转化生长因子β及其受体在小鼠精子发生过程中的表达

目的观察转化生长因子β（TGFβs）及其受体（TGFβ-R）在小鼠睾丸中的表达,证实TGFβs及其受体在小鼠精子发生过程中的定位和作用机理.方法免疫组织化学染色结合显微图像定量分析和免疫荧光双重染色.结果免疫组织化学显示：TGFβ1和TGFβ2主要表达于生精小管内Ⅵ-Ⅷ期圆形精子细胞以及长形精子细胞和变态精子细胞的胞质,位于睾丸间质中的Leydig细胞胞质呈弱表达;TGFβ3仅表达于Leydig细胞,各级生精细胞未见表达.晚期精母细胞（Ⅷ期）至第Ⅹ期长形精子细胞均表达TGFβ-RⅠ;TGFβ-RⅡ仅表达于第Ⅺ期后的变态精子细胞期;而TGFβ-RⅢ则主要表达于Ⅶ-Ⅷ期圆形精子细胞和变态精子细胞胞质.免疫荧光双重染色显示：TGFβ1和TGFβ2与TGFβ-RⅢ在生精上皮最早共表达于晚期圆形精子细胞顶体极.结论 TGFβs及其受体在小鼠精子发生过程中的表达具有细胞和周期特异性,提示其在精子发生中具有重要意义.     

Smad2和Smad4蛋白在成年大鼠睾丸的表达

目的 探讨转化生长因子－β超家族肽类的细胞内信号转导分子Smad2和Smad4蛋白在成年大鼠睾丸的表达及在精子发生中的作用机理。方法 用免疫组织化学ABC法结合葡萄糖氧化酶－DAB－硫酸镍铵增强技术,检测成年大鼠睾丸Smad2和Smad4蛋白的表达和定位。结果Smad2蛋白主要表达在大鼠睾丸生精小管的精原细胞、初级精母细胞、次级精母细胞、精子细胞、支持细胞和间质细胞中,免疫反应物质主要定位于细胞质,胞核为阴性;而Smad4蛋白同主要表达于间质细胞的胞质中,支持细胞呈弱阳性染色,结论 Smad2蛋白主要表达于睾丸生精小管各级生精细胞,Smad4蛋白表达于间质细胞,为揭示TGF－β超家族在精子发生中的分子机理提供了直接证据。     

小鼠精子发生相关蛋白3基因在小鼠生精细胞的特异表达及对HEK 293T细胞凋亡和自噬的影响

目的检测小鼠精子发生相关蛋白3(spata3)基因在小鼠生精细胞的表达情况,并借助过表达细胞模型进一步分析该基因对人胚肾HEK 293T细胞凋亡及自噬的影响,旨在探讨spata3在精子发生过程中的意义。方法分别采用RT-PCR和免疫印迹法检测spata3基因mRNA及蛋白产物在小鼠各组织中的表达;应用免疫组织化学和免疫荧光染色观察SPATA3蛋白在生精细胞中的定位;借助脂质体将真核表达载体Plv-EGFP-2(a)purospata3瞬时转染HEK 293T细胞,进一步在蛋白水平分析细胞凋亡相关蛋白Caspase-3、多聚ADP-核糖聚合酶(PARP)、BAX和Bcl-2及自噬相关蛋白LC3A/B的变化。结果 Spata3基因及其编码产物在小鼠睾丸组织特异表达;粗线期精母细胞和圆形精子细胞的胞质与胞核均有显著SPATA3蛋白的阳性着色,长形精子细胞的胞质也有大量分布;过表达spata3的HEK 293T细胞内活化型Caspase-3和PARP降解产物的含量较对照组差异无显著性,BAX表达量0.815±0.020较裸细胞组0.469±0.012和空载体转染组0.588±0.018均有所增高,Bcl-2含量0.214±0.020低于裸细胞0.507±0.021和空载体转染组0.545±0.024,LC3A/B-Ⅱ的表达量0.741±0.037则显著高于裸细胞组0.136±0.011和空载体转染组0.169±0.012。结论 Spata3基因在小鼠生精细胞特异表达,过表达spata3对HEK 293T细胞凋亡无明显影响,但可以促进细胞自噬。     

