The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa

目的:研究精子特异性钙离子通道(CATSPER1)蛋白和 mRNA 在人类睾丸和射出精子中的表达,检测抗人 CATSPER1 抗体体外对人类精子前向运动的影响以初步探讨人 CATSPER1的功能及其作为免疫避孕靶点的可能性。方法:用逆转录聚合酶链反应(RT-PCR)和间接荧光免疫组化法分别检测液氮冻存人睾丸组织和12例正常精液标本中 CATSPER mRNA 和蛋白的表达。12例正常精液标本经 Percoll 不连续密度梯度离心法上游,上游后精子分别与终浓度为20 mg/mL、4 mg/mL、0.8 mg/mL 的抗人 CATSPER1抗体孵育,以抗体的储存液(0.01mol/L PBS,pH7.4)为对照,1 h、2 h、6 h 后用计算机辅助精液分析仪(CASA)分析前向运动精子百分率(WHO 际准,a 级 b 级)和快速前向运动精子百分率(WHO 标准,a 级)。结果:CATSPER1的 mRNA 存在于人类睾丸组织和射出精子中。CATSPER1蛋在人类睾丸主要表达于精子细胞,并定位于射出成熟精子尾部的主段。实验中用所有浓度的抗人 CATSPER1机体体外在1 h、2 h、6 h 时段均使前向运动精子百分率和快速前向运动精子百分率下降,都与对照组差异显著。结论:CATSPER1在人类睾九中呈减数分裂及减数分裂后式表达,存在于射出精子中的 mRNA 将是比睾丸穿刺方便、更易接受的用于研究 CATSPER1和不育检查的靶点。这些结果也提示人 CATSPER1是免疫避孕的有效靶点。     

补肾生精方促进少弱精症大鼠精子发生的作用机制研究

目的：研究补肾生精方对腺嘌呤诱导的少弱精症大鼠精子发生的作用机制。方法：采用腺嘌呤灌胃诱导SD大鼠制备少弱精症大鼠模型,将48只SD大鼠随机分为空白组、模型组、五子衍宗丸组（1 g/kg）和补肾生精方高、中、低剂量组（2.8 g/kg、1.4 g/kg、0.7 g/kg）,给药3周。干预结束后采用放射免疫法测定血清睾酮水平;利用计算机辅助精子分析系统评估精子密度、活动力;苏木素-伊红（HE）染色观察大鼠睾丸组织病理形态学改变;免疫组织化学（IHC）检测大鼠睾丸组织支持细胞中波形蛋白表达水平。结果：与模型组相比,给药组的大鼠睾丸病理损伤有所改善,睾丸组织部分生精小管增厚,细胞排列紧密,部分管腔缩小,生精细胞和精子数量增加。与模型组相比,五子衍宗丸组及补肾生精方各剂量组血清睾酮水平均上调（P<0.05）,其中,五子衍宗丸组、补肾生精方高剂量组上升明显（P<0.05）,差异有统计学意义。与模型组相比,给药后大鼠精子质量有所改善,精子活动力差异有统计学意义（P<0.05）,同时,补肾生精方高剂量组在改善精子密度方面优于模型组（P<0.05）;补肾生精方高剂量组在改善精子...     

Leptin and varicocele-related spermatogenesis dysfunction: animal experiment and clinical study

