精子顶体小泡蛋白-1(ACRV1)在小鼠睾丸组织中的表达与定位

目的:研究精子顶体小泡蛋白-1(ACRV1)在小鼠睾丸组织发育过程中的表达特征。方法:将4 d、9 d、18 d、35 d、54 d和6月龄小鼠睾丸组织cDNA探针与Affymetrix全基因组芯片进行杂交,筛选出差异表达的ACRV1基因。采用RT-PCR方法检测ACRV1基因在小鼠不同日龄和不同组织中的表达情况,采用免疫组织化学方法检测ACRV1蛋白在小鼠睾丸组织中的定位。结果:对Affymetrix全基因组芯片杂交结果分析后,筛选得到1个差异表达杂交点,通过在NCBI网站与小鼠全基因组序列Blast分析可知该差异表达基因是ACRV1基因。RT-PCR结果表明ACRV1 mRNA呈小鼠睾丸特异性表达,在出生31 d小鼠睾丸开始高表达,在成年前达到高峰。ACRV1蛋白主要定位在睾丸圆形精子和长形精子细胞。结论:ACRV1基因存在发育的表达调控,为小鼠年龄依赖性表达基因,其表达与小鼠精子发生的过程有很强的一致性,且具有睾丸特异性表达的特征,因此推测该基因可能在精子发生中具有关键作用。     

胱蛋白酶抑制剂相关的附睾精子发生基因在小鼠睾丸和附睾中的表达

目的:研究胱蛋白酶抑制剂相关的附睾精子发生(cystatin-related epididymalspermatogenic, Cres)基因在生后不同发育阶段小鼠睾丸及附睾中的表达规律。方法:采用实时荧光定量PCR及间接免疫荧光技术检测Cres mRNA及其蛋白在生后14 d、20 d、22 d、28 d、35 d、49 d、70 d及400 d小鼠睾丸及附睾中的表达变化。结果:在小鼠睾丸中,Cres mRNA在青春期前低水平表达,此后逐渐升高,成年期达到峰值,老年期有所下降;Cres蛋白主要定位于延长精子细胞,免疫染色强度主要取决于生精细胞的发育阶段,而与小鼠的发育阶段无关。在小鼠附睾中,Cres mRNA在青春期前表达量较低,此后逐渐升高,老年期达到峰值;Cres蛋白定位于附睾头部近端上皮细胞及头部中段管腔液和部分上皮细胞中,附睾头部远端及体、尾部均未检测到Cres蛋白。结论:Cres基因在小鼠睾丸和附睾中的表达呈现明显的年龄变化趋势,且其在睾丸和附睾中分别具有阶段特异性表达和区域特异性表达的特点,提示Cres基因可能参与精子发生、成熟过程的调控。这为今后生殖药理学与生殖毒理学的分子水平研究提供了新的线索。     

非梗阻性无精子症患者睾丸组织CREM表达及意义

目的:探讨非梗阻性无精子症(NOA)患者睾丸组织中CREM表达及意义。方法:选取NOA患者28例和生精正常的梗阻性无精子症(OA)患者19例,收集睾丸活检组织,采用RT-PCR、免疫组化法及Western blot法检测睾丸组织中CREM mRNA及CREM蛋白表达。结果:CREM蛋白主要表达于睾丸组织生精小管的精母细胞、精原细胞和早期精子细胞的细胞核。OA睾丸组织中CREM mRNA含量高于NOA睾丸组织[(0.028±0.022) vs (0.014±0.024)],OA睾丸组织中CREM蛋白表达高于NOA睾丸组织[(2.263±0.371) vs (1.145±0.535)],差异均有统计学意义(P0.001)。结论:睾丸组织中CREM mRNA及蛋白表达下调可能是NOA发病的原因之一。     

