SPAG4L在人类睾丸中的表达研究

目的:观察人类睾丸新基因SPAG4L在人类不同发育阶段睾丸及隐睾中的表达,为了解该基因在精子发生中的功能奠定基础;方法:收集流产胎儿、成年人、老年人及隐睾患者的睾丸组织,应用RT-PCR和组织原位杂交技术检测SPAG4L mRNA的表达;结果:RT-PCR和组织原位杂交技术检测结果发现,SPAG4L在胎儿睾丸中几乎检测不到,在成年及老年男性睾丸中均有高表达,主要在精母细胞和圆形精子细胞中表达;在隐睾患者的睾丸中,精母细胞大量凋亡,SPAG4L表达明显下调;结论:SPAG4L主要在精子发生减数分裂阶段表达,受生长发育调控,提示该基因可能在精子发生减数分裂阶段发挥着重要的生理功能.     

隐睾小鼠与正常成年小鼠睾丸蛋白表达谱的差异分析

为使精原干细胞（spermatogonial stem cells,SSCs）在体外大量扩增,需要阐明SSCs自增殖机制。为筛选SSCs自增殖相关因子,探索SSCs自增殖机制,本研究选取10日龄昆明乳鼠行隐睾手术,术后35d分别取小鼠两侧睾丸。组织学分析结果显示,实验性隐睾中生殖细胞的分化停滞在精母细胞阶段,且只有少量的精母细胞出现,精原细胞的比例高于正常成年雄性小鼠（45日龄）。应用双向凝胶电泳分析隐睾小鼠与正常成年小鼠睾丸差异表达蛋白。结果显示,与正常成年小鼠相比,隐睾小鼠睾丸中有9种蛋白表达发生了显著变化,其中6种蛋白表达下调,3种上调。对9种差异表达蛋白点胶内酶切后进行质谱分析,其中4种蛋白分别鉴定为磷脂酰乙醇胺结合蛋白1（phosphatidylethanolamine-binding protein1,PEBP1）,HES—related basic helix-100p-helix protein（HERP）,Stathmin蛋白和一种未命名蛋白。本研究通过制作有效的隐睾动物模型,运用蛋白组学的技术方法,成功筛选并鉴定了4种隐睾相关蛋白,有助于探讨SSCs自增殖及隐睾引起雄性不育的机制。     

出生后小鼠睾丸不同发育期TIMP-4的表达

金属蛋白酶组织抑制因子-4(TIMP-4)是TIMP家族的最新成员。已有研究表明,TIMP-4mRNA大量表达于成年小鼠的睾丸中。为了证实TIMP-4基因在出生后小鼠睾丸中的表达是否具有发育依赖性,本实验利用RT-PCR、Western blotting和间接免疫荧光染色三种方法,分别检测了TIMP-4mRNA和蛋白在出生后小鼠睾丸不同发育期中的时空表达方式。RT-PCR和Western blotting结果分别显示,TIMP-4mRNA和蛋白均只在成年小鼠睾丸中表达,而在出生后的其它各阶段都不表达;间接免疫荧光染色进一步证实TIMP-4蛋白只定位在成年小鼠睾丸的Leydig细胞中。结果提示,TIMP-4在出生后小鼠睾丸中的表达具有显著的发育依赖性.     

丝氨酸/精氨酸蛋白特异激酶SRPK2在小鼠睾丸组织中的表达及意义

目的通过研究丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因mRNA及其编码蛋白产物在小鼠睾丸组织中的表达特征,探讨该基因在精子发生过程中的作用及意义。方法分别采用半定量逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹杂交(Western blotting)分析该基因mRNA及蛋白产物在小鼠多种组织中的表达;利用实时定量PCR(real-time quantitative PCR)分析SRPK2 mRNA在不同发育阶段小鼠睾丸组织中的差异表达;应用免疫组织化学染色和间接免疫荧光技术观察SRPK2蛋白在小鼠曲精小管中的细胞定位和生精细胞内的亚细胞定位。结果半定量RT-PCR和Western blotting分析显示SRPK2 mRNA和蛋白在小鼠睾丸组织中均大量表达;实时定量PCR分析发现SRPK2 mRNA在5周及8周龄雄性小鼠睾丸组织中显著表达,具有明显的阶段特异性表达特征。免疫组织化学染色结果表明SRPK2蛋白阳性着色主要位于曲精小管中的长形精子细胞核;间接免疫荧光分析显示SRPK2蛋白定位于长形精子细胞核表面。结论 SRPK2基因在小鼠睾丸组织中大量表达,并且具有显著的阶段特异性表达特征和明确的细胞核定位,极有可能在小鼠精子发生的变态成形期参与mRNA前体分子的剪接过程,其作用机制值得进一步深入研究。     

人、鼠原肠形成蛋白基因的克隆和初步分析

目的：分析原肠形成蛋白（TSG）在几个发育阶段的人和小鼠睾丸中的差异表达。方法：应用cDNA微阵列技术对成人和胚胎睾丸进行基因表达图谱分析,获得在成人高表达、胚胎低表达的人TSG的全长基因;利用RT-PCR,从4周龄小鼠睾丸中克隆出小鼠TSG基因,用地高辛标记的探针进行原位杂交,检测小鼠TSG在睾丸中的表达。结果：人TSG在成人睾丸中高表达,在胚胎中低表达;小鼠TSG仅在小鼠睾丸生殖细胞中表达,在间质细胞中不表达。结论：小鼠睾丸TSG与骨形态发生蛋白（BMP）可能同步表达,提示在小鼠精子发生中也可能存在一条由TSG信号激活BMP的传导途径。     

ADAM19 在小鼠睾丸发育中的时空表达

目的 阐明含有去整合素和金属蛋白酶结构域的跨膜蛋白19(ADAM19)在小鼠睾丸发育中的作用.方法 采用半定量RT-PCR和免疫组化两种实验方法,分别检测ADAM19 mRNA和蛋白质在小鼠睾丸发育中的时空表达.结果 ①最早在胚胎发育的15.5 d才能检测到ADAM19 mRNA的表达,后其表达随着胚胎发育天数的增加而逐渐升高,到围产期表达水平达到最高.出生后,ADAM19 mRNA的表达呈现显著下降的趋势,到成体睾丸中就几乎检测不到ADAM19的表达.②和其mRNA表达变化趋势一样,ADAM19蛋白也是首次在胚胎发育的15.5 d被检测到,一直持续存在到出生后一周,一周后则几乎检测不到;阳性表达信号主要定位在睾丸的曲细精管(睾索)中.结论 ADAM19 在小鼠睾丸中的表达具有显著的发育依赖性.     

神经生长因子在不同周龄小鼠睾丸组织中的表达

目的研究神经生长因子在小鼠不同周龄睾丸组织中的定量和定位表达。方法分别剖取不同周龄雄性小鼠的睾丸组织,部分提取总RNA,real-time PCR相对定量分析神经生长因子mRNA的表达量;另外部分组织固定、包埋,进行SABC法免疫组化分析,以观察神经生长因子蛋白在各周睾丸组织中的定位。结果Real-timePCR定量分析表明:小鼠生后1周龄睾丸组织有神经生长因子mRNA的表达,生后3周龄表达量达峰值,5周之后随鼠龄的增加呈下降趋势,成年小鼠睾丸组织的神经生长因子mRNA表达维持在一定水平。免疫组化定位分析显示:睾丸组织的神经生长因子蛋白表达于小鼠出生后的各个时期内,1周龄睾丸组织免疫阳性反应主要位于支持细胞,精原细胞也有着色;3周龄睾丸组织的间质细胞、各级生精细胞、支持细胞、管周肌样细胞表达均呈现阳性;5周后的睾丸组织内神经生长因子呈低水平表达,主要表达于间质细胞和生精细胞内。结论神经生长因子mRNA的表达量随着小鼠睾丸的生长发育期存在着一定的规律性变化;神经生长因子蛋白的表达在小鼠睾丸生长发育的不同时期其主要表达部位不同。     

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。     

小鼠睾丸生精细胞凋亡相关基因SRG2的分子克隆及其在隐睾中的表达特征

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段(BE644542)入手,利用网上生物信息学克隆了SRG2基因全长,GenBank登录号为AF395083。从小鼠睾丸cDNA文库中分离出该基因完整阅读框cDNA,SRG2基因的cDNA全长为1088bp,为编码295个氨基酸、分子量为33579kD、等电点为9．64的蛋白质,与人类同源基因TSARG2相似性为78％,而与其他已知蛋白质无明显同源性。RT-PCR结果表明：该基因只在睾丸中有高表达。应用新型的分子信标检测该基因在不同时期隐睾中的mRNA表达水平,发现该基因呈明显上调,证明该基因在隐睾的发生发展中具重要作用。     

