锌指蛋白185 在小鼠睾丸支持细胞中的表达与定位

目的:检测锌指蛋白185在小鼠睾丸支持细胞中的表达,探讨其与精子发生的关系。方法:提取睾丸和支持细胞总RNA,经半定量RT-PCR法检测ZNF185的转录水平;提取睾丸和支持细胞的蛋白质,利用Western blot分析ZNF185的表达量;制备支持细胞爬片,采用免疫荧光技术检测ZNF185的定位。结果:(1)半定量RT-PCR结果显示:在睾丸支持细胞中扩增出ZNF185基因条带。(2)Western blot结果显示:ZNF185在支持细胞中的表达量显著低于在睾丸组织中的表达量。(3)免疫荧光结果显示:ZNF185主要定位于支持细胞胞质中。结论:ZNF185可能参与支持细胞结构与功能的调控,进而影响精子发生过程。     

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.     

Gamma-ray exposure accelerates spermatogenesis of medaka fish,Oryzias latipes

To examine the spermatogenesis (and spermiogenesis) cell population kinetics after gamma-irradiation, the frequency and fate of BrdU-labeled pre-meiotic spermatogenic cells (spermatogonia and pre-leptotene spermatocytes) and spermatogonial stem cells (SSCs) of the medaka fish (Oryzias latipes) were examined immunohistochemically and by BrdU-labeling. After 4.75 Gy of gamma-irradiation, a statistically significant decrease in the frequency of BrdU-labeled cells was detected in the SSCs, but not in pre-meiotic spermatogenic cells. The time necessary for differentiation of surviving pre-meiotic spermatogenic cells without delay of germ cell development was shortened. More than 90% of surviving pre-meiotic spermatogenic cells differentiated into haploid cells within 5 days after irradiation, followed by a temporal spermatozoa exhaust in the testis. Next, spermatogenesis began in the surviving SSCs. However, the outcome was abnormal spermatozoa, indicating that accelerated maturation process led to morphological abnormalities. Moreover, 35% of the morphologically normal spermatozoa were dead at day 6. Based on these results, we suggest a reset system; after irradiation most surviving spermatogenic cells, except for the SSCs, are prematurely eliminated from the testis by spermatogenesis (and spermiogenesis) acceleration, and subsequent spermatogenesis begins with the surviving SSCs, a possible safeguard against male germ cell mutagenesis.     

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.     

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.     

Testicular mitosis, meiosis and apoptosis in mink (Mustela vison) during breeding and non-breeding seasons

Testes of mink were compared between the breeding (March) and non-breeding seasons with the start (November) and cessation (May) of spermatogenic activity. Testicular mass and spermatozoa per gram testis were assessed. Percentages of haploid (1C), diploid (2C) and tetraploid (4C) cells were monitored using DNA flow cytometry and the proportions of somatic and spermatogenetic cells were determined after selective labelling of somatic cells with a vimentin antibody. Apoptosis was examined by cell death detection ELISA, and testosterone concentrations were measured with an enzyme-immunoassay. The significantly higher testis mass during the breeding period coincided with higher numbers of testicular spermatozoa per gram testis and peak of testicular testosterone concentration in comparison with non-breeding periods. The proportions of 1C, 2C and 4C cells showed corresponding strong differences between these periods with the maximum of 1C cells during breeding. The proportions of testicular cells in G2-M phase of mitosis were very low during the period of peak spermatogenesis; they were markedly increased in the time of autumnal resumption in November but were even higher during testis involution in May. However. the meiotic transformation (1C:4C ratio) is maximal in March. The total as well as the relative proportions of spermatogenic and somatic cells differed significantly not only between breeding and non-breeding periods but also between the periods at the start and at the end of active spermatogenesis. The intensity of apoptosis was also seasonally dependent. The highest level in March indicates a stimulated apoptosis even during the breeding period. In conclusion, the production of spermatozoa in mink is intensified by enlargement of gonads as well as enhanced efficiency of spermatogenesis during breeding. In this time, the testosterone concentration and the meiotic transformation show high levels, but the mitotic activity of spermatogenic cells is already significantly diminished and an intensified apoptosis seems to precede the forthcoming testis involution after breeding. The results suggest that the regulation of seasonal testicular activity is characterised by co-ordinated shifts in the relationships between mitosis, meiosis, apoptosis and testosterone production.     

Apoptosis during spermatogenesis: the thrill of being alive

Emerging clues about the apoptotic molecular mechanisms operating during spermatogenesis indicate that the activation mechanism of executioner caspases may diverge from the traditional signaling operating in the immune system. Two issues are now been debated: (1) Whether the massive apoptosis of spermatogenic cells observed during the first spermatogenic wave represents a mechanism for ensuring the steady state sperm output in a given segment of a seminiferous tubule. (2) Whether apoptosis just represents a local remodeling process commanded by the Sertoli cell constraints to satisfy the differentiation needs of a juxtaposed cellular domain of spermatogonial, spermatocyte, and spermatid cohorts in the adult testis.     