The objective of this study was to explore the relationships between varicocele-related spermatogenesis dysfunction and the expression of leptin and leptin receptors. In rats with experimental varicocele, the function of spermatogenesis, the expression of leptin and leptin receptors in testes were analysed; and in patients with varicocele-related male infertility, serum and seminal plasma levels of leptin, gonadal hormones and semen parameters were evaluated. In the testes of rats, leptin was expressed in seminiferous tubules and intersitium, leptin receptor was predominantly expressed in interstitium. The expression of leptin and its receptor in the testis of rats was not related to the weight of rat, but was inversely related to the weight of testis (r = -0.408, p = 0.009 and r = -0.433, p = 0.005, respectively), the Johnsen scores (r = -0.916, p = 0.000 and r = -0.863, p = 0.000, respectively), the seminiferous tubules diameter (r = -0.853, p = 0.000 and r = -0.870, p = 0.000, respectively) and the thickness of seminiferous epithelium (r = -0.929, p = 0.000 and r = -0.948, p = 0.000, respectively). In varicocele patients (N = 40), the sperm concentration and motility were significantly lower (p = 0.000) than those in the control group (N = 25), and the leptin level in seminal plasma was significantly higher (p = 0.000) than that in the control group. The leptin in serum and seminal plasma was positively related (r = 0.223, p = 0.002). The seminal plasma leptin level was inversely related to sperm concentration (r = -0.632, p = 0.000) and motility (r = -0.635, p = 0.000). There was no significant relation between serum leptin and seminal parameters and between leptin and gonadal hormone values. The dysfunction of spermatogenesis in varicocele-related infertile male is associated with increase in leptin and leptin receptors. Leptin may have local effects on the function of testis and spermatogenesis.     

Oyster peptide prevents the occurrence of exercise-hypogonadal male condition by improving the function of pituitary gonadal axis in male rats

This study evaluates the protective role of oyster peptide (OP) on the occurrence of Exercise-Hypogonadal Male Condition. Male rats were given heavy-load swimming training and / or OP was supplemented for 6 consecutive weeks. After heavy-load training, sperm count, sperm viability and sperm motility in epididymis, testosterone in serum and testis, glutathione peroxidase (GSH-px) and androgen receptor (AR) in testis and mating times were remarkably decreased, malondialdehyde (MDA), capture latency and mating latency were significantly increased, mRNA expression of steroidogenic acute regulatory (StAR) and P450 cholesterol side-chain cleavage enzyme (P450scc) were obviously down-regulated, but serum follicle-stimulating hormone (FSH) and luteinising hormone (LH) were not statistically changed. Conversely, when OP was supplemented at heavy-load training, sperm count, sperm viability and sperm motility in epididymis, serum FSH, LH, testosterone, GSH-px, superoxide dismutase (SOD), testosterone, AR in testis and mating times were dramatically increased, while testicular MDA, capture latency and mating latency were significantly decreased, and mRNA expression of StAR, StARD7, P450scc and 3β-hydroxysteroid dehydrogenase (3β-HSD) were significantly up-regulated. In conclusion, heavy-load training causes testicular spermatogenic and steroidogenic disorders by enhancing the generation of reactive oxygen species (ROS), which can be protected by the co-administration of OP by enhancing the function of pituitary gonad axis and lowering ROS generation.     

11β-hydroxysteroid dehydrogenase types 1 and 2. in postnatal development of rat testis： gene express,on, localization and regulation by luteinizing hormone and androgens

11β-hydroxysteroid dehydrogenase type 1 （11β-HSD1） and type 2 （11β-HSD2） are expressed in rat testis, where they regulate the local concentrations of glucocorticoids. Here, we investigated the expression and localization of 11β-HSD in rat testis during postnatal development, and the regulation of these genes by luteinizing hormone （LH） and androgens, mRNA and protein levels were analyzed by quantitative real-time-polymerase chain reaction and western blotting, respectively, in testes collected from rats at postnatal day （PND） 7, 14, 21, 35, and 90, and from rats treated with LH, 7α.methyl-19-nortestosterone （MENT） and testosterone at PND 21 and PND 90. Immunohistochemical staining was used to identify the localization of the 11β-HSD in rat testis at PND 7, 14, and 90. We found that 11β-HSD1 expression was restricted to the interstitial areas, and that its levels increased during rat testis development. In contrast, whereas 11β-HSD2 was expressed in both the interstitial areas and seminiferous tubules at PND 7, it was present only in the interstitial areas at PND 90, and its levels declined during testicular development. Moreover, 11β-HSD1 mRNA was induced by LH in both the PND 21 and 90 testes and by MENT at PND 21, whereas 11β-HSD2 mRNA was induced by testosterone and MENT in the PND 21 testis and by LH in the PND 90 testis. In conclusion, our study indicates that the 11β-HSD1 and 11β-HSD2 genes have distinct patterns of spatiotemporal expression and hormonal regulation during postnatal development of the rat testis.     