SEPT11基因在小鼠睾丸精子发生过程中的表达特征

目的初步探讨Septin11(SEPT11)基因在精子发生中的作用。方法通过实时定量PCR、蛋白免疫印迹及免疫荧光等方法检测SEPT11在不同周龄野生型小鼠以及成年睾丸支持细胞激素受体(Ar)特异性敲除小鼠(SCARKO)和Ar全敲除(ARKO)小鼠睾丸中的表达特征。结果 SEPT11基因小鼠出生2周内高表达,随后表达水平降低,出生6周后出现并与未释放的成熟精子共定位且高表达。与野生型小鼠相比,SEPTIN11在SCARKO小鼠和ARKO小鼠睾丸中表达降低(P0.01),m RNA水平显著升高(P0.01);SEPTIN11在成熟精子中定位于尾部。结论 SEPT11基因在小鼠出生时表达最高;SEPTIN11在小鼠成熟精子中定位于尾部;Ar的敲减提高了SEPT11的表达。     

非梗阻性无精子症患者的表皮生长因子及受体表达

目的:探讨血清表皮生长因子(EGF)和睾丸组织表皮生长因子受体(EGF-R)与非梗阻性无精子症睾丸生精功能障碍的关系。方法:采用放免法对20例无精子症患者和20例已育男性志愿者进行血清EGF测定;无精子症患者接受睾丸活检,病理检查按Johnson评分法评价睾丸精子发生及障碍的程度;采用免疫组化SABC法对睾丸3种主要细胞进行EGF-R表达的检测。结果:无精子症患者血清EGF浓度明显低于男性对照者(P<0.05)。睾丸活检生精功能评为8分者14例;3分者2例;6、5、4和 2分者各1例。在20例无精子症睾丸的间质细胞、支持细胞和生精细胞均有EGF-R的表达,间质细胞EGF-R强阳性和阳性表达明显低于支持细胞和生精细胞(P<0.05)。结论:无精子症患者血清EGF浓度下降、间质细胞EGF-R强阳性和阳性表达的减少与睾丸生精功能障碍的程度是相一致的,说明血清EGF及睾丸EGF-R在调节睾丸生精功能方面有重要的作用。     

青春期雄性小鼠双酚A暴露对生殖功能及子代性别比的影响

目的:研究双酚A(BPA)暴露对青春期雄性小鼠成年后生殖功能及对子代的影响。方法:21日龄C57BL/6J雄性小鼠每日腹腔注射BPA 50 mg/kg连续7 d,35 d后检测成年后雄鼠附睾尾精子数量、精子畸形率、睾丸组织学变化;与正常雌性小鼠配种,观测生育力指标以及仔鼠的出生情况。结果:BPA暴露能引起小鼠附睾尾精子数量下降20.6%(P0.01);精子畸形率增加9.65%(P0.05);睾丸组织结构异常;BPA暴露对雄性小鼠成年后的生育力没有明显影响;但能引起子代雄∶雌出生性别比升高。结论:青春期雄鼠BPA暴露能引起成年雄鼠生精功能下降,仔小鼠雄性比例增加。     

人与大小鼠精子中精蛋白mRNA的分析

目的 :研究人与大小鼠精子中精蛋白 m RAN存在情况。方法 :应用 RT-PCR法分别从人、大鼠和小鼠精子总 RNA中扩增得到精蛋白 (protamine) c DNA片段。结果 :人、大鼠和小鼠精子中均存在精蛋白基因相应的 m RNA,并发现头部畸形率高的精子中精蛋白 m RNA明显减少。结论 :精子的 m RNA有可能代表精子发生过程中某些基因的表达情况     

Ropporin基因在人睾丸和精子中的鉴定与蛋白定位

目的:对人睾丸和精子中的Ropporin基因表达进行鉴定和蛋白定位,为进一步研究其在精子中的作用奠定基础。方法:应用免疫组织化学Western blotting和RT-PCR及间接免疫荧光方法检测Ropporin在正常人睾丸组织射出精子中的表达特征。结果:在睾丸组织中,Ropporin主要表达在圆形精子细胞阶段;在精液精子中,mRNA和蛋白水平上均检测出Ropporin的表达,且蛋白主要定位在精子鞭毛的主段和末段上。结论:人睾丸圆形精子细胞和精液精子中均有Ropporin mRNA和蛋白的表达,其可能与精子运动有关。     

