SRG4在出生后小鼠睾丸及隐睾中的表达特性

研究小鼠生精新基因SRG4在出生后小鼠睾丸及手术隐睾中的表达特性, 为了解SRG4在精子发生中的作用奠定基础。取出生后1, 3, 12 w小鼠睾丸进行免疫组化检测, 观察SRG4蛋白在出生后小鼠不同发育阶段睾丸中的表达; 制备单侧手术隐睾模型, 取术后0~18 d 的隐睾组织进行半定量RT-PCR检测, 观察SRG4 mRNA在隐睾病变过程中的表达变化, 并对隐睾术后18 d 睾丸进行组织原位杂交分析。免疫组化分析结果表明, SRG4蛋白在出生1 w的小鼠睾丸中几乎检测不到, 在出生3 w的小鼠睾丸中有明显表达, 在出生12 w的小鼠中大量表达, 主要分布在精母细胞和圆形精子细胞胞浆及胞膜, 呈不均匀分布。半定量RT-PCR结果发现, SRG4 mRNA在小鼠隐睾术后0~6 d表达没有明显下调, 9 d 开始表达下调, 第18 d表达最低。组织原位杂交结果表明, 术后18 d隐睾睾丸生殖细胞大量凋亡, 精曲小管中仅见到个别的SRG4阳性信号, 而对照则不受影响。上述结果说明, SRG4蛋白表达受小鼠生长发育调控; 隐睾模型中, 随着生殖细胞的大量凋亡, SRG4基因表达下调, 提示SRG4基因可作为一个精子发生特定阶段的分子标记用以研究精子发生过程。     

Uchl1及其关联蛋白参与小鼠实验性隐睾中精母细胞凋亡(英文)

以往研究显示Uchl1参与调节生理状况下小鼠精母细胞的凋亡。本文以小鼠实验性单侧隐睾为研究模型,以切除单侧睾丸和假手术作为对照,用苏木精-伊红(HE)染色和DNA末端标记(TUNEL)观察生精细胞的形态和凋亡情况;用免疫组化分析Uchl1及其相关蛋白Jab1和p27kip1在隐睾症的热应激反应导致精母细胞损失过程中的变化情况,并用亲和分析(pull-down)和免疫荧光共定位检测三种蛋白在精母细胞的关联性。结果显示,Jab1和p27kip1,与Uch1平行,在具凋亡形态的精母细胞中响应热应激而含量增加,而在多核巨细胞中无类似变化。Jab1可以与睾丸蛋白提取物中Uchl1结合,并与Uchl1和p27kip1特异性地在具凋亡形态的精母细胞中共定位。以上结果提示,Uchl1蛋白的积累参与隐睾中热应激诱导的生精细胞凋亡过程,但不影响接下来的多核巨细胞的形成,且Uchl1蛋白的作用机制涉及Jab1和p27kip1参与的一种新途径。     

多肽∶N-寡糖酶在小鼠人工隐睾中表达下调

真核多肽∶N-寡糖酶（peptide∶N-glycanase或PNGase）可切除错误折叠糖蛋白上的N-寡糖链,并可与内质网关联降解（endoplasmic reticulum-associated degradation, ERAD）途径中的多种关键成分相结合.然而,对于PNGase的生理功能及其与疾病的关系尚无明确报道.本研究利用重组技术表达和纯化了包含人PNGase N末端片段的融合蛋白,并经融合蛋白免疫与亲和层析纯化家兔抗血清,制备了PNGase的特异性抗体.利用该抗体和Western 印迹技术研究了PNGase在小鼠组织中的表达.结果显示PNGase在7种小鼠组织（脑、心、肺、肝、脾、肾、睾丸）中均有不同程度的表达,其中表达量最高者为睾丸;PNGase表达水平在不同品系小鼠（C57BL/6N、BALB/cAnN和昆明小鼠）间有显著差异.在小鼠单侧隐睾模型中首次观察到,与对照侧阴囊内的正常睾丸相比,隐睾内PNGase含量明显下降,提示PNGase在睾丸生精过程中可能有重要作用.     

