Summary. Sperm concentration, morphology, DNA condensation and nuclear protein pattern as well as sperm adenylate cyclase were evaluated in semen samples provided by 7 volunteers every 8 h for 2 days. During the observation period, sperm concentration and total sperm number decreased but began to return towards normal after a 3-day abstinence period. No increase in the proportion of immature sperm cells was observed by light microscopy. Total adenylate cyclase (EC 22.214.171.124) increased significantly per cell, due to an increase in the soluble component while the particulate component remained relatively constant. Microflow fluorometry revealed no consistent alterations in the DNA or nuclear protein distribution. We conclude that although a high frequency of ejaculation does not disturb the conventional measures of sperm integrity, such as DNA condensation, there are major changes in at least one biochemical measurement, the activity of soluble adenylate cyclase.
Nan B. Oldereid, J. O. Gordeladze, B. Kirkhus and K. Purvis
Nan Li, Elizabeth I Tang and C Yan Cheng
The blood–testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood–tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases.
Ya’nan Yang, Biqi Zheng, Chenchang Bao, Huiyang Huang and Haihui Ye
As the precursor of vitellin (Vn), vitellogenin (Vg) has initially been considered as a female-specific protein involved in vitellogenesis, while it was also present in males induced by hormones or organs manipulation. Distinct from vtg1 we previously found in female mud crab Scylla paramamosain, vtg2 was intriguingly detected in male testis under normal physiological conditions in this study. Sequence analysis showed that vtg2 and vtg1 were actually two isoforms of Vg caused by different types of alternative splicing. PCR and in situ hybridization analysis revealed that vtg2 was localized only in the testicular spermatozoa, while Vn was detected in both the spermatozoa of the testis and seminal vesicle. Therefore, we speculated that Vn was initially translated in testicular spermatozoa, then migrated with spermatozoa, and finally stored in the seminal vesicle, where spermatozoa gradually accomplished maturation. We presumed that vtg2/Vn might act as an immune-relevant molecule in the male reproduction system. In the subsequent experiment, the expression of vtg2/Vn in testis was significantly induced in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) injection at both transcriptional and translational levels. In the light of the results presented above, we deemed that vtg2/Vn is a novel candidate of immune-relevant molecules involved in immunoprotection during the spermatozoon maturation, and this research helps to open a new avenue for further exploring the role of Vg.
Yongmei Chen, Huizhen Wang, Nan Qi, Hui Wu, Weipeng Xiong, Jing Ma, Qingxian Lu and Daishu Han
Mice lacking TYRO3, AXL and MER (TAM) receptor tyrosine kinases (RTKs) are male sterile. The mechanism of TAM RTKs in regulating male fertility remains unknown. In this study, we analyzed in more detail the testicular phenotype of TAM triple mutant (TAM−/−) mice with an effort to understand the mechanism. We demonstrate that the three TAM RTKs cooperatively regulate male fertility, and MER appears to be more important than AXL and TYRO3. TAM−/− testes showed a progressive loss of germ cells from elongated spermatids to spermatogonia. Young adult TAM−/− mice exhibited oligo-astheno-teratozoospermia and various morphological malformations of sperm cells. As the mice aged, the germ cells were eventually depleted from the seminiferous tubules. Furthermore, we found that TAM−/− Sertoli cells have an impaired phagocytic activity and a large number of differentially expressed genes compared to wild-type controls. By contrast, the function of Leydig cells was not apparently affected by the mutation of TAM RTKs. Therefore, we conclude that the suboptimal function of Sertoli cells leads to the impaired spermatogenesis in TAM−/− mice. The results provide novel insight into the mechanism of TAM RTKs in regulating male fertility.
Xihua Chen, Bin Wu, Shufang Wang, Jianbing Liu, Haijun Gao, Fang Zhou, Nan Nan, Bonan Zhang, Jiedong Wang, Xiangbo Xu and Bin He
Menstruation is a specific physiological phenomenon that occurs in women. However, molecular mechanisms underlying this phenomenon are still unclear. According to the classical theory, tissue hypoxia resulting from vasoconstriction of the spiral arteries after progesterone (P4) withdrawal initiates the breakdown of the endometrium at the earliest stage of menstruation. However, this theory has been challenged by previous studies that have questioned the function and even the existence of hypoxia during menstruation. In this study, we not only provide convincing evidence that hypoxia exists during endometrial breakdown, but also further explore the role of hypoxia and hypoxia-inducible factor 1 (HIF1) in this process. Based on mouse menstrual-like model and experiments with human decidual stromal cells, we observed that P4 withdrawal induced both hypoxia and HIF1 activation; however, endometrial breakdown was triggered only by P4 withdrawal. Hypoxia significantly enhanced the mRNA expression of specific matrix metalloproteinases (MMPs) under the conditions of P4 withdrawal. In conclusion, hypoxia is involved but not an essential component of endometrial breakdown during menstruation.