锌指蛋白185(ZNF185)在小鼠睾丸精子细胞、间质细胞和支持细胞中的表达和定位北大核心CSCD

目的研究锌指蛋白185(ZNF185)在小鼠睾丸精子、间质和支持细胞中的表达变化。方法应用免疫荧光组织化学染色法检测ZNF185在精子、间质、支持细胞和睾丸组织中的定位;实时定量PCR和Western blot法分别检测三种细胞中ZNF185 mRNA和蛋白表达水平的差异。结果免疫荧光细胞分析显示ZNF185在小鼠睾丸精子、间质和支持细胞中均表达,主要分布于间质和支持细胞胞质、精子细胞头部和尾部;实时定量PCR和Western blot结果显示,支持细胞ZNF185 mRNA和蛋白表达水平显著低于间质和精子细胞。结论 ZNF185分布于小鼠睾丸不同细胞,表达量存在差异。     

锌指蛋白185(ZNF185)在小鼠睾丸精子细胞、间质细胞和支持细胞中的表达和定位

目的研究锌指蛋白185(ZNF185)在小鼠睾丸精子、间质和支持细胞中的表达变化。方法应用免疫荧光组织化学染色法检测ZNF185在精子、间质、支持细胞和睾丸组织中的定位;实时定量PCR和Western blot法分别检测三种细胞中ZNF185 mRNA和蛋白表达水平的差异。结果免疫荧光细胞分析显示ZNF185在小鼠睾丸精子、间质和支持细胞中均表达,主要分布于间质和支持细胞胞质、精子细胞头部和尾部;实时定量PCR和Western blot结果显示,支持细胞ZNF185 mRNA和蛋白表达水平显著低于间质和精子细胞。结论 ZNF185分布于小鼠睾丸不同细胞,表达量存在差异。     

衰老大鼠睾丸生精细胞增殖凋亡及相关因素的变化

目的:观察衰老大鼠睾丸生精细胞增殖、凋亡及相关因素的变化。方法:D-半乳糖连续腹腔注射制作亚急性衰老的大鼠模型。SP免疫组织化学法观察睾丸生精细胞衰老过程中增殖细胞核抗原(PCNA)和端粒酶的表达,TUNEL法观察生精细胞的凋亡,原位杂交、Western印迹分析观察生精细胞p53基因的表达。结果:与正常大鼠相比,衰老大鼠睾丸PCNA阳性细胞率显著下降,端粒酶阳性细胞数明显减少,凋亡管数和凋亡阳性细胞率显著升高,p53 mRNA阳性细胞率和p53蛋白含量均有所升高。结论:衰老大鼠睾丸生精细胞增殖能力下降、凋亡增多,睾丸机能减退。     

砷染毒对大鼠睾丸凋亡诱导因子表达及生精细胞凋亡的影响

目的:探讨砷染毒对大鼠睾丸凋亡诱导因子(AIF)表达及生精细胞凋亡的影响。方法:雄性SD大鼠被随机分为对照组和染砷组。采用自由饮用方式进行连续染毒6个月。采用免疫印迹及实时荧光定量PCR检测AIF表达水平;采用TUNEL法观察生精细胞凋亡的情况。结果:免疫印迹及实时荧光定量PCR结果显示,与对照组比较,染砷组大鼠睾丸内AIF蛋白及mRNA表达水平均显著增高。TUNEL法检测结果显示与对照组比较,染砷组生精上皮凋亡细胞平均灰度值显著增高。结论:砷染毒时AIF介导的非caspase依赖性凋亡途径可能参与了大鼠生精细胞凋亡。     

