Smads蛋白在大鼠睾丸发育不同阶段的表达和定位

探讨转化生长因子－β超家族肽类的细胞内信号转导分子Smads蛋白在发育不同阶段大鼠睾丸的表达及在精子发生中的作用机理。分别选用出生后3、7、14和28d以及成年大鼠,应用蛋白质免疫印迹杂交技术及免疫组织化学ABC法结合葡萄糖氧化酶－DAB－硫酸镍铵增强技术,检测Smad1、Smad2、Smad4和Smad5蛋白在大鼠睾丸的表达、定位和发育变化,并通过图像分析技术对免疫组织化学结果进行统计学分析。免疫印迹杂交发现,Samd1、Smad2和Smad4在3、7、14和28d以及成年大鼠睾丸的生精细胞中即可见免疫阳性反应;Smad2和从7d起开始表达,免疫阳性产物位于各级生精细胞的胞质内;Smad4在发育的各个阶段的间质细胞中均有较强的表达;Smad5仅在28d和成年大鼠的间质细胞中有弱的表达。结果提示：Smads蛋白在睾丸中的分布不同,表达量存在差异,为揭示TGF－β超家族在粗子发生和发育过程中的分子机制提供了直接证据。     

大鼠睾丸发育过程中促甲状腺激素释放激素受体mRNA的表达

为研究促甲状腺激素释放激素受体(TRHR)在大鼠睾丸组织中的表达规律和在生殖发育调节中的作用,依据大鼠垂体中的TRH-RcDNA设计引物,采用RT-PCR法从大鼠睾丸组织中获得了TRH-R的cDNA克隆,测序表明其核苷酸序列与大鼠垂体中的TRH-RcDNA序列完全一致.应用非放射性原位杂交(NR-ISH)技术观察TRH-RmRNA在大鼠睾丸中的定位,结果显示杂交信号集中在间质细胞中,生精细胞无杂交信号.利用实时动态定量RT-PCR法观察了TRH-R在不同发育阶段大鼠睾丸中的表达变化,发现在睾丸间质细胞发育的初期阶段(第8天),没有TRH-R的表达,但从第15天起能观察到TRH-R的表达,并且表达量在20天、35天、60天、90天逐渐增加.这些结果表明,大鼠睾丸组织间质细胞能特异性表达TRH-R,并且表达量与发育过程相关.     

Smad4蛋白在不同龄大鼠睾丸的定位研究

探讨转化生长因子-β超家族肽类的细胞内信号转导分子Smad4蛋白在发育不同阶段大鼠睾丸的表达与分布。分别选用出生后3、7、14、28天以及成年大鼠,应用免疫组织化学ABC法结合葡萄糖氧化酶-DAB-硫酸镍铵增强技术及蛋白质免疫印迹技术,检测Smad4蛋白在大鼠睾丸的表达、定位和发育变化,并通过图像分析技术对免疫组织化学结果进行统计学分析。结果显示,发育各个阶段的间质细胞中都有较强的表达,免疫阳性产物位于细胞质内,而各级生精细胞则无阳性反应,且随着大鼠睾丸发育阶段的变化而蛋白表达量逐渐增多,为TGF-β超家族成员在精子发生和发育过程中的分子机理提供了直接证据。     

Smad4蛋白在不同龄大鼠睾丸的定位研究

探讨转化生长因子－β超家族肽类的细胞内信号转导分子Smad4蛋白在发育不同阶段大鼠睾丸的表达与分布。分别选用出生后3、7、14、28天以及成年大鼠,应用免疫组织化学ABC法结合葡萄糖氧化酶－DAB－硫酸镍铵增强技术及蛋白质免疫印迹技术,检测Smad4蛋白在大鼠睾丸的表达、定位和发育变化,并通过图像分析技术对免疫组织化学结果进行统计学分析。结果显示,发育各个阶段的间质细胞中都有较强的表达,免疫阳性产物位于细胞质内,而各级生精细胞则无阳性反应,且随着大鼠睾丸发育阶段的变化而蛋白表达量逐渐增多,为TGF－β超家族成员在精子发生和发育过程中的分子机理提供了直接证据。     