表皮生长因子受体(EGFR)在大鼠和小鼠睾丸中的表达和分布

为了解大鼠和小鼠事丸EGFR的表达和分布规律及其与精子发生的关系，本试验应用BALB／c小鼠抗人A431细胞的EGFR单克隆抗体及ABC免疫组织化学方法，研究了大鼠和小鼠EGFR的表达和分布情况。免疫组织化学观察的结果显示：1．出生后，大鼠和小鼠睾丸中的间质细胞即表达EGFR。此时间质细胞体积小，细胞数量也少，所以EGFR的表达是有限。2．性成熟期，表达EGFR的睾丸间质细胞增多；此外，少数精原细胞、精母细胞及管周肌样细胞也表达EGFR。进入性成熟期后，睾丸内EGFR表达量增加。3．成熟期睾丸中，部分精原细胞和精母细胞分别表达EGFR或EGF，可通过自分泌或旁分泌机制，实现其局部调节。此机制有利于精子的发生。这些结果提示：在大鼠和小鼠睾丸中，EGFR的这一与性成熟有关的表达和分布规律与EGF是相似的。EGF通过EGFR作用于生精过程，与性成熟和精子发生关系密切。     

甲胎蛋白在不同发育时期的大鼠胃组织中的表达及定位

目的:研究大鼠正常发育的各个阶段胃组织中甲胎蛋白(AFP)的表达情况及定位。方法:提取大鼠胚胎发育18.5天(e18.5d),e20.5d,新生大鼠(p0d),p7d,p14d,p21d以及成年大鼠的胃组织。检测各时期AFP的蛋白与AFP mRNA的表达以及在胃组织中的定位。结果:AFP在e18.5d大鼠的胃组织中蛋白和mRNA表达量均显著高于其他各时期,出生后表达量稳步下降,成年后消失;AFP在大鼠的胃组织中与间充质细胞标志物波形蛋白(vimentin)呈共表达。结论:AFP在发育的各个时期均特异性表达在大鼠胃间充质细胞,可能对胃结构的形成和功能的完善起着重要作用。     

哺乳动物P1精蛋白基因表达的研究

目的:研究哺乳动物P1精蛋白基因在大鼠和小鼠中的表达。方法:用RT-PCR法制备大鼠P1精蛋白(rP)cDNA,用小鼠P1精蛋白(mP1)DNA重组质粒(pUC8)制备mP1cDNA,rPcDNA和mP1cDNA经迪高辛标记后作为探针,用Northern杂交等技术分析基因表达情况。结果:rPcDNA与大鼠睾丸RNA有杂交反应,而与肝、脑RNA无交叉反应;rPcDNA和小鼠睾丸RNA有交叉反应;mP1cDNA探针与性成熟不同阶段小鼠睾丸RNA杂交结果表明:小鼠性成熟过程中,在睾丸出现圆形精子细胞后mP1基因开始转录,而翻译成蛋白质是在性成熟小鼠睾丸内出现长形精子细胞后。结论:哺乳动物P1精蛋白的表达有器官特异性,是睾丸特异表达的基因,P1精蛋白基因在进化上保守,因而在大、小鼠睾丸中均能检测到杂交讯号;精蛋白基因的表达表现为翻译延迟,该基因在圆形精子细胞阶段开始转录,而在长形精子细胞阶段翻译成蛋白质。     

Bisphenol A induces oxidative stress and decreases levels of insulin receptor substrate 2 and glucose transporter 8 in rat testis

Bisphenol A (BPA), a monomer present in plastics, is known to impair male reproductive functions. Testis executes high-energy-demanding processes such as spermatogenesis and steroidogenesis, the successful accomplishment of which requires several factors including glucose. In this context, we sought to investigate the effects of low doses of BPA on glucose metabolism in the testis of rats and to delineate whether oxidative stress has any role to play in mediating the effects. Bisphenol A was orally administered to rats at dose levels of 0.005, 0.5, 50, and 500 μg/kg body weight for 45 days. A positive control was maintained by orally administering 17β-estradiol at a dose of 50 μg/kg body weight. The levels of plasma glucose and insulin were significantly increased, whereas the testicular glucose level significantly decreased following exposure to BPA and estradiol. A dose-dependent increase in the level of hydrogen peroxide (H?O?) and a significant decline in the activities of hexokinase and phosphofructokinase was observed in the testis of rats treated with BPA. Western blot analyses of insulin receptor substrate 2 (IRS-2) and glucose transporter 8 (GLUT-8) in the testis showed a decline in the levels of these proteins following BPA administration. Immunolocalization of GLUT-8 protein in the testis revealed decreased expression of this protein in spermatocytes and developing spermatids of rats exposed to BPA. The results suggest that persistent exposure to low doses of BPA could disturb glucose homeostasis in the testis and thereby impair testicular functions.     

