HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

P16蛋白和生精细胞凋亡对热压和11酸睾酮诱导的恒河猴无精子症和少精子症中的作用

探讨了P16蛋白和生精细胞凋亡在热压和11酸睾酮诱导恒河猴无精子症和少精子症中作用间的关系。3^末端标记分析(TUNEL)结果显示热应激和超生理剂量睾酮能够诱导生精细胞出现凋亡信号,它分别于处理后d5和d30达到最强。免疫组化结果显示,热压或TU主要诱导精原细胞和其它生精细胞以及Sertoli细胞P16的表达。P16蛋白的表达在生精细胞凋亡晚期,即隐睾手术d10或注射TUd60后迅速升高并维持高表达,该蛋白在生精细胞凋亡晚期可能通过抑制精原细胞的有丝分裂,扰乱正常的精子发生。

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from I h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly. (C) 2009 Elsevier Ltd. All rights reserved.

Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish

SOX3 has been suggested to play significant roles in gametogenesis and gonad differentiation of vertebrates, but the exact cellular localization evidence is insufficient and controversial. In this study, a protogynous hermaphrodite fish Epinephelus coioides is selected to analyze EcSox3 differential expression and the expression pattern in both processes of oogenesis and spermatogenesis by utilizing the advantages that gonad development undergoes transition from ovary to intersexual gonad and then to testis, and primordial germ cells and different stage cells during oogenesis and spermatogenesis are synchronously observed in the transitional gonads. The detailed and clear immunofluoresence localization indicates that significantly differential expression and dynamic changes of Sox3 occur in the progresses of gametogenesis and sex reversal, and EcSOX3 protein exists in the differentiating primordial germ cells, oogonia, and different stage oocytes of ovaries, and also in the differentiating primordial germ cells and the Sertoli cells of testis. One important finding is that the EcSox3 expression is a significant time point for enterable gametogenesis of primordial germ cells because EcSOX3 is obviously expressed and localized in primordial germ cells. As EcSox3 continues to express, the EcSOX3-positive primordial germ cells develop toward oogonia and then oocytes, whereas when EcSox3 expression is ceased, the EcSOX3-positive primordial germ cells develop toward spermatogonia. Therefore, the current finding of EcSOX3 in the differentiating primordial germ cells again confirms the potential regulatory role in oogenesis and germ cell differentiation. The data further suggest that SOX3, as a transcription factor, might have more important roles in oogenesis than in spermatogenesis.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT] gene from orange-spotted grouper (Epinephelus coioides), and prepared corresponding anti-EcDMRT1] antiserum to study the relationship of DMRT] to sex reversal. One important finding is that the grouper DMRT] is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1, genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. (c) 2006 Published by Elsevier Ireland Ltd.

小剂量~(16)O~(8+)离子预辐射减轻大剂量辐射所致小鼠睾丸组织损伤(英文)

为了了解小剂量重离子辐射诱导小鼠睾丸结构的适应性反应,采用小剂量(0.05Gy)~(16)O~(8+)离子照射B6C3F_1雄性小鼠睾丸。4h后,再给予2Gy~(16)O~(8+)离子照射。照射后第35天取材在光镜下观察睾丸结构。结果显示,大剂量(2Gy)照射明显损伤睾丸组织,主要表现为曲精细管直径几乎减小一半,精管内各发育阶段的生殖细胞减少或消失,特别是精原细胞几乎完全消失。而Leydig细胞和Sertoli细胞仅有轻度核固缩及胞浆减少。提示睾丸生殖细胞的辐射敏感性明显高于其间质组织细胞。预先给予小剂量(0.05Gy)照射可明显减轻随后大剂量(2Gy)辐射对睾丸组织的损伤。提示小剂量重离子辐射可诱导小鼠睾丸结构明显的适应性反应。