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。     

PTIP相关蛋白1在小鼠睾丸发育过程中的表达及定位

目的探索PTIP相关蛋白1(PTIP associated protein 1,PA1)在小鼠睾丸发育过程中的表达定位。方法采用RTPCR、实时定量PCR和免疫组织化学方法,对PA1在小鼠睾丸不同发育阶段的表达及定位进行检测。结果 RT-PCR和实时定量PCR结果显示,PA1 m RNA在1w、2w、4w、8w、12w、18w和24w的小鼠睾丸中均有表达,且其表达量在2w时达到最高峰,在小鼠性成熟(8w)以后,PA1的表达量趋于平稳。ABC法免疫组织化学染色显示PA1在1w、2w、4w和8w小鼠睾丸各级生精细胞、支持细胞和间质细胞的胞核中均有表达,免疫荧光双标进一步确定PA1表达于支持细胞和间质细胞。结论 PA1可能在维持生精细胞的正常分化及调节睾丸内分泌的平衡中起重要作用。     

环境高温促进猪睾丸Apaf-1和Caspase-9的表达

高温热应激条件下,凋亡蛋白表达量升高,生殖细胞凋亡增加。凋亡蛋白酶活化因子1（apoptosis protease activating factor 1,Apaf-1）和凋亡蛋白酶活化起始者含半胱氨酸的天冬氨酸蛋白水解酶9,(cysteine aspartic acid specific protease 9, Caspase-9）是细胞凋亡内源途径中的重要调节蛋白,热应激条件下猪睾丸Apaf-1和Caspase 9的表达未见报道。本研究发现,夏季畜舍高温使Apaf-1和Caspase-9表达量升高。qRT-PCR和Western印迹结果显示,与对照组（正常舍温20℃）相比,短时热应激组（40～42℃,1 h/d, 7 d）和长时热应激组（40～42℃,1 h/d, 42 d）,Apaf-1和Caspase-9 mRNA和蛋白的相对表达量均显著升高。免疫组织化学研究发现,Apaf-1在猪睾丸组织中免疫反应阳性物定位于间质细胞、支持细胞和各个发育阶段生精细胞。热应激处理导致精母细胞和精子细胞Apaf-1表达量升高。在各实验猪睾丸组织中,Caspase-9定位于间质细胞、支持细胞和各个发育阶段生精细胞的胞质中。与对照组相比,热应激处理导致减数分裂以后的生精细胞和支持细胞Caspase-9表达量升高。上述结果表明,高温热应激促进Apaf-1和Caspase-9的表达,提示Apaf-1和Caspase-9表达的变化可能与猪舍高温导致的猪精液品质下降存在关联。     

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。     

p38 MAPK在小鼠睾丸不同发育阶段的表达和定位

为探讨丝裂原活化蛋白激酶p38 MAPK在小鼠睾丸不同发育阶段的表达，应用蛋白质免疫印迹杂交技术和免疫组织化学SABC法检测1至7周龄小鼠睾丸p38 MAPK的表达、定位及发育变化，并通过图像分析技术对免疫组织化学结果进行统计学分析。免疫印迹杂交发现，p38 MAPK在2～7周龄小鼠睾丸中均有表达。免疫组织化学结果显示，在2周龄小鼠睾丸曲细精管上皮中即可观察到p38 MAPK免疫阳性反应，免疫反应阳性细胞为精原细胞；3、4、5周龄小鼠睾丸仅有个别曲细精管上皮可见p38 MAPK免疫阳性反应；6、7周龄小鼠睾丸中p38 MAPK表达较丰富，免疫反应阳性细胞为精原细胞和初级精母细胞，免疫阳性反应物均主要位于细胞核内。在7周龄小鼠睾丸中还可见到部分间质细胞的细胞质亦呈p38 MAPK阳性。这些结果提示，p38 MAPK可能对生精细胞的增殖分化具有调控作用。     

