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.     

ARHGEF15 is expressed in undifferentiated spermatogonia but is not required for spermatogenesis in mice

Spermatogenesis is a continual process that relies on the activities of undifferentiated spermatogonia, which contain spermatogonial stem cells (SSCs) that serve as the basis of spermatogenesis. The gene expression pattern and molecular control of fate decisions of undifferentiated spermatogonia are not well understood. Rho guanine nucleotide exchange factor 15 (ARHGEF15, also known as EPHEXIN5) is a guanine nucleotide-exchange factor (GEF) that activates the Rho protein. Here, we reported that ARHGEF15 was expressed in undifferentiated spermatogonia and spermatocytes in mouse testes; however, its deletion did not affect spermatogenesis. Arhgef15^-/- mice were fertile, and histological examination of the seminiferous tubules of Arhgef15^-/- mice revealed complete spermatogenesis with the presence of all types of spermatogenic cells. Proliferation and differentiation of the undifferentiated spermatogonia were not impacted; however, further analysis showed that Arhgef15 deletion resulted in decreased expression of Nanos2, Lin28a and Ddx4. Together, these findings suggest that ARHGEF15 was specifically enriched in undifferentiated spermatogonia and regulated gene expression but dispensable for spermatogenesis in mice.     

Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

Bis-(2-ethylexhyl) phthalate impairs spermatogenesis in zebrafish (Danio rerio)

Bis-(2-ethylhexyl) phthalate (DEHP) is a widely used industrial additive for increasing plastic flexibility. Its metabolites are known to exert toxic effects on reproduction and development of mammals. The aim of this study was to evaluate the effects of environmentally relevant concentrations of DEHP (0.2 and 20 μg/L) on the reproductive biology of adult male zebrafish (Danio rerio). The effects of DEHP and 17β-ethynylestradiol (a positive control) were determined after one or three weeks of exposure by TUNEL assay, histomorphometric analysis and evaluation of reproductive performance. DEHP impaired reproduction in zebrafish by inducing a mitotic arrest during spermatogenesis, increasing DNA fragmentation in sperm cells and markedly reducing embryo production (up to 90%). In conclusion, relatively short-term exposure to environmentally relevant concentrations of DEHP is able to alter spermatogenesis and affect reproduction in zebrafish.     

The essential roles of Mps1 in spermatogenesis and fertility in mice

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.Subject terms: Cell division, Spermatogenesis     

Suppression of spermatogenesis for cell transplantation in adult mice

Summary Spermatogenesis occurs within the testis of adult males by a complex and very well organized process. Breakthroughs in techniques such as cryopreservation and culture of spermatogenic cells and the maturation of these cells in exogenous testes after transplantation renewed the interest in this process. Transplantation of spermatogenic cells from a donor to a recipient animal needs a preparatory step that consists in the elimination of the endogenous population of spermatogenic cells. The most common method used to empty the seminiferous tubules is the treatment with busulfan (1,4-butanediol dimethanesulfonate). Busulfan partially eliminates stem cells because of its alkylating nature, but a residual component of stem cells survives the treatment and competes in the regeneration of the testis with transplanted cells. Estradiol has also been used as an agent that causes a delay in the process of spermatogenesis by altering its hormonal stimulation, although it does not affect the spermatogonia population. Therefore, we have tested different treatments with busulfan, estradiol benzoate, and also an agonist of the chorionic gonadotrophin-releasing hormone, leuprolide acetate, for the inhibition of endogenous spermatogenesis. We have found that a combination of estradiol, busulfan, and leuprolide can destroy the population of endogenous spermatogenic cells without altering Sertoli cells and maintains the optimal environment needed to allow the development of transplanted cells.     

Impaired spermatogenesis and elevated spontaneous tumorigenesis in xeroderma pigmentosum group A gene (Xpa)-deficient mice

We have reported that xeroderma pigmentosum group A (Xpa) gene-knockout mice [Xpa (−/−) mice] are deficient in nucleotide excision repair (NER) and highly sensitive to UV-induced skin carcinogenesis. Although xeroderma pigmentosum group A patients show growth retardation, immature sexual development, and neurological abnormalities as well as a high incidence of UV-induced skin tumors, Xpa (−/−) mice were physiologically and behaviorally normal. In the present study, we kept Xpa (−/−) mice for 2 years under specific pathogen-free (SPF) conditions and found that the testis diminished in an age-dependent manner, and degenerating seminiferous tubules and no spermatozoa were detected in the 24-month-old Xpa (−/−) mice. In addition, a higher incidence of spontaneous tumorigenesis was observed in the 24-month-old Xpa (−/−) mice compared to Xpa (+/+) controls. Xpa (−/−) mice provide a useful model for investigating the aging and internal tumor formation in XPA patients.     

