Effect of heat stress on expression of junction-associated molecules and upstream factors androgen receptor and Wilms' tumor 1 in monkey sertoli cells.

Sertoli cells are important in determining the fate of spermatogenic cells by providing nutrition and structural support via cell junctions. In this study, we sought to examine the effect of 43 C warming on cell junctions in seminiferous epithelium and the expression of junction-associated molecules in Sertoli cells. Electron microscopy showed the appearance of large vacuoles between Sertoli and germ cells and adjacent Sertoli cells, leading to disruption of corresponding cell junctions 24 h after terminating the heat treatment. Using primary Sertoli cells isolated from pubertal monkey testes, we demonstrated that expression of adherens junction-associated molecules, such as N-cadherin and beta-catenin, and tight junction-associated molecule zonula occludens protein 1 was significantly reduced in 24-48 h after heat treatment. In contrast, intermediate filament vimentin expression was up-regulated in 6-48 h. Androgen receptor (AR) and Wilms' tumor gene 1 expression dramatically decreased after heat treatment. Both proteins completely disappeared immediately after terminating heat treatment and began to recover after 6 h. Treatment of the monkey Sertoli cells with an AR antagonist, flutamide, could mimic the heat-induced changes in the expression of junction-associated molecules in Sertoli cells. Furthermore, overexpression of AR in the Sertoli cells up-regulated the expression of N-cadherin, beta-catenin, and zonula occludens protein 1 and down-regulated vimentin expression. Their expression after heat treatment could be rescued by the AR overexpression. These results indicate that the decreased AR expression after heat treatment is involved in heat-induced cell junction disruption.

Signal pathway of GnRH-III inhibiting FSH-induced steroidogenesis in granulosa cells.

Gonadotrophin-releasing hormone type 1 and type 2 have been demonstrated to inhibit follicle-stimulating hormone (FSH)-induced granulosa cell (GC) steroidogenesis. A third type of GnRH (GnRH-III) was also purified from salmon, its action on the FSH-regulated GC function, however is not clear. In the present study we demonstrated that the FSH-induced estrogen and progesterone production in cultured DES-treated GCs was significantly inhibited by GnRH-III. Furthermore, the FSH-stimulated steroidogenic acute regulatory protein and the enzymes for steroidigenesis, such as HSD3B2,aromatase and cytochrome P450 side-chain cleavage were also significantly suppressed by this peptide. The inhibitory action of GnRH-III on the FSH-induced steroidogenenisis was demonstrated via Akt and p38 mitogen-activated protein kinase signaling pathways through suppressing its own receptor expression. Further studies indicated that FSH could stimulate NR5A2 and upstream stimulatory factor (USF) activation, and their induction was significantly suppressed by the GnRH-III. Therefore, it is suggested that GnRH-III inhibiting FSH-induced steroidogenenisis in GCs might be by suppressing FSH-induced its own receptor expression via NR5A2 and USF transcriptional factors.

Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.

Increasing evidence has shown that excess androgen may be a main cause of polycystic ovary syndrome (PCOS). However, the molecular mechanism of androgen action on the ovary is unclear. To investigate the possible impacts of androgen on early follicular development, neonatal mouse ovaries mainly containing primordial follicles were cultured with testosterone. We demonstrated that the number of primary follicles was increased after 10 d culture with testosterone treatment via phosphatidylinositol 3-kinase/Akt pathway. Androgen induced Forkhead box (Foxo)-3a activation, and translocation of Foxo3a protein from oocyte nuclei to cytoplasm, which might be a key step for primordial follicle activation. Interestingly, testosterone was also capable of down-regulating growth and differentiation factor-9 expression via its receptor. In summary, we infer that intraovarian excess androgen in PCOS might result in excess early follicles by inducing oocyte Foxo3a translocation and follicular arrest by down-regulating growth and differentiation factor-9 expression.

Acrosome formation-associated factor is involved in fertilization.

OBJECTIVE: To investigate the effects of a novel acrosome formation-associated factor (Afaf) on fertilization by its regulation of acrosomal exocytosis and endosomal trafficking. DESIGN: Controlled laboratory study. SETTING: Institution-affiliated state key laboratory. SUBJECTS: ICR mice. INTERVENTION(S): Sperm penetration assay and in vitro fertilization experiment were performed to study the effects of the Afaf antibody on acrosome reaction and fertilization. Acrosome exocytosis (AE) with streptolysin O (SLO) permeabilization was conducted to test the Afaf's action in calcium events. Colocalization and coimmunoprecipitation was done to determine the interaction between Afaf and SNAP25 (synaptosome-associated protein of 25,000 daltons). Transferrin (Tf) uptake assay was performed to demonstrate the impact of Afaf on endosomal pathway. RNAi was used to rescue the inhibition of Afaf on Tf uptake. MAIN OUTCOME MEASURE(S): Number of penetrated sperms, in vitro fertilization rate. Acrosomal exocytosis index, relative Tf fluorescence. RESULT(S): The Afaf antibodies were capable of significantly inhibiting sperm penetration of the eggs, therefore reducing the rate of in vitro fertilization. Acrosome formation-associated factor was involved in calcium-triggered AE by acting upstream of the calcium efflux from the acrosome inside. Acrosome formation-associated factor might exert an interaction with SNAP25, which is a crucial component in both exocytosis and endosomal trafficking. Acrosome formation-associated factor was also involved in the endocytic pathway by down-regulating Tf endocytosis in the HeLa cells, and the miRNA-mediated RNAi could rescue this alternation induced by Afaf. CONCLUSION(S): Acrosome formation-associated factor might play an important role in membrane trafficking during acrosome formation and participate in fertilization.