小鼠睾丸生精细胞凋亡相关基因mTSARG3的克隆

从在小鼠隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 4 4 5 37)出发 ,利用GeneScan软件分析该片段所在染色体基因组序列 ,获得一个包含该EST的新基因序列。设计该基因特异性引物从小鼠睾丸cDNA文库中进行PCR扩增 ,分离出小鼠睾丸生精细胞凋亡相关基因mTSARG3(GenBank登录号为AF4 192 92 )。该基因定位于小鼠 7号染色体 7E1 E2区带 ,全长为 11kb ,cDNA全长为 132 8bp ,包含 8个外显子 ,编码由 316个氨基酸组成的、分子量为 36kD的蛋白质。该蛋白质含有DnaJ区和DnaJ- c区 ,与热激蛋白 4 0家族多种蛋白质有较高相似性 ,其中与小鼠DJB4 - MOUSE在 336aa的范围内有 4 6 %的相似性 ,属热激蛋白 4 0家族新成员。多组织RT PCR和Northern印迹结果显示 ,该基因在小鼠睾丸组织高表达 ,转录本大小约为 1.35kb ;Southern杂交结果显示 ,该基因在小鼠正常睾丸和隐睾组织无缺失和重排。实验结果证明成功克隆到了一个小鼠睾丸生精细胞凋亡相关基因mT SARG3。     

Ghrelin在正常及X线辐射损伤小鼠睾丸中的表达

目的探讨ghrelin在正常及X射线辐射损伤后小鼠睾丸中的表达变化及其意义。方法采用辐射剂量1·0GyX线对成年小鼠进行全身照射,于照射后16h取睾丸组织制备标本,免疫组织化学ABC法检测ghrelin在辐射损伤小鼠及正常对照组小鼠睾丸中的表达,探讨ghrelin表达的变化及意义。结果正常小鼠睾丸ghrelin主要表达在间质细胞的胞浆中,呈强阳性。X线照射后16h,ghrelin的表达主要集中于生精小管内各级生殖细胞中,特别是在精原细胞和初级精母细胞中有强阳性反应,且其表达具有生精周期特异性,而间质细胞中呈阴性。结论Ghrelin在小鼠正常及辐射损伤睾丸中的特异性表达和变化,提示其可能与睾丸精子发生、精子形成及辐射损伤修复有关,具有重要的睾丸生物学调节功能。     

多肽∶N-寡糖酶在小鼠人工隐睾中表达下调

真核多肽∶N-寡糖酶(peptide∶N-glycanase或PNGase)可切除错误折叠糖蛋白上的N-寡糖链,并可与内质网关联降解(endoplasmic reticulum-associated degradation,ERAD)途径中的多种关键成分相结合.然而,对于PNGase的生理功能及其与疾病的关系尚无明确报道.本研究利用重组技术表达和纯化了包含人PNGase N末端片段的融合蛋白,并经融合蛋白免疫与亲和层析纯化家兔抗血清,制备了PNGase的特异性抗体.利用该抗体和Western印迹技术研究了PNGase在小鼠组织中的表达.结果显示PNGase在7种小鼠组织(脑、心、肺、肝、脾、肾、睾丸)中均有不同程度的表达,其中表达量最高者为睾丸;PNGase表达水平在不同品系小鼠(C57BL/6N、BALB/cAnN和昆明小鼠)间有显著差异.在小鼠单侧隐睾模型中首次观察到,与对照侧阴囊内的正常睾丸相比,隐睾内PNGase含量明显下降,提示PNGase在睾丸生精过程中可能有重要作用.     

GST pull-down联合质谱筛选（肌）营养不良短小蛋白结合蛋白1在小鼠睾丸组织中的相互作用蛋白质

（肌）营养不良短小蛋白结合蛋白1（dystrobrevin binding protein 1,dysbindin-1）是溶酶体相关细胞器生物发生复合体-1(biogenesis of lysosome related organelles complex 1, BLOC-1)的1个亚基,在多种组织细胞中广泛表达;然而,其在睾丸组织中的作用至今尚不明确。为寻找（肌）营养不良短小蛋白结合蛋白1在睾丸组织中的相互作用蛋白质,以进一步研究（肌）营养不良短小蛋白结合蛋白1在睾丸中的作用,本研究首先在Rosetta(DE3)菌种中表达可溶性GST-dysbindin-1融合蛋白,经谷胱甘肽 琼脂糖珠亲和纯化后,与小鼠的睾丸组织蛋白质孵育进行GST pull-down实验,并通过液相色谱串联质谱（LC MS/MS）分析筛选（肌）营养不良短小蛋白结合蛋白1在睾丸组织中的相互作用蛋白质。利用BioGPS数据库聚类在睾丸组织中高表达和特异性表达的互作蛋白质,运用DAVID6.8在线分析工具从细胞组分、分子功能、生物学过程和KEGG通路等方面对筛选出的互作蛋白质进行GO（gene ontology）富集分析。本实验共筛选出108个（肌）营养不良短小蛋白结合蛋白1在睾丸组织中的潜在互作蛋白质,其中98个为尚未报道的（肌）营养不良短小蛋白结合蛋白1相互作用蛋白质,7个为睾丸高表达蛋白质,5个为睾丸特异性表达的蛋白质。这些候选蛋白质主要分布在细胞质、细胞核、细胞膜、细胞外泌体等细胞组分中,通过与蛋白质、核酸等分子结合参与蛋白质翻译和转运、囊泡运输及凋亡等生物学过程以及氨基酸生物合成、溶酶体及蛋白酶体等生物学通路。我们推测,在睾丸组织中（肌）营养不良短小蛋白结合蛋白1可能通过与多种蛋白质相互作用参与精子的发生和受精等过程。     

