Testis expressed 50 is essential for maintaining sperm acrosome integrity during epididymal transit.

In mammals, sperm formation is completed in the seminiferous tubules within the testis, and sperm maturation occurs during the epididymal transit of the spermatozoa. Sperm morphology drastically changes when abnormal spermatozoa migrate from the testis to the epididymis. Detailed molecular mechanisms for sperm survival in the epididymis have not been determined yet.

Ovochymase 2 is a key regulatory factor modulating proteolytic pathways and sperm maturation in the mammalian epididymis†.

Spermatozoa acquire fertilizing competence during epididymal transit through proteolytic, chaperone-mediated, and post-translational modifications. Ovochymase 2, an epididymis-specific trypsin-like serine protease, has emerged as a central regulator of this maturation process. Here, we integrate targeted gene disruption, comprehensive proteomic profiling, and affinity-based proteome enrichment to delineate how Ovochymase 2 influences sperm functionality.

Large-scale CRISPR/Cas9 deletions within the WFDC gene cluster uncover gene functionality and critical roles in mammalian reproduction.

Despite 96 million years of evolution separating humans and rodents, 11 closely related reproductive tract-specific genes in humans-, , , , , , , , , , and -and the 13 reproductive tract-specific orthologous genes in mice, form highly conserved syntenic gene clusters indicative of conserved, combined critical functions. Further, despite significant progress toward a nonhormonal male contraceptive targeting the protein encoded by one of these genes, epididymal peptidase inhibitor (EPPIN), and associations found between mutations in and an increased risk of male infertility, neither EPPIN nor any closely related whey acidic protein four-disulfide core (WFDC) gene have been explored functionally. To clarify the involvement of WFDC genes in male fertility, we strategically used CRISPR/Cas9 to generate mice lacking 13, 10, 5, or 4 genes within the cluster and demonstrated that males with deletions of 13, 10, or 4 genes (Wfdc6a, Eppin, Wfdc8, and Wfdc6a) were sterile due to an arrest in spermatogenesis, preventing formation beyond round spermatids.

Affinity-tagged SMAD1 and SMAD5 mouse lines reveal transcriptional reprogramming mechanisms during early pregnancy.

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci ( and ).

knockout male mice are sterile secondary to sperm head malformations and failure to penetrate through the zona pellucida.

Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. It is assumed that sperm head shape is important for sperm-zona pellucida (ZP) penetration but research has yet to elucidate why. We generated testis expressed 46 () knockout mice to investigate the essential roles of TEX46 in mammalian reproduction.

CUB domains are not required for OVCH2 function in sperm maturation in the mouse epididymis.

Background: Ovochymase 2 (Ovch2) is an epididymis-specific gene that is required for male fertility. While a multitude of reproductive tract-specific genes required for male fertility have been identified, OVCH2 is thus far the first protein required for male fertility that contains Complement C1r/C1s, Uegf, Bmp1 (CUB) domains located in tandem in the C-terminus of the protein. Identifying the functional significance of this unique domain has implications in better understanding fertility and infertility and as a potential contraceptive target.

Oocyte-specific Wee1-like protein kinase 2 is dispensable for fertility in mice.

Wee1-like protein kinase 2 (WEE2) is an oocyte-specific protein tyrosine kinase involved in the regulation of oocyte meiotic arrest in humans. As such, it has been proposed as a candidate for non-hormonal female contraception although pre-clinical models have not been reported. Therefore, we developed two novel knockout mouse models using CRISPR/Cas9 to test loss-of-function of Wee2 on female fertility.

Molecular dissection and testing of PRSS37 function through LC-MS/MS and the generation of a PRSS37 humanized mouse model.

The quest for a non-hormonal male contraceptive pill for men still exists. Serine protease 37 (PRSS37) is a sperm-specific protein that when ablated in mice renders them sterile. In this study we sought to examine the molecular sequelae of PRSS37 loss to better understand its molecular function, and to determine whether human PRSS37 could rescue the sterility phenotype of knockout (KO) mice, allowing for a more appropriate model for drug molecule testing.

Testis-specific serine kinase 3 is required for sperm morphogenesis and male fertility.

Background: The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis-specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known.

Objective: The goals of our studies were to understand the mechanism of action of TSSK3.

MRNIP interacts with sex body chromatin to support meiotic progression, spermatogenesis, and male fertility in mice.

Meiosis has a principal role in sexual reproduction to generate haploid gametes in both sexes. During meiosis, the cell nucleus hosts a dynamic environment where some genes are transcriptionally activated, and some are inactivated at the same time. This becomes possible through subnuclear compartmentalization.

The testis-specific E3 ubiquitin ligase RNF133 is required for fecundity in mice.

Background: Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin-proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control.

Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis.

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre.

Knockout of family with sequence similarity 170 member A (Fam170a) causes male subfertility, while Fam170b is dispensable in mice†.

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.

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.

Knockout of mouse receptor accessory protein 6 leads to sperm function and morphology defects†.

Receptor accessory protein 6 (REEP6) is a member of the REEP/Ypt-interacting protein family that we recently identified as essential for normal endoplasmic reticulum homeostasis and protein trafficking in the retina of mice and humans. Interestingly, in addition to the loss of REEP6 in our knockout (KO) mouse model recapitulating the retinal degeneration of humans with REEP6 mutations causing retinitis pigmentosa (RP), we also found that male mice are sterile. Herein, we characterize the infertility caused by loss of Reep6.

The testis-specific serine proteases PRSS44, PRSS46, and PRSS54 are dispensable for male mouse fertility†.

High-throughput transcriptomics and proteomics approaches have recently identified a large number of germ cell-specific genes with many that remain to be studied through functional genetics approaches. Serine proteases (PRSS) constitute nearly one-third of all proteases, and, in our bioinformatics screens, we identified many that are testis specific. In this study, we chose to focus on Prss44, Prss46, and Prss54, which we confirmed as testis specific in mouse and human.

Sperm-borne phospholipase C zeta-1 ensures monospermic fertilization in mice.

Sperm entry in mammalian oocytes triggers intracellular Ca oscillations that initiate resumption of the meiotic cell cycle and subsequent activations. Here, we show that phospholipase C zeta 1 (PLCζ1) is the long-sought sperm-borne oocyte activation factor (SOAF). Plcz1 gene knockout (KO) mouse spermatozoa fail to induce Ca changes in intracytoplasmic sperm injection (ICSI).

Viable offspring after imaging of Ca2+ oscillations and visualization of the cortical reaction in mouse eggs.

During mammalian fertilization, egg Ca 2+ oscillations are known to play pivotal roles in triggering downstream events such as resumption of the cell cycle and the establishment of blocks to polyspermy. However, viable offspring have not been obtained after monitoring Ca 2+ oscillations, and their spatiotemporal links to subsequent events are still to be examined. Therefore, the development of imaging methods to avoid phototoxic damage while labeling these events is required.

CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.

Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals.