In vitro responses to known in vivo genotoxic agents in mouse germ cells

Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n‐ethyl‐n‐nitrosourea (ENU), 6‐mercaptopurine (6‐MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration‐dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration‐related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6‐MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real‐time PCR results showed that the mRNA level expression of p53 increased and bcl‐2 decreased significantly with the increasing ENU, 6‐MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6‐MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6‐MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99–107, 2017. © 2017 Wiley Periodicals, Inc.     

The expression pattern of PARP-1 and PARP-2 in the developing and adult mouse testis

Although the importance of the PARP family members in the adult testis has already been acknowledged, their expression in the developing testis has not been addressed. We performed immunohistochemistry by using PARP-1 and PARP-2 antibodies on the developing mouse testis at embryonic day (E) 15.5, E17.5, postnatal day (PN) 0, PN3, PN9, PN20 and adult. Our results showed that at embryonic and early postnatal days, the expression of PARP-1 was in the nuclei of gonocytes and spermatogonia. PARP-1 was positive in interstitial cells with nuclear localization at all studied ages. At embryonic and early postnatal days, the expression of PARP-2 was in the cytoplasm of gonocytes and spermatogonia. During the progress of spermatogenesis, PARP-2 was localized in the cytoplasm of pre-leptotene spermatocytes on PN9, in the cytoplasm of pachytene spermatocytes on PN15 and in the cytoplasm of round spermatids on PN20. In the adult, PARP-2 staining can still be observed in the cytoplasm of spermatogonia, but to a much lesser degree than in the round and elongating spermatids. For all the studied ages, PARP-2 was positive in Sertoli cells and interstitial cells with cytoplasmic localization. Our results indicate that PARP proteins are present in germ and somatic cells during testis development in mice.     

Death receptor and mitochondrial pathways are involved in germ cell apoptosis in an experimental model of autoimmune orchitis

BACKGROUND: Studies on experimental autoimmune orchitis (EAO) have helped to elucidate immunological mechanisms involved in testicular damage. We previously demonstrated that EAO is characterized by lymphomononuclear cell infiltrates and apoptosis of spermatocytes and spermatids expressing Fas and TNFR1. The aim of this work was to characterize the pathways involved in germ cell apoptosis in EAO and to determine the involvement of the Bcl-2 protein family in this process. METHODS AND RESULTS: EAO was induced in rats by immunization with testicular homogenate (TH) and adjuvants, whereas control (C) rats were injected with saline solution and adjuvants. Testis of EAO rats showed procaspase 8 cleavage products (western blot) with high caspase 8 activity. Cytochrome c content increased in the cytosol and decreased in the mitochondrial fraction of testis from EAO rats compared with C, concomitant with increased caspase 9 activity. Bax was mainly expressed in spermatocytes and spermatids and Bcl-2 in basal germ cells (immunohistochemistry). Baxbeta isoform content increased in EAO rat testis compared with C, whereas content of Baxalpha remained unchanged (western blot). However, Baxalpha content decreased in the cytosol and increased in the mitochondrial and endoplasmic reticulum (ER)-enriched fractions of testis from EAO rats compared with C (western blot). Bcl-2 content also increased in the testes of EAO rats. CONCLUSIONS: Our results demonstrated that extrinsic, mitochondrial and possibly ER pathways are inducers of germ cell apoptosis in EAO and that Bax and Bcl-2 proteins modulate this process.     

Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate.

The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alphaVasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of vasa and its protein changes dynamically during oogenesis. The alphaVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, Cagvasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alphaVasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation.     

低剂量电离辐射诱导小鼠睾丸生精细胞凋亡及P53基因和蛋白表达

目的:研究低剂量电离辐射对小鼠睾丸生精细胞凋亡及 P53 基因和蛋白表达的影响。方法:应用密度梯度离心法分离不同种类生精细胞, 流式细胞术检测其细胞凋亡, 免疫组化法观察生精细胞P53蛋白表达, 原位杂交法观察其 P53 mRNA水平。结果:0.025-0.2 Gy X射线全身照射后, 生精细胞凋亡具有明显的细胞种类规律性。在较低剂量照射(0.025和0.05 Gy)时, 以精原细胞凋亡为主, 随照射剂量增加(0.075-0.2 Gy)逐渐累及精母细胞, 并且前者凋亡率明显高于后者, 很少累及精子细胞和精子。P53蛋白表达主要见于精原细胞和精母细胞, 并且前者阳性率高于后者, 随照射剂量增加, 其阳性率逐渐升高, 而精子细胞和精子阳性率较低; P53 mRNA表达在较低剂量照射(0.025 Gy)时, 主要以精母细胞和精子细胞为主, 随剂量增加(0.05-0.2 Gy)逐渐累及精原细胞。精原细胞和精母细胞 P53 mRNA表达呈明显的剂量依赖性关系, 但精子细胞表现不明显。结论:低剂量电离辐射可选择性诱导小鼠睾丸生精细胞凋亡, 具有明显的剂量和时程效应关系。提示, 这种选择性诱导凋亡调控机制可能与 P53 基因和蛋白表达相关联。     

CD147 is required for matrix metalloproteinases-2 production and germ cell migration during spermatogenesis

Spermatogenesis is a highly programmed process that requires the degradation of the extracellular matrix and the remodeling of tight junctions (TJ) to facilitate differentiating germ cell migration. Matrix metalloproteinases (MMPs) are essential in regulating Sertoli cell TJ in the testis. CD147 is known to stimulate the production of MMPs in tumor metastasis and its knockout mice are infertile. However, the functional relationship between CD147 and MMPs in spermatogenesis has not been investigated. In the present study, we examined the expression profile of CD147 and MMPs during mouse testicular development by RT-PCR, western blot and immunofluorescence staining. We also examined CD147 involvement in the production of MMP-2 and the migration of germ cells (GC-1 and GC-2 cells) using CD147 antibody or synthetic microRNA mimics-mediated knockdown. The results showed that CD147 was present at all stages of testicular development from 7 to 56 days post-partum (dpp). CD147 expression was found to increase after 21 days from moderate levels in 7 and 14 days. Of the eight MMPs studied, MMP-2, MMP-7, MMP-9 and MMP-23 were detected to have changes in expression during testicular development, with MMP-2 showing the largest change. CD147 and MMP-2 were co-localized in spermatogonia, spermatocytes and round spermatids in mouse testis, while in human testis, they were co-localized in spermatocytes and round spermatids. MMP-2 expression and migration of GC-1 and GC-2 cells were reduced by interfering with CD147 expression and function in vitro. These data suggest that CD147 regulates migration of spermatogonia and spermatocytes via induction of MMP-2 production during spermatogenesis.     

Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide,adriamycin, and diepoxybutane

In testis, apoptosis is a way to eliminate damaged germ cells during their development. In this study, we evaluated the ability of three germ cell mutagens to induce apoptosis (or programmed cell death) at specific stages of rat seminiferous epithelial cycle. These chemicals include the cancer chemotherapy drugs etoposide and adriamycin and the butadiene metabolite diepoxybutane. According to our results, etoposide is a very potent inducer of apoptosis in male rat germ cells and the cell types most sensitive to it include all types of spermatogonia, zygotene, and early pachytene spermatocytes and meiotically dividing spermatocytes. Also, adriamycin causes an increase in apoptosis at specific stages of seminiferous epithelial cycle and the most sensitive cell types are type A_3–4 spermatogonia, preleptotene, zygotene, and early pachytene spermatocytes. Die poxybutane does not cause any significant increase in the frequency of apoptosis in rat testis. In addition, we studied whether p53 is taking part in the apoptotic response of spermatogenic cells by studying the levels of p53 protein in testis before and after chemical treatment. No accumulation of p53 in testis was seen after treatment with these three chemicals. The expression of two p53-regulated genes, p21^WAF1 and mdm2, was also studied but no increase in the levels of mRNA of these genes was observed after treatment. The results indicate that apoptosis should be taken into consideration when the genotoxic effects of chemicals are evaluated in germ cells. Environ. Mol. Mutagen. 31:133–148, 1998 © 1998 Wiley-Liss, Inc.     

人绒毛膜促性腺激素对切除颌下腺小鼠静止期精母细胞的一些影响

目的：探讨人绒毛膜促性腺激素(HCG)对切除颌下腺小鼠静止期精母细胞表皮生长因子(EGF)的表达和生精功能的影响。方法：采用组织化学和免疫组织化学技术。结果：EGF、分布在小鼠睾丸间质细胞、精原细胞、静止期精母细胞、细线期精母细胞和精子细胞。去颌下腺组EGF阳性反应静止期精母细胞数量明显减少，同时相应的生精小管的各级生精细胞也显著减少。与去颌下腺组相比，去颌下腺给药组EGF阳性反应静止期精母细胞数量明显增多，同时相应的生精小管的生精细胞也显著增多。结论：睾丸间质细胞和部分生精细胞能分泌EGF。EGF在生精小管上皮的分布与生精周期有密切关系。静止期精母细胞EGF表达减弱是切除颌下腺导致小鼠少精症的原因之一。HOG可促进静止期精母细胞表达EGF，以调节精子发生过程。     

Importin alpha mRNAs have distinct expression profiles during spermatogenesis.

Importin proteins control access to the cell nucleus by mediating the nuclear transport of specific cargoes. We hypothesized that developmental regulation of gene expression may be partially effected by changes in the nuclear transport machinery complement, manifested as regulated expression of importin alpha family genes. We first clarified the identity of the five known mouse importin alpha genes relative to those for human and then determined their expression throughout postnatal rodent testis using PCR and in situ hybridization. Distinct expression patterns were observed for each. At 10 dpp, all importin alpha mRNAs were detected in spermatogonia. In the adult mouse testis, importins alpha1 and alpha3 were detected in spermatogonia and early pachytene spermatocytes. Importin alpha4 mRNA was identified in pachytene spermatocytes, alpha6 mRNA in round spermatids, and alpha2 mRNA in both of these. The distinct importin alpha expression patterns are consistent with their having specific roles and transport cargoes during spermatogenesis.     

Regulated expression of the ubiquitin protein ligase, E3(Histone)/LASU1/Mule/ARF-BP1/HUWE1, during spermatogenesis.

A ubiquitin protein ligase (E3), E3(Histone)/LASU1 (Mule/ARF-BP1/HUWE1), was recently identified that mediates ubiquitination of core histones, the Mcl-1 anti-apoptotic protein, and the p53 tumor suppressor protein. However, the expression of E3(Histone)/LASU1 remains poorly studied. Because we identified E3(Histone)/LASU1 from the testis, we explored its regulation during spermatogenesis. In the first wave of rat spermatogenesis, E3(Histone)/LASU1 mRNA and protein had peak expression at days 10 and 20, respectively, and decreased with age. Consistent with these findings, immunohistochemistry revealed that E3(Histone)/LASU1 was highly expressed in nuclei from spermatogonia to mid-pachytene spermatocytes. There was no obvious staining in spermatids, when histones are ubiquitinated and degraded. E3(Histone)/LASU1 was also expressed in other tissues. However, except in neuronal cells of the brain, expression was cytoplasmic. Thus, E3(Histone)/LASU1 may play a role in chromatin modification in early germ cells of the testis, but also has functions in other tissues.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

二乙基己烯雌酚诱发成年仓鼠睾丸生精异常及对生精细胞表达bcl-2、P53和cadherin的影响

目的探讨在二乙基己烯雌酚(DES)诱发成年动物生精异常过程中,原癌基因bcl-2、p53及黏附分子cadherin在生精细胞中表达的变化,旨在阐明DES诱发生精异常的作用机理。方法成年雄性仓鼠皮下注射DES连续7d后,取其睾丸,进行光镜及电镜的观察和原癌基因bcl-2、p53及黏附分子cadherin的免疫组织化学染色。结果DES处理组的成年仓鼠睾丸生精细胞发育异常,bcl-2和p53表达量均比对照组有显著增加,并以精母细胞和圆形精子细胞较为明显。生精上皮中cadherin的表达比对照组有明显减少。结论DES增加精母细胞和圆形精子细胞表达bcl-2和p53;同时抑制cadherin表达,是诱发成年仓鼠生精异常的原因之一。     

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.     

The contribution of RNA-binding motif (RBM) antibody to the histopathologic evaluation of testicular biopsies from infertile men

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

Apurinic/apyrimidinic endonuclease immunoreactivity in germ cells of experimental varicocele-induced rat testes

Increased germ cell apoptosis is related to oxidative DNA damage; therefore, we investigated whether there was a significant change in apurinic/apyrimidinic endonuclease (APE) in varicoceles. Experimental varicoceles were created by partial ligation of the left renal vein of adult male Sprague-Dawley rats, which were sacrificed at 1, 3 and 6 weeks after varicocele creation. Testicular tissues were sampled for TUNEL, Western blotting and immunohistochemistry. There was a significant increase in apoptotic germ cells in the ipsilateral testes 6 weeks after varicocele creation. Increased activation of p53, Bax and cleaved caspase-3 in the left testes was also noted. APE increased activation until 3 weeks after varicocele creation, and then decreased at 6 weeks after varicocele surgery. The spermatocytes were immunostained for both 8-hydroxy-2′-deoxyguanosine and APE, but the spermatogonia revealed only APE immunopositivity in the defective tubules. These results suggest that repression of APE is an underlying mechanism of augmented p53-dependent apoptosis in varicocele-induced rat testes and that remaining APE in the spermatogonia plays a decisive role in regaining testicular spermatogenic function after varicocelectomy.     

Induction of apoptosis involving multiple pathways is a primary response to cyclin A1-deficiency in male meiosis.

The meiotic arrest in male mice null for the cyclin A1 gene (Ccna1) was associated with apoptosis of spermatocytes. To determine whether the apoptosis in spermatocytes was triggered in response to the arrest at G2/M phase, as opposed to being a secondary response to overall disruption of spermatogenesis, we examined testes during the first wave of spermatogenesis by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining. We observed enhanced apoptosis coinciding with the arrest point in postnatal day 22 tubules, with no overt degeneration. Along with activation of caspase-3, an increase in the levels and change of subcellular localization of Bax protein was observed in cyclin A1-deficient spermatocytes, which coincided with the detection of apoptosis. As p53 is implicated in the activation of Bax-mediated cell death, we generated mice lacking both cyclin A1 and p53. Although the absence of p53 did not rescue the meiotic arrest, there was a decrease in the number of apoptotic cells in the double-mutant testes. This finding suggested that p53 may be involved in the process by which the arrested germ cells are removed from the seminiferous tubules but that other pathways function as well to ensure removal of the arrested spermatocytes.     

Human spermatogenesis: basic research and clinical issues]

Histological evaluation of human spermatogenesis suffers from the hazy border line between normal and pathological germ cell development. This border line needs better definition for histological fertility diagnosis and the early detection of germ cell tumors. Testicular biopsies from more than 2,900 patients with fertility disturbances and more than 1,900 patients with testicular tumors were investigated by means of semithin sectioning, different immunocytochemical methods and transmission electron microscopy. Cellular systems of the human testes possess a degree of autonomy from the body. Their morphological and functional heterogeneity reveals characteristics of cells that are not terminally differentiated. In the testis of an adult, fertile man not only the proliferation of spermatogonia, maturation divisions of spermatocytes and differentiation of spermatids take place, but also abortive germ cells, as well as apoptotic and degenerative cells appear. Disturbances of spermatogenesis are defined by the evaluation of quantity and quality of germ cell alterations. Compensatory and non compensatory defects of spermatogenesis may be distinguished. Deficiency of spermatogonial cell types, multilayered spermatogonia, megalospermatocytes, malformed spermatids and single tumor cells in the face of sufficient development of mature spermatids are considered compensatory defects of spermatogenesis. Dominating malformed germ cells or tumor cells accompanied by an arrest or lack of spermatogenesis, however, represent non-compensatory defects of spermatogenesis. In addition, normal organization and function of the microvasculature, Leydig cells and compartmentalizing cells in the intertubular space are prerequisites for spermatogenesis. The neuroendocrine function of Leydig cells may be responsible for regulating the blood flow rate and the permeability to hormones and nutritive substances. Finally, for patients a successful definition of the border line between normal and pathological events of germ cell development may be essential for early detection of germ cell tumors. Therefore, anatomical sciences not only contribute to basic research, advanced diagnostics and therapeutic concepts related to diseases of the male gonad, but also to the improvement of assisted reproduction.