二乙基己烯雌酚诱发成年仓鼠睾丸生精异常及对生精细胞表达bcl-2、P53和cadherin的影响

目的探讨在二乙基己烯雌酚(DES)诱发成年动物生精异常过程中,原癌基因bcl-2、p53及黏附分子cadherin在生精细胞中表达的变化,旨在阐明DES诱发生精异常的作用机理。方法成年雄性仓鼠皮下注射DES连续7d后,取其睾丸,进行光镜及电镜的观察和原癌基因bcl-2、p53及黏附分子cadherin的免疫组织化学染色。结果DES处理组的成年仓鼠睾丸生精细胞发育异常,bcl-2和p53表达量均比对照组有显著增加,并以精母细胞和圆形精子细胞较为明显。生精上皮中cadherin的表达比对照组有明显减少。结论DES增加精母细胞和圆形精子细胞表达bcl-2和p53;同时抑制cadherin表达,是诱发成年仓鼠生精异常的原因之一。     

哺乳动物精子发生中转录调控机制的特点

在哺乳动物精子发生中,存在着特异而精细的转录调控。生精细胞中活跃的转录活动,终止于圆形精子细胞向长型转变的过程之初。睾丸特异性转录调控包括:转录装置的过度表达、睾丸特异性转录因子及启动子的利用,特异性的转录调控途径。转录后调控的作用也十分重要。这些分子事件与卵子发生中的转录调控互有异同。     

磷酸化的丝裂原活化蛋白激酶在大鼠睾丸的表达

目的研究丝裂原活化蛋白激酶(MAPKs)的3个亚家族成员细胞外信号调节激酶(ERK)、c-Jun N-端激酶(JNK)和p38 MAPK的活化形式在睾丸中的定位,了解MAPKs在生精过程中所起的作用。方法免疫组织化学方法检测正常大鼠睾丸中磷酸化的p-ERKJ、NK、p38 MAPK的表达情况。结果正常大鼠睾丸中p-ERK主要分布于精原细胞、细线前期到粗线期的初级精母细胞以及9~12期长形精子细胞的细胞核,p-JNK则主要位于支持细胞与支持细胞、支持细胞与生精细胞(尤其是19期精子细胞)之间,而p-p38 MAPK除了在生精小管的部分细胞胞质中有分布外,其表达最明显的部位是在间质细胞的细胞质。结论ERKJ、NK和p-38 MAPK分别定位于正常大鼠睾丸内的不同部位,提示MAPKs不同的亚家族成员分别在精子发生的不同环节中发挥主要作用。ERK可能参与生精细胞增殖、分化的信号转导,JNK则可能通过调节细胞的黏附而最终影响生精细胞的迁移与精子释放过程,而p38 MAPK除了可能与JNK一起参与精子释放的调节外,最主要的作用可能是睾酮合成分泌的调节。     

Expression of the diaphanous-related formin proteins mDia1 and mDia2 in the rat testis.

Cytoskeletal alterations in both Sertoli cells and germ cells are important during many facets of mammalian spermatogenesis. Diaphanous-related formin proteins are known to control many aspects of actin-based cytoskeletal rearrangements, yet nothing is known regarding the expression of formins in the testis. Accordingly, here we present the first data describing mDia1 and mDia2 mRNA and protein expression in primary Sertoli cell isolates, established tissue culture cell lines often used as models for Sertoli cell analysis, and mixed populations of adult rat male germ cells. Furthermore, we have examined intact sections of rat testis. The results suggest strongly that mDia1 and mDia2 are indeed involved in the regulation of Sertoli cell and germ structure during mammalian spermatogenesis, and provide strong indications of the future directions for mechanistic studies.     

Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature.