大鼠睾丸前促甲状腺激素释放激素原及其受体的表达与发育变化

为研究促甲状腺激素释放激素（thyrotrophin-releasing hormone,TRH)及其受体（TRH receptor,TRHR)在大鼠睾丸组织中的表达规律和在生殖发育调节中的作用,依据大鼠下丘脑中的前TRH原（PreproTRH,ppTRH)和垂体中的TRH－R cDNA设计引物,采用RT－PCR法从大鼠睾丸组织中获得了ppTRH和TRH－R的cDNA克隆,测序后构建表达载体,在大肠杆菌中表达了可溶性的pTRH t TRH-R融合蛋白,利用实时动态定量RT－PCR（real time quantitative RT-PCR)法观察了ppTRH和TRH－R在不同发育阶段大鼠睾丸中的表达变化,发现在睾丸间质细胞发育的初期阶段（第8天）,没有ppTRH和TRH－R的表达,但从第15天起能观察到pp-TRH和TRH－R的表达,并且表达量在20天,35天,60天和90天逐渐增加,这些结果表明：大鼠睾丸组织能特异性表达ppTRH和TRH－R,并且表达量与发育过程相关,ppTRH和TRH－R体外表达产物的获得为后续研究其功能奠定了基础。     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Presenilin-1 protein specifically expressed in Leydig cells with its expression level increased during rat testis development

Presenilin-1, mutations of which cause the early-onset of Alzheimer's disease, was shown to be abundantly expressed in the testis as well as the brain. In spite of the high expression level of this protein in the testis, no further analysis has been undertaken. We aimed to study the distribution and developmental changes in presenilin-1 protein, and to provide clues so as to elucidate the role of this protein in the rat testis. To evaluate the specificity of the anti presenilin-1 antibody, rat presenilin-1 protein was expressed in COS-7 cells and the recombinant protein was used for western blot analysis. A positive band of approximately 20 kDa corresponding to the C-terminal fragment of proteolyzed presenilin-1 protein was observed. Using testis and brain tissue samples, a 20 kDa band was detected in both tissues suggesting a similar proteolytic process, but the expression level in the testis was higher than that in the brain. The expression level increased significantly during postnatal testis development. By an immunohistochemical analysis of the rat testis, a strong signal was observed in interstitial cells and further study with cultured TM3 murine Leydig cells revealed an abundant expression of presenilin-1 in Leydig cells. Our study suggests that presenilin-1 expression in Leydig cells may play an important role in Leydig cell function and testis development.     

Structure and expression of the rat relaxin-like factor (RLF) gene.

The relaxin-like factor (RLF) is a novel member of the insulin-IGF-relaxin family of growth factors and hormones, and its mRNA is expressed very specifically in the Leydig cells of the testis and in the theca and luteal cells of the ovary. Here we report the cloning of the RLF gene and cDNA from the rat. The 0.8kb mRNA is produced from a small gene comprising two exons situated less than 1 kb downstream of the gene for the signalling factor JAK3. Northern hybridization confirms high RLF mRNA expression in the adult rat testis, and low expression in the ovary, but in no other tissues examined. Northern analysis of fetal and neonatal gonadal tissues showed that RLF mRNA is highly upregulated in the testes of day 19 embryos, but not in later neonatal stages, nor in any ovarian tissue from this period. This would indicate that RLF is a marker for the mature fetal as well as the adult-type Leydig cell, but is not expressed in premature, precursor, or dedifferentiated Leydig cells of either cell type. Finally, RNA was analysed from the testes of rats which had been treated with ethylene dimethane sulfonate (EDS), an alkylating agent that specifically destroys rat Leydig cells. RLF mRNA was absent from the acutely treated testes, but became detectable between 15 and 20 days post-treatment, concomitant with the repopulation of the testes by new Leydig cells. Continuous testosterone substitution of EDS-treated rats suppressed the production of gonadotropins, and LH-dependent Leydig cell differentiation, with the result that RLF mRNA remained undetectable throughout the study period. In conclusion, RLF is a very specific marker for the mature Leydig cell phenotype in both the adult-type and fetal Leydig cell populations of the rat testis.     