Sperm structural and motility changes during aging in the Brown Norway rat

The Brown Norway rat provides a useful model to study aging of the male reproductive tract because of the selective age-dependent pathological changes that are found in the testis, epididymis, and prostate. In the testis, there is a clear age-dependent decrease in both steroidogenesis and spermatogenesis. In the epididymis, some striking segment-specific changes occur at the histological and biochemical levels prior to the major loss of spermatogenesis. We hypothesized that formation of spermatozoa in the testis and maturation of spermatozoa in the epididymis (ie, acquisition of motility and loss of the cytoplasmic droplet) may be altered during aging. Changes in the morphology of spermatozoa were assessed by light and electron microscopy. Using computer-assisted sperm analysis, the motility parameters of spermatozoa obtained from the caput and cauda epididymidis of young and old Brown Norway rats were compared. In old animals, we also compared the motility of spermatozoa from epididymides adjacent to regressed testes with those from epididymides adjacent to nonregressed testes. There was a marked increase with age in the number of spermatozoa with abnormal flagellar midpieces; the nature of these defects did not change with age. In caput epididymidis, the percentage of motile sperm was similar in young and old rats. In contrast, the percentage of motile spermatozoa was significantly decreased in cauda epididymidis of old rats; spermatozoa from the regressed testis side had altered motility characteristics. Furthermore, in the cauda epididymidis on the regressed testis side of aged Brown Norway rats, the proportion of spermatozoa that retained their cytoplasmic droplet was markedly elevated. Some of these effects are likely due to changes taking place in spermatozoa during the process of spermatogenesis in the testis (eg, formation of the flagellum), whereas others could occur during sperm maturation in the epididymis (eg, acquisition of motility). The multiple effects of aging on sperm morphology, the acquisition of motility, and the shedding of the cytoplasmic droplet clearly indicate that the quality of spermatozoa is affected by aging.     

荧光定量聚合酶链反应检测去颌下腺大鼠睾丸Bcl 2和Bax mRNA的改变

目的观察切除颌下腺对大鼠睾丸Bax和Bcl 2 mRNA表达的影响。方法切除大鼠颌下腺,分别于术后14、28和42 d处死大鼠,提取睾丸总RNA,反转录,设计特异性引物和Taqman探针,荧光定量聚合酶链反应(PCR)检测Bax和Bcl 2 mRNA的改变。结果随着颌下腺切除时间的延长,实验组大鼠睾丸Bax和Bcl 2 mRNA的比值显著升高,与对照组比较,有显著性差异(P<0.01)。结论颌下腺切除大鼠睾丸Bax和Bcl 2 mRNA比值明显升高,提示颌下腺切除所致的大鼠睾丸生精细胞的凋亡可能是由Bax和Bcl 2介导的线粒体途径进行,但Fas/FasL是否参与该凋亡过程还有待进一步研究。     

Changes in the Acetylcholinesterase Activity of Mammalian Spermatozoa during Maturation

Acetylcholinesterase (AChE) activity of sperm recovered from the testes and several epididymal sites was studied in the boar, bull, and rat. AChE was highest in the bull spermatozoa followed by those of the rat and the boar. Between the testis and caput epididymis washed spermatozoa lost about 86% (bull), 60% (boar) or 32% (rat) in AChE activity while between the caput and cauda epididymides, a further loss of 28, 10, and 27% in the enzyme activity occurred in the respective species. Sperm AChE activity was negatively related to the development of sperm motility during sperm maturation and with sperm abnormality.     