小鼠生精相关基因SRG4的cDNA克隆及在小鼠各发育阶段的表达

从大鼠精子尾部基因Spag4出发,在dbEST数据库中同源搜寻与大鼠Spag4基因编码氨基酸同源性较高的表达序列标签(Expressed Sequence Tag,EST),找到两个在小鼠精母细胞中表达的ESTs,BG101130和BG100990。通过电子杂交得到1155bp的片段,包含了小鼠假想基因AK006225的全部序列,定位于2H1-H2,其开放阅读框为87～1133bp,并被RT-PCR所证实,将该基因命名为小鼠生精相关基因4(Sperrnatogenesis Related Gene4,SRG4,GenBank登录号为AY307077)。SRG4基因推定编码348个氨基酸,有一个coiled-coil区,可能是一个跨膜蛋白。该基因与人类同源基因TSARG4同源性为74％(277／374),与大鼠Spag4同源性为45％(103／224)。多组织RT-PCR和Northern blot结果显示,SRG4在睾丸中特异性表达。RT-PCR结果发现,小鼠出生后2周内,SRG4表达量极低;3周开始时SRG4大量表达,到4∽5周时表达量最高。该结果提示。SRG4基因可能在小鼠精子形成过程中发挥着重要的作用。     

Germ cell removal after induction of cryptorchidism in adult rats

Cryptorchidism is associated with male infertility due to germ cell loss in response to elevated temperature. However, there is a great deal of contradictory information prevalent on the status of germ cells and their process of removal in the cryptorchid testis. In the present study, we investigate the cell removal from cryptorchid rat testis by the methods of morphology and stereology. The testis weight is reduced according to previous reports after surgical induction of cryptorchidism. Interestingly, the epididymal weight is significantly increased in 7 days after surgery, and the caput epididymis tubules show filling with countless round germ cells. We found that the elongating spermatids (steps 10-13), newborn spermatids (step 1) and the dividing spermatocytes are the most susceptible cells to elevated temperature, and are the first disappeared cells from the seminiferous tubules after surgery. Germ cell removal followed the order, starting first with elongating spermatids and newborn spermatids, followed by round spermatids and elongated spermatids and later extending to spermatocytes.     

Efficiency of the process of meiosis in scrotal testes of healthy boars and unilateral abdominal cryptorchid boars.

Unilateral abdominal cryptorchidism has usually been correlated with abnormalities in the spermatogenic activity of the scrotal testis. The present study describes the effects of unilateral abdominal cryptorchidism on the meiotic process in scrotal testes from postpubertal boars. The percentage of primary spermatocytes, secondary spermatocytes, and round spermatids was evaluated in testicular smears from scrotal testes of healthy boars and of right-sided unilateral abdominal cryptorchid boars. As compared to the scrotal testes of healthy boars, the scrotal testes of unilateral abdominal cryptorchid boars showed low transformation from primary to secondary spermatocytes (meiosis I), but normal transformation from secondary spermatocytes to round spermatids (meiosis II). The data obtained indicate that spontaneous unilateral abdominal cryptorchidism on the right side induced partial arrest of spermatogenesis at the primary spermatocyte stage that was attributed to anomalies in Sertoli-cell activity. Abnormal paracrine signals from altered Sertoli cells could have resulted in either disturbed mitosis, which led to the formation of spermatocytes with an abnormal DNA content, or abnormalities in the metabolic activity and the organization of the cytoskeleton of primary spermatocytes.     

Expression of Hsf1, Hsf2, and Phlda1 in cells undergoing cryptorchid-induced apoptosis in rat testes

Surgery‐induced cryptorchidism, in which the testes are prevented from descending into the scrotal sac, results in testicular germ cell death, and it is commonly used as an experimental tool in the study of spermatogenesis. However, the molecular events underlying the activation of germ cell death remain poorly understood. In the present study, we investigate selective cell loss from cryptorchid rat testis by using DNA flow cytometry and by determining protein and mRNA expression of Hsf1, Hsf2, and Phlda1. The hypo‐haploid cell fraction is significantly decreased as early as 3 days after surgical induction of cryptorchidism (from 42.01 ± 5.74% to 15.98 ± 3.88%), followed by a significant decrease in the haploid cell fraction at Day 7. At the latter time point, an apoptotic peak of spermatocytes appears in DNA histograms just before the tetraploid peak; the percentage of aneuploid cells between diploid and tetraploid rises as high as 14.05 ± 2.98% of the total cells in 7‐day cryptorchid testis, suggesting that a large number of spermatocytes are undergoing apoptosis. The expression of Phlda1 mRNA is significantly elevated 3 days after induction of cryptorchidism. After 7 days of cryptorchidism, Hsf1 and Phlda1 are strongly expressed in the nucleus and cytoplasm, respectively, of primary spermatocytes. Numerous apoptotic spermatocytes are also observed at this time point. These results suggest that the Hsf1/Phlda1 pathway plays an important role in the apoptosis of primary spermatocytes in cryptorchid testis. We present evidence suggesting that Hsf2 is also involved in germ cell removal in cryptorchid testis. Mol. Reprod. Dev. 78:283–291, 2011. © 2011 Wiley‐Liss, Inc.     