Characteristics of boys with the so-called true undescended testis diagnosed at the third postnatal month – a population-based case–control study

Objective: Undescended testis (cryptorchidism) is a common congenital abnormality of male genital organs diagnosed at birth followed with frequent postnatal descensus. However, the so-called isolated true undescended testis (ITUT) diagnosed at the third postnatal month seems to be an independent defect-entity, and this hypothesis was planned to confirm or reject in the study.

Method: The evaluation of birth outcomes and maternal socio-demographic data of cases with ITUT in the population-based large dataset of the Hungarian Congenital Abnormality Registry.

Results: There was a higher rate of preterm birth and particularly of low birthweight in 2052 cases with ITUT compared to 24?814 population male controls without any defects. The rate of twins was not higher in cases with older mothers, higher birth order and lower socio-economic status. The comparison of data of boys with undescended testis diagnosed at birth found in the previous study and with ITUT in this study confirmed our hypothesis.

Conclusions: Undescended testis can be differentiated into two subgroups: boys with frequent postnatal descensus mainly after preterm delivery and boys with ITUT without postnatal testis descensus with frequent intrauterine growth restriction, older mothers with higher birth order and low socio-economic status.     

Evaluation of meiotic impairment of azoospermic men by fluorescence in situ hybridization

OBJECTIVE: To identify predictive criteria for the existence of spermatogenesis in nonobstructive azoospermic men. DESIGN: Prospective study. SETTING: Andrology laboratory at a teaching hospital. PATIENT(S): Twenty-two azoospermic men were divided into three groups by qualitative testicular histopathology and the presence of spermatozoa in minced biopsies. INTERVENTION(S): Testicular biopsies evaluation.Main OUTCOME MEASURE(S): The presence of spermatozoa and/or mature spermatids, the percentage of sex vesicle formation (X and Y chromosomes in proximity), and the pairing of the two 18 homologous chromosomes. RESULT(S): Spermatozoa and mature spermatids were found in 17 study patients. Whenever few mature spermatids and/or spermatozoa were found, the rates of X-Y and 18 bivalents were significantly higher (mean +/- SD, 73% +/- 13. 3% and 91% +/- 7.1%) than those in cases of spermatocyte maturation arrest (23% +/- 8.0% and 60% +/- 11.8%, respectively). CONCLUSION(S): Pairing of chromosomes during meiosis is apparently related to the progression of spermatogenesis. Consequently, high rates of bivalent formation increase the prospect of focal spermatogenesis in the testis, despite the failure to identify mature spermatids in the specific testicular biopsy under examination.     

Characterization of D1Pas1, a mouse autosomal homologue of the human AZFa region DBY, as a nuclear protein in spermatogenic cells

OBJECTIVE: To gain insight into the function of D1Pas1 in spermatogenesis. DESIGN: The cellular and subcellular distribution of D1Pas1 protein were examined. SETTING: Academic research laboratory. ANIMALS: Swiss Webster and C57B1/6J mice. INTERVENTION(S): Antibodies were generated against a D1Pas1 fusion protein. Immunoblot analysis was performed on lysates of testicular cells separated into enriched populations of spermatogenic cells and fractionated into nuclear and cytoplasmic compartments. Immunohistochemistry was performed on histological sections of testis from adult and postnatal day 17 mice. MAIN OUTCOME MEASURE(S): D1Pas1 protein distribution. RESULT(S): D1Pas1 was expressed in germ cells, and its expression was developmentally regulated because it was detected specifically in the meiotic and postmeiotic haploid stages of spermatogenesis. D1Pas1 protein was predominantly localized in the nucleus, with weak cytoplasmic staining. CONCLUSION(S): Nuclear localization of D1Pas1 in the testis and its sequence homology to putative RNA helicases suggests a role of D1Pas1 in pre-mRNA processing during spermatogenesis. Germ cell expression of D1Pas1 and homology to the Y chromosome gene DBY, which is located in an area deleted in azoospermia, suggests a potential role for an autosomal gene in the regulation of spermatogenesis.     