Attractin蛋白和mRNA在不同日龄大鼠睾丸中的表达

目的:已知成熟大鼠睾丸组织中有Attractin蛋白的广泛表达,本文旨在进一步探讨Attractin蛋白和AttractinmRNA在各年龄段大鼠睾丸组织中的分布。方法:取出生第1d(新生鼠)、第5d(青春前期)、第20d(青春中期)、第50d(青春后期)及第70d(成年期)大鼠睾丸和附睾组织固定,采用酶免疫组织化学和原位杂交方法检测Attractin和AttractinmRNA在大鼠睾丸组织中的表达。结果:免疫组化显示各年龄段大鼠睾丸生精小管和间质细胞、管周肌样细胞上均有Attractin蛋白的表达,分布于胞膜和胞质,精子和附睾上未见表达。随着日龄的增加,间质细胞的表达逐渐增强。原位杂交显示各年龄段大鼠睾丸生精小管和间质细胞、管周肌样细胞、支持细胞上也均有AttractinmRNA阳性棕色颗粒杂交信号,主要表达于胞核及胞质。且随着日龄的增加,睾丸生精细胞表达略强于间质细胞。精子和附睾上未见表达。结论:AttractinmRNA和Attractin蛋白在各日龄大鼠睾丸组织中均有广泛的分布,且分布一致。提示各日龄大鼠睾丸组织都具有合成Attractin蛋白的能力。其生理功能和机制尚有待进一步探讨。     

Regulation of Sertoli cell myotubularin (rMTM) expression by germ cells in vitro

Recent studies have shown that rat myotubularin (rMTM), the homolog of human myotubularin, which is a putative protein tyrosine phosphatase (PTP), is expressed by Sertoli cells in the rat testis. In addition, a significant increase in its steady-state mRNA level was detected in Sertoli cells at the time of inter-Sertoli tight junction (TJ) assembly in vitro. Since the interplay of protein kinases and phosphatases that determines the intracellular phosphoprotein content can, in turn, regulate the assembly and maintenance of TJ and anchoring junctions (AJ) in vitro, as demonstrated in different cell types, such as Madin-Darby canine kidney (MDCK) cells, endothelial cells, and Sertoli cells, rMTM may be an important molecule in regulating the assembly and maintenance of inter-Sertoli TJs during spermatogenesis. We thus sought to characterize its regulation. During testicular maturation, it was shown that the rMTM steady-state mRNA level increased drastically with aging. The expression of rMTM increased by as much as 2-4-fold in the rat testis at 45-60 days of age versus 20 days of age, coinciding with the onset of spermiation. This result seemingly suggests that rMTM may participate in the release of spermatids by disassembling the Sertoli-spermatid AJs, since PTP inhibitor was shown to perturb the inter-Sertoli TJ permeability barrier in vitro. Unexpectedly, when Sertoli cells were isolated from 20-, 45-, and 90-day-old rats and the steady-state rMTM level was quantified, it was shown that there is a drastic reduction in rMTM expression in adult Sertoli cells. Studies that used Sertoli-germ cell cocultures and Sertoli cells incubated with increasing germ cell-derived proteins have shown that the high level of testicular rMTM expression in the testis might be maintained by germ cells. Although work remains to be done to delineate the role of rMTM in the testis, these results illustrate that germ cells play a very active role in regulation testicular function by altering the phosphoprotein content.     