Physiological scrotal hypothermia is necessary for normal spermatogenesis and fertility in mammals. Cirp is a recently identified cold-inducible RNA-binding protein that is inducible at 32 degrees C in mouse somatic cells in vitro. Cirp is constitutively expressed in the testis of mouse and structurally highly similar to RBM1, a candidate for the human azoospermia factor. To elucidate the role played by Cirp in spermatogenesis, we investigated its expression levels during spermatogenesis and after heat stress. In the mouse testis, cirp mRNA was detected in the germ cells, and the level varied depending on the stage of differentiation. Also, a high level of Cirp protein was detected immunohistochemically in the nucleus of primary spermatocytes. Expression of Cirp was decreased in the GC-2spd(ts) mouse germ cell line when culture temperature was raised from 32 degrees C to 37 degrees C. When mouse testis was exposed to heat stress by experimental cryptorchidism or immersion of the lower abdomen in warm (42 degrees C) water, the expression of Cirp was decreased in the testis within 6 hours after either treatment. In human testis with varicocele analyzed immunohistochemically, germ cells expressed less Cirp protein than those in the testis without varicocele. These results demonstrated that CIRP expression is down-regulated at elevated temperature in male germ cells of mice and humans. Analysis of Cirp expression in the testes will help elucidate the molecular mechanisms leading to male infertility.     

E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis

Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis.     

Molecular chaperone calmegin localization to the endoplasmic reticulum of meiotic and post-meiotic germ cells in the mouse testis.

Calmegin is a testis-specific Ca(2+)-binding protein that is homologous to calnexin. Recently, sperm from transgenic mice lacking calmegin have been shown to be infertile. To further characterize calmegin, we analyzed the precise stage of expression and the intracellular localization of this protein in germ cells during mouse spermatogenesis by an immunoperoxidase technique using the anti-calmegin monoclonal antibody TRA369. Light microscopic immunocytochemistry showed that calmegin appeared in early pachytene spermatocytes, with the highest expression in round and elongating spermatids, and disappeared in the maturation phase of spematids at step 15. Immunoelectron microscopy showed that selective localization was found at the endoplasmic reticulum membrane and the nuclear envelope of spermatogenic cells. During the maturation phase, a dramatic reduction in calmegin occurred in the endoplasmic reticulum of the spermatids, suggesting that the major function of calmegin has been completed by the time spermatids reach step 14. In addition, although the immunoreactivity was completely absent in the calmegin-deficient mutant mouse testis, ultrastructural analysis showed that mature sperm from the knockout mice were normal. This suggests that calmegin is not required for the morphogenesis of male germ cells. Thus, our results suggest that calmegin has a major role in mouse spermatogenesis, and also indicate that this protein would be useful as a maker molecule to study the functional role of the endoplasmic reticulum in the process of spermatid differentiation.     

Expression of a Y-box protein, YB2/RYB-a, precedes protamine 2 expression during spermatogenesis in rodents.

Y-box binding proteins, a large family of proteins, are involved in a variety of functions. The present study describes the expression of YB2, a rat Y-box binding protein, and/or RYB-a, an alternatively spliced product of the YB2 gene during spermatogenesis. YB2/RYB-a is thought to be the rat orthologue of mouse Y-box protein 3 (MSY3). An antibody which recognizes YB2/RYB-a was developed and applied in an immunochemical study of rat and mouse testes. We also carried out an in-situ hybridization study and Northern blot analysis of YB2/RYB-a and protamine 2 mRNA expression. Both YB2/RYB-a mRNA and the proteins appeared in prepubertal mouse testes, prior to the expression of the mouse protamine 2 mRNA. The mRNA and protein were present at high levels in spermatocytes, decreased in round to elongated spermatids, and were absent in spermatozoa. Since the protamine 2 mRNA was present at high levels in round and elongating spermatids, the proposed function of the YB2/RYB-a protein as a translational repressor of the mRNA was supported in mouse. The level and localization of YB2/RYB-a mRNA and protein expression in the rat testis was comparable to that in mouse testis, although rat testis is known to express a very low level of protamine 2, but is also likely to affect the expression of other proteins (including protamine 1) during spermatogenesis.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules

 The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I–III and IX–XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell. Accepted: 27 Oct 1998