Effect of gossypol on the fertility of male rats.

Thirty male rats were grouped into 5 groups of 6 animals each. Animals in groups II-V were given gossypol at a dose of 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight per day for 45 days respectively. Animals of group I served as control. A significant decrease in body weight after administration of 40 mg/kg body weight of gossypol was observed; low doses of gossypol, however did not affect the body weight. Testis, epididymis, prostate and seminal vesicles weights decreased gradually with the increasing doses of gossypol. With the increasing doses of gossypol, a marked decrease in the vas deferens sperm motility was observed. At 40 mg/kg dose there was a total inhibition of sperm motility. Histological studies after 5 mg/kg revealed no apparent sign of degeneration, while after 10 mg/kg dose the changes in the individual cell types were accompanied by overall disorganisation of the germinal epithelium involving displacement of the spermatocytes. The rats treated with 20-40 mg/kg gossypol showed a pronounced deleterious effect on the histological structure of the testis. The drug effect was dose dependent developing sequentially; from the uppermost layer of elongated spermatids affecting round spermatids and finally spermatocytes. Quantitatively the ratios of pachytene spermatocytes: resting spermatocytes, stage 7 spermatids: pachytene spermatocytes, and stage 19 spermatids: stage 7 spermatids and tubular diameter and germinal height decreased significantly. The activities of glucose-6-phosphatase, fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase in testis decreased significantly at high dose (40 mg/kg), while the activity of amylase and glycogen content increased significantly with the increasing doses of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermatogenic events in rat and mice following intratesticular administration of optical isomers of gossypol

Male rats and mice were administered racemic and dextrorotatory gossypol intratesticularly at the dose of 200 micrograms/testis. In a separate experiment, 100 micrograms of dextrorotatory and levorotatory gossypol was administered to male mice. Animals were sacrificed 72 hours after drug treatment. In another experiment, mice were sacrificed 3, 10, 20, 30 and 60 days after racemic gossypol (200 micrograms/testis) administration. Marked degenerative changes in rat and mice tests were observed in the animals receiving racemic gossypol. Dextrorotatory gossypol had less pronounced effect, both in rat and mice. Stage 7 and 16 spermatids were most susceptible to the deleterious effects of racemic gossypol. In mice, 57.52% and 85.40% decrease in stage 7 and 16 spermatids were observed 72 hrs after drug administration. A progressive recovery was observed after drug treatment; the damage (stage 7 & 16) was reduced to 7.92% and 21.30% after 60 days. Histopathological changes in mice testis following 100 micrograms of levorotatory gossypol were distinctly different from those of the dextrorotatory (100 micrograms/testis) gossypol. On the basis of observations made by us, it can be suggested that mice equally respond to the antifertility effect of gossypol following intratesticular administration. The dextrorotatory gossypol, both in rat and mice, had less pronounced effect on the histoarchitecture of the testis in comparison to racemic and levorotatory gossypol. Our observations further suggest that this animal model provides meaningful information on the mechanism of action of gossypol, and recovery of spermatogenesis is possible after termination of drug treatment.     

Histologic changes in the seminiferous tubules after vasectomy

4 different studies on the effect of vasectomy on the testis in canines and humans are reported. Testicular biopsies were performed at certain intervals and the results in both humans and dogs were nearly identical. It was found that spermatogenesis 2 to 3 weeks after vasectomy remained unchanged with accumulation of spermatozoa in the tubules. Between 3 and 6 weeks, progressive spermatogenic arrest with few spermatozoa and decreased spermatids were observed. Between 100 and 300 days, occasional mature sperm were found in the tubules indicating a return of spermatogenesis. Meiotic studies showed this to be an arrest in early prophase. It is theorized that spermatogenesis may be sensitive to pressure changes in the tubular system.