香螺精子发生及精子超微结构

本文采用透射电镜技术对香螺(NpatunedecumingiCrosse)精子发生过程进行了观察。结果表明,精原细胞胞质中含有大量的线粒体;初、次级精母细胞的细胞核和大量的线粒体呈极性分布;精子细胞分化过程中,细胞核形态、核内物质以及线粒体的形态发生显著变化;细胞核的核质由不均匀颗粒状浓缩成纤丝状,再浓缩成细线形,最后呈致密均匀状态,细胞核由近圆形伸长为粗线形,具有核后窝;在细胞核后端有8个膨大的线粒体,由卵圆形变为螺旋形,弯曲盘绕在轴丝外部,形成精子的中段;根据细胞核和线粒体的变化特点,将精子形成分为早、中、后三个时期。香螺典型性精子属于进化型,头部呈线形,中段加长,糖原颗粒包围轴丝构成主段。在精子发生过程中,细胞质内没有发达的高尔基复合体和前顶体池,没有观察到香螺精子的顶体。在成熟个体的精巢内,同时存在不具有受精能力的畸变精子。     

Long glucocorticoid-induced leucine zipper (L-GILZ) protein interacts with ras protein pathway and contributes to spermatogenesis control

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.     

Stages and duration of spermatogenesis in the domestic ferret (Mustela putorius furo)

Classification of seminiferous tubules is the basis for understanding normal and abnormal spermatogenesis. The aim of the present study was to determine spermatogenic stages and the duration of the cycle in the domestic ferret using bromodeoxyuridine (BrdU) as a tracer. Eleven adult male ferrets that were maintained in a breeding condition were used. Testicular sections were stained with the periodic acid-Schiff reaction for light microscopy. To determine the cycle duration, six ferrets were injected intraperitoneally with BrdU, and testes were collected 3h later and 10 days and 3h later. BrdU was detected by immunohistochemistry. Seminiferous tubules were classified into eight stages, and frequencies of stages I-VIII were 10.6, 2.2, 7.9, 13.1, 22.3, 21.9, 14.0 and 8.0%, respectively. The most advanced BrdU-labeled cells at 3h post-injection were leptotene spermatocytes in stage VI and those at 10 days and 3h were pachytene spermatocytes in stage V. From differences in stage frequency and BrdU staining frequency between two time points, the duration of one cycle was estimated to be 13.0 days. The present observations indicate that stages and the cycle duration of the ferret spermatogenesis are similar to those reported in other carnivores.     

隆线溞精子发生及成熟精子的超微结构

用透射电镜和扫描电镜观察隆线精子的发生过程及成熟精子的超微结构。隆线精子发生经历精原细胞、精母细胞、精子细胞和成熟精子四个时期。精原细胞核染色质凝集成团,细胞质内有线粒体、粗面内质网分布。精母细胞核染色质分散,不均匀地分布于核中,细胞质内粗面内质网聚集。在精子细胞分化形成精子的早期,细胞纵向拉伸,核物质开始浓缩;中期精子细胞呈明显的长条形,精子细胞逐渐移入精巢管腔中央,外围包裹一厚层精子鞘;后期精子细胞已进入管腔中,核物质呈高度浓缩状,细胞质层较少,精子细胞间通过外围精子鞘相互粘连成片。成熟精子分散在精巢管腔中央,外形呈棒状,一端稍钝,一端稍尖,无鞭毛、棘突等附属物;核内染色质解聚,均匀分布在核中,具双层核膜,细胞质层很少,精子鞘为单层,无法确认顶体端。隆线雄性生殖细胞的结构及其发生过程均较高等甲壳动物简单和原始,但在功能上表现出相对的适应性,使以隆线为代表的枝角类能适应复杂多变的生活环境     

Sertoli cell Dicer is essential for spermatogenesis in mice

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.     

Rab11 is essential for fertility in Drosophila

Rab11, a small GTP binding protein involved in vesicular trafficking, has emerged as a key player in regulating various cellular events during Drosophila development and differentiation. In our earlier study a P-insertion line, Rab11(mo), was established as a new hypomorphic allele of Rab11 gene, showing degenerated eye phenotype, bristle abnormalities and sterility. We show here that Rab11 is expressed in the entire testis, more prominently in the secretory cells, and in ovary it is localized at the posterior pole. Rab11(mo) males and females are sterile. The sterility in males has been attributed to defects in the sperm individualization process, while in females, cytoskeleton disruption and reduction/loss of the posteriorly localized protein, Vasa, as a consequence of loss/mislocalization of Rab11 might be the cause of sterility. Fertility as well as the posterior localization of Rab11 and Vasa or cytoskeleton integrity was restored in pCaSpeR4-Rab11/+; Rab11(mo)/Rab11(mo) egg chambers, confirming the requirement of Rab11 in these events.