PA1 Participates in the Maintenance of Blood-Testis Barrier Integrity via Cooperation with JUN in the Sertoli Cells of Mice.

BackgroundThe blood-testis barrier (BTB) is essential to the microenvironment of spermatogenesis, and Sertoli cells provide the cellular basis for BTB construction. Numerous nuclear transcription factors have been identified to be vital for the proper functioning of Sertoli cells. PA1 has been reported to play important roles during diverse biological processes, yet its potential function in male reproduction is still unknown. ResultsHere, we show that PA1 was highly expressed in human and mouse testis and predominantly localized in the nuclei of Sertoli cells. Sertoli cell-specific Pa1 knockout resulted in an azoospermia-like phenotype in mice. The knockout of this gene led to multiple defects in spermatogenesis, such as the disorganization of the cytoskeleton during basal and apical ectoplasmic specialization and the disruption of the BTB. Further transcriptomic analysis, together with Cut-Tag results of PA1 in Sertoli cells, revealed that PA1 could affect the expression of a subset of genes that are essential for the normal function of Sertoli cells, including those genes associated with actin organization and cellular junctions such as Connexin43 (Cx43). We further demonstrated that the expression of Cx43 depended on the interaction between JUN, one of the AP-1 complex transcription factors, and PA1. ConclusionOverall, our findings reveal that PA1 is essential for the maintenance of BTB integrity in Sertoli cells and regulates BTB construction-related gene expression via transcription factors. Thus, this newly discovered mechanism in Sertoli cells provides a potential diagnostic or even therapeutic target for some individuals with azoospermia.

The Non-hormonal Male Contraceptive Adjudin Exerts its Effects via MAPs and Signaling Proteins mTORC1/rpS6 and FAK-Y407

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a nonhormonal male contraceptive, since it effectively induces reversible male infertility without perturbing the serum concentrations of follicle stimulating hormone (FSH), testosterone, and inhibin B based on studies in rats and rabbits. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli–spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively inducing spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell–cell AJ called basal ectoplasmic specialization (basal ES), which, together with tight junction constitute the blood-testis barrier (BTB), unless an acute dose of adjudin was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to not only study adjudin-mediated male infertility but also the biology of spermatogenesis.

CFAP43-mediated intra-manchette transport is required for sperm head shaping and flagella formation

Summary Mutation in CFAP43 leads to severe asthenozoospermia and multiple morphological abnormalities of the sperm flagellum (MMAF) in both human and mouse. Previous studies have shown that disruption of intra-manchette transport (IMT) caused failure of flagellum assembly and sperm head shaping. In a previous study, therefore, we postulated that disruption of IMT may contribute to the failure of sperm flagellum formation and result in MMAF, however the mechanisms underlying these defects are still poorly understood. Cfap43-deficient mice were studied here to reveal the cellular mechanisms of abnormal sperm head morphology and MMAF. Depletion of Cfap43 led to abnormal spermiogenesis and caused MMAF, sperm head abnormality and oligozoospermia. Furthermore, both abnormal manchette and disorganized ectoplasmic specialization (ES) could be observed at the elongated spermatids in Cfap43-deficient mice. Therefore, our findings demonstrated that, in mice, CFAP43-mediated IMT is essential for sperm head shaping and sperm flagellum formation.

Filamentous actin disorganization and absence of apical ectoplasmic specialization disassembly during spermiation upon interference with retinoid signaling†

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.

Novel localizations and interactions of intercellular bridge proteins revealed by proteomic profiling†

Intercellular bridges (ICBs) connecting germ cells are essential for spermatogenesis, and their deletion causes male infertility. However, the functions and component factors of ICBs are still unknown. We previously identified novel ICB-associated proteins by proteomics analysis using ICB enrichment. Here, we performed immunoprecipitation–proteomics analyses using antibodies specific to known ICB proteins MKLP1, RBM44, and ectoplasmic specialization-associated protein KIAA1210 and predicted protein complexes in the ICB cores. KIAA1210, its binding protein topoisomerase2B (TOP2B), and tight junction protein ZO1 were identified as novel ICB proteins. On the other hand, as well as KIAA1210 and TOP2B, MKLP1 and RBM44, but not TEX14, were localized at the XY body of spermatocytes, suggesting that there is a relationship between ICB proteins and meiotic chromosomes. Moreover, small RNAs interacted with an ICB protein complex that included KIAA1210, RBM44, and MKLP1. These results indicate dynamic movements of ICB proteins and suggest that ICB proteins could be involved not only in the communication between germ cells but also in their epigenetic regulation. Our results provide a novel perspective on the function of ICBs and could be helpful in revealing the biological function of the ICB.