Shu-Fang Wang, Xi-Hua Chen, Bin He, De-Dong Yin, Hai-Jun Gao, Hao-Qi Zhao, Nan Nan, Shi-Ge Guo, Jian-Bing Liu, Bin Wu and Xiang-Bo Xu
Stress impacts the reproductive axis at the level of the hypothalamus and the pituitary gland, which exert an effect on the ovary. Menstruation is regulated by the hypothalamic–pituitary–ovary (HPO) axis. However, the role of stress in menstruation remains unclear. The objective of this study was to explore the role of stress in endometrial breakdown and shedding, using the pseudopregnant mouse menstrual-like model. Female mice were mated with vasectomized males and labeled day 0.5, upon observation of a vaginal seminal plug. On day 3.5, decidualization was induced in pseudopregnant mice using arachis oil. On day 5.5, pseudopregnant mice with artificial decidualization were placed in restraint tubes for 3 h. The findings indicated that acute restraint stress resulted in the disintegration of the endometrium. While corticosterone concentration in the serum increased significantly due to restraint stress, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and progesterone (P4) levels in the serum decreased significantly. An endometrial histology examination indicated that progesterone implants may rescue P4 decline caused by acute stress and block endometrium breakdown and shedding. In addition, mice were treated with metyrapone, an inhibitor of corticosterone synthesis, 1 h prior to being subjected to restraint stress. Interestingly, metyrapone not only inhibited stress-induced endometrium breakdown and shedding, but also prevented stress-induced reduction of P4, LH and FSH. Furthermore, real-time PCR and western blot showed that mRNA and protein expression of CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and steroidogenic acute regulatory protein (StAR), the two rate-limiting enzymes for progesterone synthesis in the ovary, decreased following acute stress. But metyrapone prevented the reduction of StAR expression induced by restraint stress. Overall, this study revealed that acute stress results in an increase in corticosterone, which may inhibit LH and FSH release in the serum and CYP11A1 and StAR expression in the ovary, which finally leads to the breakdown and shedding of the endometrium. These experimental findings, based on the mouse model, may enable further understanding of the effects of stress on menstruation regulation and determine the potential factors affecting stress-associated menstrual disorders.
Li-Ying Yan, Jun-Cheng Huang, Zi-Yu Zhu, Zi-Li Lei, Li-Hong Shi, Chang-Long Nan, Zhen-Jun Zhao, Ying-Chun OuYang, Xiang-Fen Song, Qing-Yuan Sun and Da-Yuan Chen
The assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. α-Tubulin was localized around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around the introduced nucleus in most reconstructed embryos and formed a transient spindle 2–4 h post-fusion (hpf). A mass of microtubules surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMAwas detected in the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase. In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit oocyte meiosis and cloned embryo mitosis.
Xue-Min Qiu, Zhen-Zhen Lai, Si-Yao Ha, Hui-Li Yang, Li-Bing Liu, Yan Wang, Jia-Wei Shi, Lu-Yu Ruan, Jiang-Feng Ye, Jiang-Nan Wu, Qiang Fu, Xiao-Fang Yi, Kai-Kai Chang and Ming-Qing Li
Immune cells and cytokines have important roles in the pathogenesis of endometriosis. However, the production and role of cytokines of T helper type 1 (Th1) and Th2 cells in the progress of endometriosis have remained to be fully elucidated. The present study reported that the interferon (IFN)-γ levels and the percentage of IFN-γ+CD4+ cells were significantly increased in the peritoneal fluid (PF) at the early stage and maintained at a higher level at the advanced stage of endometriosis; furthermore, interleukin (IL)-10 and IL-10+CD4+ cells were elevated in the advanced stage of endometriosis. In addition, IL-2 levels in the PF at the advanced stage of endometriosis were elevated and negatively associated with IFN-γ expression. In a co-culture system of ectopic endometrial stromal cells (ESCs) and macrophages, elevated IL-2 was observed, and treatment with cytokines IL-2 and transforming growth factor-β led to upregulation of the ratio of IL-2+ macrophages. IL-27-overexpressing ESCs and macrophages were able to induce a higher ratio of IL-10+CD4+ T cells. Blocking of IL-2 with anti-IL-2 neutralizing antibody led to upregulation of the ratio of IFN-γ+CD4+ T cells in the co-culture system in vitro. Recombinant human IL-10 and IFN-γ promoted the viability, invasiveness and transcription levels of matrix metalloproteinase (MMP)2, MMP9, and prostaglandin-endoperoxide synthase 2 of ESCs, particularly combined treatment with IL-10 and IFN-γ. These results suggest that IL-2 and IL-27 synergistically promote the growth and invasion of ESCs by modulating the balance of IFN-γ and IL-10 and contribute to the progress of endometriosis.