Characterization of the protein expression and localization of hnRNP family members during murine spermatogenesis.

Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.

Identification of programmed cell death 1 and its ligand in the testicular tissue of mice.

PROBLEM: This study aims to determine the expression and localization of programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) in the testes of mice at different developmental stages. METHOD OF STUDY: By means of RT-qPCR, Western blot and immunofluorescence, the expression and localization of PD-1 and PD-L1 were detected in the testicular tissues of mice at different postnatal times: P7, P14, P21, P28, P35, and adulthood. Meanwhile, the level of soluble PD-L1 (sPD-L1) was evaluated by ELISA in the testicular interstitial fluid (IF) of the adult mice, culture supernatants of TM4 cell lines (Sertoli cells lines), and primary Sertoli cells at P14. RESULTS: Pd-1 mRNA levels were unexpectedly low. From P7 to P21, there was limited PD-1 protein detected while PD-1 was evident at P28 and afterward at significantly higher levels than at P14 and P21 (P < 0.05). Despite being found in the interstitial area at P7, P14, and P21, PD-1 was also detected in the germ cells of the seminiferous tubules after P28. Pd-l1 mRNA exhibited age-related changes, peaking at P21, while PD-L1 protein was constitutively expressed at any stage, specifically localized in the nucleus of Sertoli cells. Moreover, the level of sPD-L1 in IF was significantly higher than that in the culture supernatants of both TM4 and primary Sertoli cells at P14. CONCLUSIONS: PD-1 and PD-L1 were present in the testicular tissue of adult mice. The expression and localization of PD-1 fluctuated with age, and PD-1 was mainly localized to advanced germ cells, suggesting that it may play a role in spermiogenesis. PD-L1 was constitutively expressed in the nucleus of Sertoli cells, which could secrete sPD-L1 into the testicular interstitial space and thus may be involved in testicular immune privilege.

The cytoplasmic droplet may be indicative of sperm motility and normal spermiogenesis.

Although the cytoplasm of spermatids is removed at the end of spermiogenesis, a tiny portion is usually retained in the sperm flagellum, which is termed the cytoplasmic droplet (CD) in mammals. CDs are believed to play a role in sperm volume adaptation. However, we have noticed that epididymal spermatozoa that display initial (flagellation in situ) and progressive motility mostly possess CDs, whereas spermatozoa without CDs are rarely motile, suggesting that CDs have a role in motility development during sperm epididymal maturation. In the present study, we analyzed the relationship between the presence or absence of CDs, motility development and positional changes of CDs during sperm epididymal maturation in mice and monkeys. We also examined CDs on spermatozoa of three knockout mouse lines with late spermiogenic defects. Our data suggest that the CD is a normal organelle transiently present exclusively on epididymal spermatozoa, and normal CD morphology and location are associated with normal motility development during epididymal maturation of spermatozoa. Abnormal CD formation, e.g., a complete lack of CDs or ectopic CDs, is indicative of defective spermiogenesis. If CDs are essential for sperm motility development, then CDs may represent an ideal drug target for the development of non-hormonal male contraceptives.