Detection of platelet-derived growth factor-alpha (PDGF-A) protein in cells of Leydig lineage in the postnatal rat testis

Platelet-derived growth factor-A (PDGF-A) is a locally produced growth factor in the rat testis secreted by both Sertoli cells and Leydig cells. It has been suggested that PDGF-A may be involved in modulation of testosterone production and may be essential to Leydig cell differentiation, however it is not known at what stage of differentiation PDGF-A begins to be expressed in the cells of Leydig lineage in the postnatal rat testis. Therefore, the objectives of this research were to determine at what postnatal age and in which cell type is PDGF-A first expressed in cells of the adult Leydig cell lineage, and does PDGF-A expression coincide with expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), an indicator of steroid hormone synthesis. Male Sprague Dawley rats of postnatal day 1, 7, 9-14, 21, 28, 40, 60, and 90 were used (n=6). Animals were euthanized and their testicles removed, fixed in Bouin's solution, embedded in paraffin, and 5 micrometers sections were prepared. Immunolocalization of PDGF-A and 3beta-HSD was carried out using a peroxidase-streptavidin-biotin method. PDGF-A was first detected in cells of the Leydig cell lineage at postnatal day 10 in progenitor cells, which were surrounding the seminiferous tubules (peritubular). These cells were confirmed to be the progenitor cells and not the mesenchymal or any other spindle-shaped cells in the testis interstitium by immunolocalization of 3beta-HSD and PDGF-A in the cells in adjacent sections of testis tissue from rats of postnatal days 10-14. After postnatal day 10, PDGF-A was continued to be expressed in subsequent cells of the Leydig lineage through day 90 (adult), however, was not present in peritubular mesenchymal precursor cells of the Leydig cell lineage or any other spindle-shaped cells in the testis interstitium at any tested age. These results revealed that PDGF-A first appears in Leydig progenitor cells in the postnatal rat testis at the onset of mesenchymal cell differentiation into progenitor cells at postnatal day 10 and suggest that a functional role(s) of PDGF-A in postnatally differentiated Leydig cells in the rat testis is established at the time of the onset of postnatal Leydig stem cell differentiation. It is suggested that the significance of the first expression of PDGF-A in the Leydig progenitor cells may be associated with inducing cell proliferation and migration of this cell away from the peritubular region during Leydig cell differentiation.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

Change in expression of a 90-kDa glycoprotein GP90-MC301 during prenatal and postnatal testicular development: a differentiation marker for rat germ cells

GP90-MC301, a 90-kDa glycoprotein recognized by the monoclonal antibody MC301, is a reliable stage-specific marker for preleptotene to pachytene spermatocytes in adult rat testes. In this study we confirmed that the glycoprotein is also useful as a marker for germ cells in prenatal and postnatal testes. Immunohistochemical analysis showed a dramatic change in GP90-MC301 expression in germ cells during testis development. Strong expression was detected in primordial germ cells at embryonic day (E) 13 and in gonocytes at E16, and the expression was then markedly reduced at around the time (E18) gonocytes undergo G1/G0 arrest, and was not restored in gonocytes or spermatogonia afterward. Thereafter, it reappeared in primary spermatocytes in the prepubertal period. Testicular somatic cells such as Sertoli cells, Leydig cells, and peritubular myoid cells expressed GP90-MC301 during specific periods which were largely correlated with periods of active proliferation of these testicular somatic cells. Western blotting showed that GP90-MC301 was expressed during testis development without a change in its molecular size. Thus, GP90-MC301 is potentially useful for the analysis of not only spermatogenesis but also early testis development.     

Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis

Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.     

Expression and distribution of trihydrophobin 1 in postnatal developing mouse testis

The human trihydrophobin 1 (TH1) is a highly conserved and widely expressed protein. It is clear that TH1 serves as a new specific negative regulator of A-Raf kinase. In this study, we found that TH1 associated with A-Raf in mouse testis by using coimmunoprecipitation analysis. Then we characterized the gene expression of TH1 in mouse testis and analyzed the changes of TH1 protein during postnatal development. The protein expression of TH1 in mouse testis was further analyzed by immunohistochemistry staining. Strong signals were detected in the seminiferous tubules and the distribution patterns varied with the different ages of postnatal mouse testis. TH1 was distributed in spermatocytes and Sertoli cells at 2 weeks postnatal, and was abundant in spermatogonia at 8 weeks postnatal. Leydig cells were positive to TH1 throughout testicular development. A high expression of TH1 in both Leydig cells and mouse Leydig tumor cells (mLTC-1cells) was found to be concentrated in the cytoplasm. The colocalization of TH1 and A-Raf in mLTC-1 cells or in adult testis was also observable.