Improvement of spermatogenesis in adult cryptorchid rat testis by intratesticular infusion of lactate.

In order to test the hypothesis that a lack of energy could be a cause of germ cell death at high temperatures, cryptorchid rats testes were infused with lactate, delivered by osmotic pumps over 3-15 days. In cryptorchid testes, the spermatids and spermatocytes were lost between 3 and 8 days. In cryptorchid testes supplemented with lactate, elongated spermatids persisted in a few seminiferous tubules at Day 15. Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. The loss of spermatocytes increased after 8 days, and 30% of seminiferous tubules still contained meiotic or meiotic plus spermiogenetic cells at Day 15. After 8 days, the chromatin of step 8 round spermatids was abnormal and nuclear elongation did not commence. The Sertoli cell cytoplasm that was retracted toward the basal compartment of the seminiferous epithelium could not hold the germ cells of the adluminal compartment. Therefore, attachment of germ cells to Sertoli cells and the supply of lactate seem necessary for the development of germ cells at high temperatures. The improvement in spermatogenesis in cryptorchid supplemented testes for several days is a new finding.     

Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis

Spermatogenesis is a complex process. Duringspermatogenesis, the production of sperm occurs withinthe testicular seminiferous tubules through three separatedphases. First of all, diploid germ cells, primitivespermatogonia, will self renew to amplify and producetypes A and B spermatogonia. Type B spermatogonia willdifferentiate into primary spermatocytes. Then, meioticdivisions of spermatocytes will produce round spermatids.Finally, after a series of biochemical and morphologicalchanges, sper…     

Effect of the W mutation, for white belly spot, on testicular germ cell differentiation in mice.

The effect of the mutation for white belly spot controlled by the dominant gene W on spermatogenesis in mice was examined by experimental cryptorchidism and its surgical reversal. The course of spermatogenesis from spermatogonia to spermatid was normal in intact testes of W/+ mice. In cryptorchid testes, there was no difference in the number and activity of Type A spermatogonia between the testes of W/+ and +/+ mice, in mitotic and labelling indices. Although surgical reversal of the cryptorchid testis resulted in regenerative differentiation of germ cells in both genotypes, the recovery of cell differentiation in the W/+ testis was slower than in the +/+ testis. There were fewer germ cells, such as intermediate-Type B spermatogonia or more advanced ones, in W/+ testes. On Day 17 after surgical reversal, cell associations in W/+ testes were abnormal and the numbers of intermediate-Type B spermatogonia, spermatocytes and spermatids were approximately 70, 50 and 15%, respectively, of those in +/+ testes. These results indicate that the W gene affects spermatogenic cell differentiation in adult mice.     

Heat-shock resistance in experimental cryptorchid testis of mice

Cryptorchidism is commonly used for research on spermatogenesis. However, there are few comparative investigations about the strain differences in mice, especially in long-term experiments. In the present study, the authors demonstrate its specific dynamics in the MRL/MpJ mouse strain, and discuss the cause of strain differences. In the mouse strains A/J BALB/c, C3H/He, and C57BL/6, after 2 weeks of experimental cryptorchidism, the ratios of the cryptorchid testis weight against the intact one were 0.38+/-0.05, 0.43+/-0.05, 0.38+/- 0.02, and 0.44+/-0.14, respectively. On the other hand, in the MRL/MpJ strain it was shifted to 0.69+/-0.08. The details of this strain difference were compared by calculation of germ cells with the Sertoli cell index at 2 weeks after operation. The indices of spermatogonia in all strains were not significantly different; however, in MRL/MpJ mice remarkable numbers of late spermatocytes and round spermatids were detected. The decrease of the testis weight ratio was similar until 10 days in the C57BL/6 and MRL/MpJ strains, but continued in C57BL/6 until 21 days, whereas in MRL/MpJ mice it plateaued after 10 days. Northern blot analysis for heat shock protein 70-2 using total RNA prepared from the cryptorchid and intact testes at 2 weeks after operation revealed that the expression was decreased in the cryptorchid testis of C57BL/6, but not MRL/MpJ mice. The results suggested that heat-resistant germ cells were present in MRL/MpJ, originating possibly from the genetic background.