以支持细胞为饲养层培养小鼠精原干细胞

为探索精原干细胞(Spermatogonialstemcells,SSCs)体外自增殖的条件以及SSCs体外快速扩增的方法,以6-8日龄昆明乳鼠为材料,分离小鼠睾丸细胞,采用Percoll梯度离心法富集SSCs;以经丝裂霉素C处理的Sertoli细胞作饲养层,以DMEM为基本培养基,加入5%胎牛血清和103u/ml的白血病抑制因子(Leukemiainhibitoryfactor,LIF),体外培养SSCs;运用免疫荧光技术,以SSCs特异性表面分子Thy1为标志,对原代培养20d和传代培养14d的细胞进行鉴定。该培养体系下,SSCs贴壁时间为6h-9h,48h后可见细胞分裂,迅速增殖出现在接种12d以后。接种后第20d形成数十至上百个细胞的细胞团,细胞总数比接种时增加了45-245倍,100倍显微镜下观察可见,单位视野内细胞团数为26±4个。传代后细胞增殖较快。原代培养20d和传代培养14d的细胞均为Thy1阳性;而传代20d后,细胞周缘不整,有伪足出现,呈现出死亡迹象。该培养条比较适合SSCs短期快速增殖。     

MPTP慢性帕金森病小鼠纹状体差异蛋白质的研究

目的分离、鉴定MPTP诱导慢性帕金森病模型小鼠纹状体差异表达的蛋白质,对MPTP慢性PD动物模型的特异性蛋白质组进行初步探讨,为PD的发病机制提供一定的蛋白质组学依据。方法成功建立MPTP诱导慢性帕金森病小鼠模型,提取模型组和对照组小鼠脑纹状体蛋白质,分别以固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向进行2-DE。图像分析软件PDQUEST8.0分析电泳图谱找出差异表达蛋白,运用MALDI-TOF MS质谱鉴定;其肽质量指纹图（PMF）经MS Fit检索。结果比较MPTP诱导慢性PD模型小鼠和正常对照小鼠纹状体二向电泳图,发现12个蛋白表达异常,最终鉴定出其中4个蛋白质：线粒体裂殖调节因子1（mitochondrial fission regulator 1）、类泛素样蛋白3前体（ubiquitin-like protein 3 precursor）表达下调;S100蛋白A10（proteinS100-A10）、Lin-7 homolog B为新出现点。结论初步鉴定出MPTP慢性PD模型小鼠纹状体部分差异表达蛋白,所发现4个表达异常的蛋白质与帕金森病线粒体的损伤和兴奋性神经毒性密切相关,与PD的发病机制相符,为深入研究帕金森病病理机制奠定了基础。     

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.     

Steel-Dickie (Sld) mutation affects both maintenance and differentiation of testicular germ cells in mice.

The effects of Steel-Dickie (Sld) mutations on testicular germ cell differentiation were investigated using experimental cryptorchidism and its surgical reversal in mutant, C57BL/6-Sld/+ and wild-type C57BL/6- +/+ mice. In Sld/+ cryptorchid testes the maintenance of undifferentiated type-A spermatogonia was impaired and their numbers decreased. In contrast, the proliferative activity of type-A spermatogonia in the cryptorchid testis of mutant mice appeared normal as judged by their progression through the cell cycle. Surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells in +/+ testes. However, the regenerative differentiation of type-A spermatogonia which remained in Sld/+ cryptorchid testes was strongly impaired, particularly at two steps of cellular differentiation, from type-A spermatogonia to intermediate or type-B spermatogonia and at meiotic division. Furthermore, in mutant mice, no significant recovery of testicular weight was observed after surgical reversal compared with +/+ mice.     