外环小切口睾丸提出显微精索静脉结扎术

目的 :探讨外环小切口显微精索静脉结扎术的疗效。　方法 :4 8例精索静脉曲张病人 ,采用外环小切口睾丸提出显微精索静脉结扎术。　结果 :6个月后 ,精液检查各项指标改善者 30例 ,6例不育者配偶已受孕。全部病例无复发 ,无睾丸萎缩及鞘膜积液。　结论 :该术式有并发症少、复发率低及近期效果显著等优点     

Acidification of epididymal fluid in the boar

The present study describes the measurement of pH made in vivo in the rete testis fluid and in different regions of the boar epididymis. Furthermore, samples of whole ejaculates, semen fractions, testicular (ductuli efferents/rete testis), epididymal and deferential fluids collected from the same fertile boars, were analysed for their acid/base status with an automatic blood gas analyser. A pH gradient of activity was found between the fluid entering the ductus epididymis (pH 7.2) and the region of sperm storage at the cauda (pH 6.5). A significantly lower concentration of bicarbonate ion was found in the cauda epididymidis (3-4 mM) compared to rete testis fluid (30 mM), which might be related to the quiescence of the spermatozoa. A significant increase in extracellular pH and bicarbonate concentration occurred at ejaculation, the bicarbonate levels being 9-10-fold higher in the semen fraction rich in seminal vesicle fluid, where sperm showed higher motility, than in the cauda epididymis.     

Suppression of androgen receptor gene expression by testosterone undecanoate in rats

Long--termtestosteroneisanidealmalecontraceptive,whichdoesnotneedtosupplyandrogentomaintainlibidoandsexfunctionasLHRHanalogueorprogestogen.WHO(1990)organizedamultiplecenterclinicaltrial.Thetrialrecuited217healthyfertilemen,whowereinjected200mgtestosteroneenanthate(TE)weekly.After12monthsexposure,azoospermiawasinducedin157men.Therewasoneofpregnancyinl,486effectivemonths(0.8/100womenyear)[IJ.Testosteroneundecanoate(TU)isalong--termtestosterone,whichcanmaintainabout60dayswithonedose.Itisapr…     

Activation of TLR signalling regulates microwave radiation‐mediated impairment of spermatogenesis in rat testis

Microwave radiation could increase the expression of pro‐inflammatory cytokines in rat Sertoli cells, which may impair spermatogenesis. However, the mechanisms that microwave radiation induces the cytokine expression in Sertoli cells remain to be clarified. The activation of TLRs by their ligands can trigger a common signalling pathway to upregulate inflammatory cytokines such as IL‐1, IL‐6, IL‐12 and TNF‐α. Microwave radiation can increase the expression of TLRs in lymphocytes. The purpose of this study was to determine the effect of microwave radiation on the TLRs in rat testis. We focus on the effect of TLR2‐5 (which is expressed relatively highly) by microwave radiation. The results showed that the expression of TLR2‐5 and the pro‐inflammatory cytokines (IL‐1β, IL‐6 and TNF‐α) was increased both in mRNA and in protein. Furthermore, p‐p38, p‐ERK1/2, p‐JNK and p‐NF‐κB p65, the key factors of TLR signalling, were also elevated by microwave exposure. And the NF‐κB can be induced more dominantly. These results suggest that TLRs signalling can be activated by microwave radiation in testis, which may provide the molecular basis for the in‐depth study.     

电磁辐射对大鼠睾丸P450scc mRNA表达及睾酮合成的影响和防护

目的:探讨电磁辐射对大鼠睾丸合成睾酮(T)的影响及其机制,评价铜丝网的防护效果。方法:采用Wistar雄性大鼠,随机分为对照组(n=60)、无屏蔽电磁辐照组(n=60)和铜丝网全身屏蔽辐射组(n=60),电磁辐照后分别取3、6、24、72h各时相点各组大鼠15只;采用放射免疫法(RIA)分别测定电磁辐照后3、6、24、72h及对照组血清T含量,反转录聚合酶链反应(RTPCR)测定各组睾丸组织中细胞色素P450胆固醇侧链裂解酶(P450scc)mRNA水平。结果:无屏蔽辐照组辐照后3h血清T含量和P450sccmRNA水平均明显低于对照组(分别较对照组降低83.9%,56.9%;P均<0.01),6h略有回升,但仍明显低于对照组(分别较对照组降低54.8%,27.3%;P均<0.01),24h恢复到正常水平,72h再次出现明显降低(分别较对照组降低60.1%,56.1%;P均<0.01);采用铜丝网全身屏蔽后,血清T和睾丸组织P450sccmRNA表达均未见明显变化。结论:电磁辐射能在转录水平影响睾丸间质细胞P450sccmRNA的表达,从而降低T的合成;铜丝网屏蔽具有较好的防护效果。     