Construction of a proteome profile and functional analysis of the proteins involved in the initiation of mouse spermatogenesis

Spermatogenesis is a complex process of terminal differentiation wherein mature sperm are produced. In the first wave of mouse spermatogenesis, different spermatogenic cells appear at specific time points, and their appearance is expected to be accompanied by changes in specific protein expression patterns. In this study, we used 2D-PAGE and MALDI-TOF/TOF technology to construct a comparative proteome profile for mouse testis at specific time points (days 0, 7, 14, 21, 28, and 60 postpartum). We identified 362 differential protein spots corresponding to 257 different proteins. Further cluster analysis revealed 6 expression patterns, and bioinformatics analysis revealed that each pattern was related to many specific cell processes. Among them, 28 novel proteins with unknown functions neither in somatic cells nor germ cells were identified, 8 of which were found to be uniquely or highly expressed in mouse testes via comparison with the GNF SymAtlas database. Further, we randomly selected 7 protein spots and the above 8 novel proteins to verify the expression pattern via Western blotting and RT-PCR, and 6 proteins with little information in testis were further investigated to explore their cellular localization during spermatogenesis by performing immunohistochemistry for the mouse testis tissue. Taken together, the above results reveal an important proteome profile that is functional during the first wave of mouse spermatogenesis, and they provide a strong basis for further research.     

Polycomb基因家族在大鼠精子发生过程中的表达

目的检测大鼠精子发生不同阶段细胞中Polycomb-group（Pc-G）家族在mRNA水平上表达是否有差异。方法提纯大鼠精子发生过程中的精原细胞、精母细胞、圆形精子细胞以及支持细胞,用荧光定量PCR方法检测Pc-G家族基因mRNA表达量。结果Pc-G基因家族中Ezh2、Eed、Bmi-1在精子发生中后期高表达;在各生精细胞中,YY1基因表达量低于支持细胞。结论Pc-G基因家族在精子发生各阶段细胞中特征性表达,与精子发生具有相关性,可能对精子发生分化和维持遗传稳定性都有重要的作用。     

Spermatogonial depletion in adult Pin1-deficient mice

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.     

Testis morphometry, duration of spermatogenesis, and spermatogenic efficiency in the wild boar (Sus scrofa scrofa)

The wild boar is a natural inhabitant of Europe, Asia, and North Africa and is phylogenetically the ancestor of the domestic pig. Because of its phylogenetic and economic importance, this species is an interesting model for studying testis function in boars. Therefore, the present study was performed to investigate the testis structure, spermatogenic cycle length, and Sertoli cell (SC) and spermatogenic efficiencies in eight adult wild boars. Each spermatogenic cycle lasted 9.05 days, and the total duration of spermatogenesis was estimated as lasting approximately 41 days. The percentages of testis volume occupied by seminiferous tubules and by Leydig cells were 87% and 6%, respectively. The mean number of SCs per gram of testis was 42 million. The SC (round spermatids per SC) and spermatogenic (daily sperm production per gram of testis) efficiencies were 6.6 cells and 28.6 million, respectively. In general, the testis structure, overall germ cell associations at the different stages of the seminiferous epithelium cycle, and duration of spermatogenesis in the wild boar were similar to those in domestic pigs. Probably because of the small size of Leydig cells (400 microm3), their number per gram of testis (157 million) was the highest among investigated mammalian species. Although the SC efficiency in wild boars was low, their spermatogenic efficiency was comparable to that observed in domestic pigs, mainly because of the higher number of SCs per gram of testis in wild boars. These data suggest that SCs became more efficient during evolution, genetic selection, and domestication in pigs.     

Proteomic analysis of testis biopsies in men treated with injectable testosterone undecanoate alone or in combination with oral levonorgestrel as potential male contraceptive

