Cabs1 Maintains Structural Integrity of Mouse Sperm Flagella during Epididymal Transit of Sperm.

The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1, was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece-principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1-/- mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.

Hippo kinases MST1 and MST2 control the differentiation of the epididymal initial segment via the MEK-ERK pathway.

Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2, but the mechanisms of ERK1/2 activation in IS are not fully understood. Here we show that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. Our results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility.

Vitamin K2-Dependent GGCX and MGP Are Required for Homeostatic Calcium Regulation of Sperm Maturation.

A low-calcium microenvironment is essential for spermatozoa to mature in the epididymis; however, it remains unclear how dysregulation of epididymal luminal calcium is associated with male infertility. Using a warfarin-induced vitamin K2 deficiency rat model, we found that vitamin-K-dependent γ-glutamyl carboxylase (GGCX) and matrix Gla protein (MGP) were essential in extracellular calcium signaling of the intercellular communication required for epididymal sperm maturation. We found that GGCX and MGP co-localized in the vesicular structures of epididymal cells and spermatozoa. Calcium-regulated MGP binds to proteins in a biphasic manner; sub-millimolar calcium enhances, whereas excessive calcium inhibits, the binding. Bioinformatic analysis of the calcium-dependent MGP-bound proteome revealed that vesicle-mediated transport and membrane trafficking underlie the intercellular communication networks. We also identified an SNP mutation, rs699664, in the GGCX gene of infertile men with asthenozoospermia. Overall, we revealed that the GGCX-MGP system is integrated with the intercellular calcium signaling to promote sperm maturation.

Correction: CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

[This corrects the article DOI: 10.1371/journal.pgen.1005485.].

CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

Cyclin Y-like 1 (Ccnyl1) is a newly-identified member of the cyclin family and is highly similar in protein sequences to Cyclin Y (Ccny). However, the function of Ccnyl1 is poorly characterized in any organism. Here we found that Ccnyl1 was most abundantly expressed in the testis of mice and was about seven times higher than the level of Ccny. Male Ccnyl1-/- mice were infertile, whereas both male and female Ccny-/- mice displayed normal fertility. These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility. Spermatozoa obtained from Ccnyl1-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of Ccnyl1-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells.

Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H(+)-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10-30 µM) and HA1077 (30 µM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 µg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 µM) or HA1077 (30 µM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.