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.     

The effects of steel mutation on testicular germ cell differentiation

The effects of artificial cryptorchidism and its surgical reversal on spermatogenesis were examined in germ cell mutant, S1/+ and wild type, +/+, mice. In cryptorchid testes no difference was found between S1/+ and +/+ mice in the number of undifferentiated type A spermatogonia. The activity of type A spermatogonia in mutant mice appeared normal as judged by its mitotic cell number and DNA synthesis. The surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells in both types of mice, but the pattern of cellular differentiation in the mutant testes was completely different from that of the wild type testes. At two steps of cellular differentiation, intermediate or type B spermatogonia and spermatid, the numbers of cells were much smaller in the S1/+ testes than those in the +/+ testes. The steel gene was therefore suggested to exert its effects on the differentiation of type A spermatogonia to intermediate or type B spermatogonia, on meiotic division and/or the survival rate of these cells, but not on the undifferentiated type A spermatogonia or stem cells.     

The Fate of Spermatogonial Stem Cells in the Cryptorchid Testes of RXFP2 Deficient Mice

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool.     

Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.     

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.     

Ultrastructural study of the boar seminiferous epithelium: changes in cryptorchidism

The present study compares the ultrastructural features of Sertoli cells and germ cells between scrotal testes of healthy boars and abdominal testes of unilateral and bilateral cryptorchid boars. In healthy boars, spermatogonia are flat cells lying in close association with the basal lamina. As differentiation progresses, spermatogonia acquire an oval profile and lose their contact with the basal lamina. Spermatocytes are round cells moving from the basal compartment of the seminiferous epithelium to the luminal compartment. Spermatids exhibit complex morphological changes leading to the formation of spermatozoa. Sertoli cells extend from the basal lamina to the tubular lumen. The nucleus encloses fine euchromatin and one or two nucleoli; the nuclear envelope has a few deep infoldings. The lateral cell membranes form junctional specializations that constitute the blood-testis barrier. The cytoplasm encloses smooth endoplasmic reticulum, vesicles, aggregates, and scattered mitochondria. The seminiferous epithelium of abdominal testes from unilateral and bilateral cryptorchid boars contains few spermatogonia with an abnormal appearance; the alteration in germ cell number is more severe in the bilateral disease. In unilateral cryptorchid boars, spermatogonia appear as either large pyramidal cells or roundish cells; in bilateral cryptorchid boars, spermatogonia show roundish profiles and degenerative patterns. Abdominal testes of both unilateral and bilateral cryptorchid boars are constituted by immature Sertoli cells that show abnormal cytoplasmic content, defective development of the blood-testis barrier, and atypical nuclear appearance; in bilateral cryptorchid boars, immature Sertoli cells exhibit degenerative signs. At postpubertal age, unilateral and bilateral cryptorchidism induce total arrest of spermatogenesis at spermatogonial stage as a result of an abnormal differentiation of the Sertoli cells. Moreover, the degeneration of abdominal testes initiates earlier in bilateral cryptorchidism than in unilateral cryptorchidism.     

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).     

Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate

Normal spermatogenesis is essential for reproduction and depends on proper spermatogonial stem cell (SSC) function. Genes and signaling pathways that regulate SSC function have not been well defined. We report that glial cell-line-derived neurotrophic factor (GDNF) signaling through the RET tyrosine kinase/GFRA1 receptor complex is required for spermatogonial self-renewal in mice. GFRA1 and RET expression was identified in a subset of gonocytes at birth, was restricted to SSCs during normal spermatogenesis, and RET expressing cells were abundant in a cryptorchid model of SSC self-renewal. We used the whole-testis transplantation technique to overcome the limitation of neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice and found that each of these genes is required for postnatal spermatogenesis and not for embryological testes development. Each mutant testis shows severe SSC depletion by Postnatal Day 7 during the first wave of spermatogenesis. These defects were due to lack of SSC proliferation and an inability of SSCs to maintain an undifferentiated state. Our results demonstrate that GDNF-mediated RET signaling is critical for the fate of undifferentiated spermatogonia and that abnormalities in this pathway may contribute to male infertility and testicular germ cell tumors.