Treatment with injectable testosterone undecanoate (TU) alone or in combination with oral levonorgestrel (LNG) resulted in marked decreases in sperm concentrations. In this study, we used proteomic analyses to examine the cellular/molecular events occurring in the human testis after TU or TU + LNG treatment. We conducted a global proteomic analysis of the human testicular biopsies before and at 2 weeks after TU alone or TU + LNG treatment. Proteins showing significant changes in expression were identified and analyzed. As a result, 17 and 46 protein spots were found with significant differential expression after the treatment with TU alone and TU + LNG, respectively. TU treatment changed the expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), proteasome inhibitor PI31 subunit (PSMF1), and superoxide dismutase [Mn] mitochondrial precursor (SOD2). These proteins inhibit "assembly", induce cell death, and promote compensatory "cell survival" in the testis. After TU + LNG treatment, "proliferation/cell survival" and "apoptosis/death" were the predominant responses in the testis. TU + LNG treatment inhibited the expression of Prolyl 4-hydroxylase beta subunit (P4HB) and Annexin A2 (Annexin II). These proteins are involved in apoptosis and cell proliferation, respectively. TU + LNG treatment also enhanced the expression of SOD2 and Parvalbumin alpha (Pvalb). These two proteins may protect testicular cells against apoptosis/death and promote cell survival. In conclusion, TU and TU + LNG treatments suppress spermatogenesis through different pathways by changing the expression of different proteins. hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb, are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.     

Detection of quantitative trait loci causing abnormal spermatogenesis and reduced testis weight in the small testis (Smt) mutant mouse.

The small testis (Smt) mutant mouse is characterized by a small testis of one third to one half the size of a normal testis, and its spermatogenesis is mostly arrested at early stages of meiosis, although a small number of spermatocytes at the late prophase of meiosis and a few spermatids can sometimes be seen. We performed quantitative trait locus (QTL) analysis of these spermatogenic traits and testis weight using 221 F2 males obtained from a cross between Smt and MOM (Mus musculus molossinus) mice. At the genome-wide 5% level, we detected two QTLs affecting meiosis on chromosomes 4 and 13, and two QTLs for paired testis weight as a percentage of body weight on chromosomes 4 and X. In addition, we found several QTLs for degenerated germ cells and multinuclear giant cells on chromosomes 4, 7 and 13. Interestingly, for cell degeneration, the QTL on chromosome 13 interacted epistatically with the QTL on chromosome 4. These results reveal polygenic participation in the abnormal spermatogenesis and small testis size in the Smt mutant.     

Expression of connexin 43 in human testis

In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency. Accepted: 10 June 1999     

Proteomic analysis of testis biopsies in men treated with transient scrotal hyperthermia reveals the potential targets for contraceptive development

Mild testicular heating safely and reversibly suppresses spermatogenesis. In this study, we attempted to clarify the underlying molecular mechanism(s) involved in heat‐induced spermatogenesis suppression in human testis. We conducted global proteomic analyses of human testicular biopsies before, and at 2 and 9 wk after heat treatment. Thirty‐one and Twenty‐six known proteins were identified with significant differential expression at 2 and 9 wk after heat treatment, respectively. These were used to characterize the cellular and molecular events in the testes when seminiferous epithelia became damaged (2 wk) and recovered (9 wk). At 2 wk post‐treatment, the changed expression of a series of proteins could promote apoptosis or suppress proliferation and cell survival. At 9 wk post‐treatment, the changed expression of proteins mainly promoted cell proliferation, differentiation and survival, but resisted cell apoptosis. Among those heat‐regulated proteins, HNRNPH1 was selected for the further functional study. We found that HNRNPH1 was an anti‐apoptosis protein that could regulate the expression of other heat‐induced proteins. In conclusion, heat‐induced reversible suppression of spermatogenesis occurred by modulating the expression of proteins related to proliferation, differentiation, apoptosis and cell survival pathways. These differentially expressed proteins were found to be key molecular targets affecting spermatogenesis after heat treatment.     

Mitochondrial proteomics reveals the mechanism of spermatogenic cells apoptosis induced by carbon ion radiation in zebrafish

The mitochondrial proteins involved in spermatogenic cells apoptosis in zebrafish after carbon ion radiation (CIR) were screened. The relative biological effectiveness (RBE) of CIR in zebrafish testes was investigated. Apoptosis of testicular cells was measured within 24 hr following 1 and 4 Gy CIR. Immunoblotting was used to assess the levels of mitochondrial apoptotic proteins in testes, and proliferative and apoptotic spermatogenic cells were detected by immunofluorescence after CIR. Label-free quantitative (LFQ) and parallel reaction monitoring-based target proteomics (PRM) were combined to screen and validate differential mitochondrial proteins in testes between 4 Gy and control groups at 24 hr after CIR. The RBE of CIR in zebrafish testes was 1.48 ± 0.04, and induction of apoptosis by CIR was higher than that of X-rays in testicular cells. Mitochondrial apoptotic pathways play a crucial role in spermatogenic cells apoptosis after CIR, with 60 differential mitochondrial proteins identified. Among 20 target proteins, 12 were significantly upregulated, 2 were significantly downregulated in the 4 Gy CIR group. The results of PRM were consistent with label-free analysis. This is the first study to screen the differential mitochondrial proteins and provide useful information to understand the underlying mechanisms of spermatogenic cell apoptosis in zebrafish following CIR.