血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞项体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和／或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

VEGF、VEGFR2在青春期大鼠睾丸、附睾及附睾精子上的表达

目的通过对血管内皮生长因子(VEGF)及其受体VEGFR2在青春期大鼠睾丸及附睾表达的研究,探讨其在雄性生殖器官中的作用。方法采用免疫组化法检测VEGF、VEGFR2在SD大鼠睾丸和附睾的表达定位,用免疫荧光法检测它们在大鼠附睾精子上的表达定位。结果VEGF及VEGFR2在青春期大鼠睾丸和附睾组织中均有表达。在睾丸中,VEGF主要表达于精原细胞胞质、精子细胞发育中的顶体、Sertoli细胞胞质及精子残余体内,Leydig细胞胞质也有阳性表达;VEGFR2主要表达于精子细胞发育中的顶体和间质细胞胞质。在附睾中,VEGF表达于附睾管上皮所有主细胞胞质内;而VEGFR2表达于附睾管头段和尾段上皮主细胞胞质内,体段免疫染色阴性。免疫荧光显示,VEGF与VEGFR2都与精子头部顶体、尾部颈段、中段和主段相结合,末段未见阳性荧光。结论VEGF及VEGFR2在大鼠的睾丸和附睾中均有表达,其表达定位具有细胞特异性和区域特异性,提示其可能在大鼠睾丸精子发生和附睾精子成熟中发挥重要作用。     

精索静脉曲张对青春期大鼠睾丸中VEGF和Flt-1蛋白表达的影响

目的研究血管内皮生长因子(VEGF)及其受体Flt-1蛋白在实验性左侧精索静脉曲张(ELV)大鼠睾丸中的表达和定位,探讨它们在精索静脉曲张(VC)致男性不育中的作用。方法建立青春期大鼠ELV模型,采用免疫组化法检测VEGF及Flt-1在ELV4周、8周组及相应对照组大鼠睾丸中的表达变化。结果 VEGF和Flt-1蛋白在大鼠睾丸中定位具有细胞特异性。VEGF蛋白表达于生精细胞、精子细胞发育中的顶体、Sertoli和Leydig细胞胞质内;Flt-1表达于精子细胞发育中的顶体及Leydig细胞胞质中。ELV4周组睾丸中VEGF蛋白的表达显著增加(P<0.01),8周时其表达量下降(P<0.01);ELV4周组与8周组睾丸中Flt-1蛋白的表达均比相应对照组下降(P<0.01),ELV8周组比4周组显著减少(P<0.01)。结论 ELV可影响青春期大鼠睾丸中VEGF和Flt-1蛋白的表达量,可能会影响精子的发生、发育,因而该变化可能是VC引起男性不育的原因之一。     

棕色田鼠睾丸和附睾雌激素α受体表达的增龄变化

应用免疫组织化学方法系统研究了初生（1日龄）到成体（60日龄）5个发育阶段各6只棕色田鼠睾丸和附睾内雌激素受体α（ERα）的表达。结果发现：①在生殖细胞中,1日龄组幼鼠的生殖母细胞和支持细胞有微弱的ERa阳性表达,10日龄组精原细胞中ERa有弱表达,25日龄组精子细胞出现了较强ERα阳性表达,发育到45日龄时精子细胞ERα阳性表达最强,成体组中各级生精细胞中均有ERα阳性表达。②在间质细胞中,1日龄组间质前体细胞有ERα阳性表达,10日龄表达减弱,而到25日龄增强,至45日龄达到峰值,而成体又减弱。③附睾中,1日龄附睾上皮细胞有ERα阳性表达,10和25日龄附睾管类肌细胞出现ERα阳性表达,45日龄和成体附睾管上皮细胞和类肌细胞中均有ERα阳性表达。上述结果表明,ERα可能作为一种特异性受体在棕色田鼠睾丸发育过程中影响睾丸间质细胞雄激素的分泌,进而调节生精过程和精子的发育成熟。     

小鼠精子表面SBA结合糖复合物的形成与变化

用ＨＲＰ标记的大豆凝集素（ＳＢＡ）对睾丸与附睾切片,以及取自附睾和子宫（交配后）内的精子进行了标记,旨在认识精子在发生、成熟和获能过程中表面糖复合物的形成与变化规律。在睾丸内,精母细胞和早期精子细胞胞质内有一强阳性颗粒,处于精子形成期的精子细胞呈弱阳性标记。附睾管内的精子团呈强阳性,附睾管上皮则仅在游离缘呈弱阳性。交配后１．５小时自子宫内洗出的精子,其顶体区的标记增至强阳性,但随着在子宫内存留时间的延长,标记强度逐渐减弱或消失。结果表明,１）精子表面的ＳＢＡ结合糖复合物出现于精子形成期;２）在成熟和获能过程中精子表面的ＳＢＡ结合糖复合物发生明显的变化     

