expressed strongly in mouse and human testis and begins expression during the haploid phase of spermatogenesis in mice. To analyze the function of CIB4 in vivo, we generated Cib4 KO mice using the CRISPR/Cas9 system. Cib4 KO male mice are sterile due to impaired haploid differentiation, phenocopying Cib1 KO male mice. Spermatogenic cells isolated from seminiferous tubules demonstrate an essential function of CIB4 in the formation of the apical region of the sperm head. Further analysis of CIB4 function may shed light on the etiology of male infertility caused by spermatogenesis defects, and CIB4 could be a target for male contraceptives because of its dominant expression in the testis., Cib4 mutant mice exhibit male infertility due to impaired spermiogenesis.

Sympathetically mediated contractions of smooth muscle cells in the vasa deferentia are mediated by neuronally released adenosine 5′-triphosphate (ATP) and noradrenaline, which stimulate P2X1-purinoceptors and α1A-adrenoceptors, respectively. This process is crucial for sperm transport, as demonstrated in knockout mouse studies where simultaneous genetic deletion of P2X1-purinoceptors and α1A-adrenoceptors resulted in male infertility. We hypothesize that dual pharmacological antagonism of these two receptors could inhibit sperm transport sufficiently to provide a novel nonhormonal method of male contraception. To generate a suitable P2X1-purinoceptor antagonist, substituents were introduced on the phenyl moiety of 2-phenyl-5,6,7,8-tetrahydroquinoxaline to create a series of analogues that were tested for P2X1-purinoceptor antagonism in isolated preparations of rat vas deferens. Novel compounds were initially screened for their ability to attenuate contractile responses to electrical field stimulation (EFS: 60 V, 0.5 ms, 0.2 Hz). The addition of polar substituents to the meta, but not ortho, position markedly increased the inhibition of contractions, as did the addition of both polar and aliphatic substituents to the para position. Di-substituted compounds were also synthesized and tested, resulting in a compound 31 (2-hydroxy, 4-fluoro), which exhibited the greatest potency, with an IC50 of 14 μM (95% confidence limits: 12–16 μM). Additionally, compound 31 noncompetitively antagonized contractions mediated by exogenously administered αß-methylene ATP (10 nM–30 μM) but had no inhibitory effect on contractions mediated by exogenously administered noradrenaline (30 nM–100 μM) or acetylcholine (30 nM–100 μM). These results have contributed to a structure–activity relationship profile for the P2X1-purinoceptor that will inform future designs of more potent antagonists., Novel small molecule P2X1-purinoceptor antagonists were able to selectively inhibit the purinergic component of contractions of the rat vas deferens., Graphical Abstract Graphical Abstract

Inhibition of the sperm transport process in the female reproductive tract could lead to infertility. We previously showed that a pan-serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), blocked semen liquefaction in vivo and resulted in a drastic decrease in the number of sperm in the oviduct of female mice. In this study, we used a mouse model to test the efficacy of AEBSF as a reversible contraceptive, a sperm motility inhibitor, and a spermicide. Additionally, this study evaluated the toxicity of AEBSF on mouse vaginal tissues in vivo and human endocervical cells in vitro. We found that female mice treated with AEBSF had significantly less pups born per litter as well as fertilization rates in vivo compared to the vehicle control. We then showed that AEBSF reduced sperm motility and fertilization capability in vitro in a dose-dependent manner. Furthermore, AEBSF also exhibited spermicidal effects. Lastly, AEBSF treatment in female mice for 10 min or 3 consecutive days did not alter vaginal cell viability in vivo, similar to that of the vehicle and non-treated controls. However, AEBSF decreased cell viability of human ectocervical (ECT) cell line in vitro, suggesting that cells in the lower reproductive tract in mice and humans responded differently to AEBSF. In summary, our study showed that AEBSF can be used as a prototype compound for the further development of novel non-hormonal contraceptives for women by targeting sperm transport in the female reproductive tract., Blockade of the sperm transport process using a pan-serine protease inhibitor can be developed as a non-hormonal contraceptive for women.

Postcoital tests (PCTs) have been used for over a century in the clinical evaluation of infertile couples, and for nearly 70 years in the evaluation of new vaginal contraceptive products. PCTs have been largely replaced by more modern methods in the study of infertility, but they remain the most useful way to obtain preliminary data on the effectiveness of vaginal contraceptive products. The World Health Organization has described important aspects of the procedure. It involves collection of cervical mucus at a certain time point after intercourse and the counting and characterization of sperm found in the mucus. A wide range of progressively motile sperm (PMS) has been associated with pregnancy rates in infertility studies. Eligibility for contraceptive trials includes the requirement that couples achieve a certain threshold number of PMS per high power field at midcycle in a baseline cycle without the test product. The primary endpoint, or definition of a satisfactory result in test cycles, is predefined. A literature review identified 10 PCT studies of vaginal contraceptives involving nine test products. Phase II trials of vaginal contraceptives have not been deemed feasible in the development of any vaginal contraceptive to date. A PCT study of a test product can be predictive of contraceptive efficacy, although ultimate contraceptive effectiveness is influenced by the ease of use of the product, along with patient compliance. PCT results similar to results seen with products that later showed satisfactory performance in efficacy trials is the best indicator of likely success of a test product., A product that performs well in a postcoital test (PCT) study goes on to demonstrate a high level of contraceptive effectiveness, although the PCT is not predictive of exact effectiveness; ultimate contraceptive effectiveness is influenced by the ease and convenience of use of the product.