Developmental expression of c-kit protooncogene in the rat testis

IntroductionInmammalianembryos,thegermcelllinebeginswithprimordialgermcelIs,andinmales,primordialgermcellsbecomegono-cytes,thece1lprecursorsofthespermatogoni-a.ThedevelopmentofanormaltestisdependsupontheproliferationofprimordialgermcellsandtheiraggregationwithSertolicellprecur-sorsLl:.Theseeventshavebeenshowntobeassociatedwiththeexpressionofthec-kitProtooncogenemappedtothe"whitespottinglocus(W)~ofthemouse[2,3j.Thec-kitPro-tooncogeneencodesatransmembranetyrosinekinasereceptor(4,5).Thestructu…     

Effects of oral zinc administration on long‐term ipsilateral and contralateral testes damage after experimental testis ischaemia–reperfusion

The purpose of this study was to examine potential long‐term post‐torsion changes that can occur in the histopathology, biochemistry and spermatogenesis of both torsioned and nontorsioned opposite testes. The study also determines the effect of zinc (Zn) administration on the testicular torsion/detorsion (T/D) damage on both testes. Forty‐eight male rats, divided equally into eight groups: (SHAM), (SHAM+,Zn+), (T/D+, Zn? 1 month), (T/D+,Zn? 2 months), (T/D+,Zn? 3 months), (T/D+,Zn+ 1 months), (T/D+,Zn+ 2 months), (T/D+,Zn+ 3 months), have been used. Drug administration was carried out by adding 100 μg (0.016 ml/rat) Zn per rat to drinking water in related groups. Testicular damage decreased superoxide dismutase (SOD) and glutathione (GSH) and increased malondialdehyde (MDA) in the testis tissues of rats, while Zn administration increased SOD and GSH and decreased MDA in the testis tissues in comparison with the SHAM group. The beneficial effect of zinc sulphate was more evident on the nonrotated testis than the rotated testis. In the histopathological study, a significant decrease in torsion and detorsion injuries was observed in the treatment groups compared to the torsion and detorsion groups. We found a protective effect of zinc sulphate on oxidative stress as a result of T/D injuries in rats, especially for the nonrotated testis; results were supported histopathologically.     

小鼠单侧睾丸损伤后环孢素A对Fas系统的影响

目的 :研究昆明小鼠 (KM小鼠 )单侧注射冰乙酸致睾丸损伤后环孢素A(CsA)对对侧睾丸生精功能和Fas系统表达的影响。　方法 :6 0只KM小鼠随机分为 4组 :A组为对照组 ,B组为单侧睾丸损伤组 ,C组为单侧睾丸损伤后 6h切除损伤睾丸组 ,D组为单侧睾丸损伤后 6h内开始腹腔注射CsA组。 4周后取对侧附睾尾 ,计数精子及其活率 ,对侧睾丸作石蜡切片苏木精 伊红染色和免疫组化链霉素抗生物素蛋白过氧化物酶连结 (SP)法检测Fas和FasL的表达。　结果 :D组附睾尾精子和活率计数显著高于B组 (P <0 .0 5 ) ,D组的FasL和Fas较B组显著降低 (2 4 .3± 7.0vs37.8± 5 .8和 17.8± 4 .3vs32 .4± 3.6 ,P <0 .0 5 )。　结论 :KM小鼠单侧睾丸损伤后CsA可以通过抑制Fas和FasL的表达 ,降低生精细胞凋亡 ,维持生精功能的稳定