棕色田鼠睾丸及附睾胚后发育的形态学变化

通过组织学方法,对产后1 d、10 d、25 d、45 d、60 d及70 d的棕色田鼠Lasiopodomys mandarinus睾丸和附睾发育进行了观察,以探讨其精子发生特点.结果 发现,1 d棕色田鼠的生殖细胞主要是生殖母细胞和前精原细胞;10 d出现大量精原细胞,睾丸间质细胞明显;25 d出现精子细胞;45 d有少量精子出现;60 d和70 d具有各级生精细胞,睾丸生精小管和附睾内出现大量成熟精子.睾丸生精小管管径和生精上皮厚度随日龄增加,于60 d达到最大;附睾管腔直径和附睾上皮厚度也于60 d达到最大.这些结果表明,棕色田鼠在生后45 d左右进入青春期,60 d左右达到性成熟,精子的产生及成熟与附睾的发育同步.     

Bcl-2和Bax在吗啡依赖雄性大鼠生殖细胞中的表达

目的：探讨B细胞淋巴瘤/白血病-2和人Bcl-2相关x蛋白（Bcl-2、Bax）在吗啡依赖大鼠睾丸生殖细胞中的表达及细胞凋亡可能机制,为治疗阿片类毒品造成的男性性功能减退提供理论依据。方法：以递增法每日给予雄性大鼠皮下注射盐酸吗啡针剂,建立吗啡依赖组。空白对照组注射等量生理盐水。实验成功后将两组大鼠睾丸组织作常规HE染色和免疫组化染色。结果：吗啡依赖组大鼠生精管壁细胞明显地出现上皮层次减少,仅有2~3层,细胞排列疏松,界限模糊,精子细胞和精子数目减少,并发现曲细精管腔内有脱落的生精细胞;免疫组化结果：吗啡依赖组大鼠生殖细胞中bcl-2的阳性表达率明显低于对照组（P〈0.01）,而生殖细胞中bax蛋白的阳性表达率明显高于对照组（P〈0.01）。结论：吗啡依赖可造成雄性大鼠生殖细胞凋亡数量显著增加,其机制可能是通过下调抑凋亡因子Bcl-2,上调促凋亡因子Bax,促进生殖细胞凋亡来实现的。     

小鼠精子表面Con A结合糖复合物的形成与变化

用辣根过氧化物酶标记的ＣｏｎＡ（伴刀豆素Ａ）对小鼠睾丸与附睾切片,以及对取自附睾和子宫（交配后）内的精子涂片进行了标记,旨在认识精子在发生、成熟和获能过程中表面糖复合物的形成与变化。本研究表明,睾丸内的生精细胞和支持细胞均呈ＣｏｎＡ标记阳性。附睾的输出小管和附睾管上皮细胞,ＣｏｎＡ标记呈中度至强阳性,有部位的差别。附睾头和附睾尾内精子表面的标记无明显差别,标记位置均主要在顶体区和尾部。精子在子宫内存留１．５小时后,顶体后区出现中度阳性标记,但存留３小时和６小时后,顶体和顶体后区的标记均减弱或消失。这些结果提示,（１）精子发生期即可合成ＣｏｎＡ结合糖复合物,（２）精子在附睾成熟过程中表面的ＣｏｎＡ结合糖复合物无明显变化,（３）精子获能后顶体后区出现的ＣｏｎＡ结合糖复合物可能与受精能力有关。     

细胞松弛素E对大鼠生精细胞发育的影响

本文采用微丝抑制剂——细胞松弛素E对大鼠生精细胞发育的影响作了形态学观察,特别对支持细胞骨架复合体的作用进行了较为详细的研究。结果表明睾丸内注射0.1ml,1000μmol/L～2000μmol/L,细胞松弛素E,6-14小时后,光镜下可见曲细精管上皮排列疏松,组合紊乱,有的生精上皮基底部出现双核和三核的圆形细胞和多核巨精子细胞,管腔内出现未成熟的精子;在第ⅤⅢ～Ⅸ期曲细精管上皮中,有许多第8、第9期的精子细胞顶体不指向基底方向,属定向不正的精子。电镜下,实验组动物可见一些面向第8-18期精子细胞顶体的支持细胞骨架复合体出现不同程度的缺如,有的断裂成小段;有的破坏仅发生在顶体上方;有的几乎全部丢失,并有类管球复合体的形成。另外,在高渗液处理下,可见精子细胞顶体和支持细胞间的间隙扩大。最后对微丝在精子发育中的作用进行了讨论。     

大鼠睾丸内皮型一氧化氮合酶表达的增龄变化

为研究雄性大鼠睾丸在发生、发育和衰老过程中内皮型一氧化氮合酶(eNOS)在生精功能中的作用及其变化规律，本实验采用了免疫组织化学染色及体视学图像分析等方法，对生后1d至生后24月龄大鼠睾丸eNOS表达的变化进行了系统研究，并统计测量了阳性血管内皮细胞及间质细胞面密度的变化。结果表明：生后1d至2周龄eNOS阳性表达极少；3周龄血管内皮细胞、间质细胞及精子细胞均出现了阳性表达；1月龄至18月龄生精小管靠近管腔的精子细胞也呈阳性，阳性血管内皮细胞、间质细胞数目差异显著；24月龄血管内皮细胞、间质细胞eNOS大量表达，部分生精细胞也有表达。本结果提示一氧化氮参与精子的生成及睾酮的分泌过程，衰老时eNOS阳性表达显著增加，这种变化可能会抑制睾酮的分泌，最终会影响睾丸的生精功能[动物学报49(3)：339—345，2003]。     

Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium

A quantitative immunohistochemical study was performed of the distribution of protein gene product 9.5 (PGP, a soluble protein localized in neurons and neuroendocrine cells as well as in some non-nervous cells) and ubiquitin along the rat epididymis. In the ductuli efferentes, PGP immunoreaction was observed in the whole cytoplasm of some columnar cells; a smaller number of columnar cells showed ubiquitin immunoreactivity with limited apical and basal cytoplasmic localization. In the proximal caput epididymidis, the whole cytoplasm of all columnar cells showed PGP immunoreactivity, ubiquitin immunostaining was negative in this region. In the middle and distal caput epididymidis and the distal cauda, the apical cytoplasm of some columnar cells and the whole cytoplasm of some basal cells showed immunoreactivity to PGP. In these regions, immunoreactivity to ubiquitin was positive in the supranuclear cytoplasm of some columnar cells but not in the basal cells. No immunoreactivity to PGP or ubiquitin was detected in the corpus epididymis and the proximal cauda. Double immunostaining revealed that all the epididymal ubiquitin immunoreactive cells were also PGP immunoreactive, whereas most PGP immunoreactive cells did not immunoreact to ubiquitin. In ubiquitin-PGP immunoreactive cells, the site of the PGP immunoreaction differed from that of the ubiquitin immunoreaction. PGP-ubiquitin immunoreactive cells also seemed to be immunoreactive to anti-AE1/AE3 keratin antibodies. The spermatozoal heads were immunoreactive to PGP antibodies in the epididymal regions from proximal caput to distal cauda but not in the ductuli efferentes. The findings suggest that non-ubiquitinated PGP immunoreactive proteins are secreted in the epididymis, mainly in the proximal caput, and attach to spermatozoa.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm

Previously, we identified a 26-kDa fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. The objective of the present study was to immunohistochemically localize this enzyme to the various cell types within the bull testis and seven subsegments of the epididymis, and on ejaculated sperm in order to gain further insight into its potential function in male reproduction. In the testis, immunoperoxidase staining was localized within the elongating spermatids and Sertoli cells of the seminiferous tubules, varying with the stage of the spermatogenic cycle. The highest level of staining occurred during stages III-VII. The cuboidal epithelial cells of the rete testis and efferent ducts were also immunoreactive. Expression of lipocalin-type prostaglandin D synthase was not uniform in the seven epididymal subsegments, suggesting a possible role in sperm maturation. In all epididymal regions, expression was limited to the epithelial principal cells; no immunoreactivity was apparent in other cell types. Lipocalin-type prostaglandin D synthase was strikingly localized in the caput epididymidis, while moderate to weak staining was observed in the remainder of the epididymis. Droplets of reaction product observed within the lumen increased progressively from the caput to cauda. Using fluorescence microscopy, we also localized lipocalin-type prostaglandin D synthase to the apical ridge of the acrosome on ejaculated sperm.     

钙调素在文昌鱼性腺和表皮分布的研究

用免疫组织化学技术首次发现雌、雄文昌鱼成熟性腺内分布有丰富的钙调素免疫反应物质。钙调素免疫阳性反应分别定位于精细胞和卵细胞的胞核内。文昌鱼表皮细胞尤其细胞顶端胞浆对钙调素抗体显示强阳性反应。作者讨论了文昌鱼性腺和表皮内钙调素的可能作用。     

Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells

To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.     

Liprin α3: a putative estrogen regulated acrosomal protein

Liprin α3 was reported for the first time using sperm proteomics. Present study reports its localization on sperm and immunochemical characterization. Liprin α3 is identified as a 133 kDa protein in testis and epididymal protein extracts. In testis, immunohistochemical localization was seen in pachytenes, diplotenes, round spermatids whereas it was localized in the epithelial cells and luminal sperm in all the three regions of epididymis. Protein was localized in acrosome of rat sperm, which was further confirmed by sequential treatment of sperm with hypertonic solution. In the spermatogenic cells the protein was found to be located in developing acrosome as evident by its co-localization with Golgi marker. Protein was found to be developmentally regulated. In silico analysis of Liprin α3 revealed presence of the estrogen responsive elements upstream to initiation site and its regulation by estrogen was experimentally validated using a tamoxifen treated rat model. Western blot analysis of epididymosomes showed the presence of Liprin α3, indicating its involvement in trafficking of vesicle. The protein expression was seen in both mouse and human sperm indicating conserved nature and a probable role in acrosome reaction.     

Localization and expression of ADAM2 in the dromedary camel testis,epididymis and sperm during rutting season

ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.