Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

Insertional mutation of the murine kisimo locus caused a defect in spermatogenesis

Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products. An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules. We cloned the disrupted locus of kisimo and identified a novel testis-specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse. The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-1epsilon, suggesting that THEG protein functions as a regulatory factor in protein assembly. Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility.     

A wild common shrew (Sorex araneus) with an XXY sex chromosome constitution

An adult male common shrew with an XXY sex chromosome constitution was found in a natural population. The external appearance of the animal and size of the accessory glands were normal but the testes were as small as those of immature shrews. Histological preparations of the testis revealed seminiferous tubules containing only one type of cell, probably Sertoli cells, and interstitial cell hyperplasia.     

乙醇在HBV DNA转染中对大鼠血睾屏障的影响及其机制探讨(英文)

目的:探讨机体摄入大量乙醇后,血睾屏障(blood-testis barrier,BTB)能否有效阻止含乙型肝炎病毒(hepatitis B virus,HBV)DNA的质粒转染生精小管生精上皮细胞和影响BTB完整性的因素。方法:取20只Wistar成熟雄性大鼠随机分为实验组(A组)和对照组(B组),应用聚合酶链反应(polymerase chain reaction,PCR)和原位杂交(in situ hybridization,ISH)检测HBVDNA的存在和生精小管的转染情况,应用透射电子显微镜(transmission electron microscopy,TEM)技术检测BTB与生精内环境的超微结构形态学变化。结果:①PCR:A组样本可见特异的HBVDNA阳性条带;②原位杂交:A组发现阳性杂交信号弥散,可被广泛发现于生精上皮基底室和近腔室的生精细胞上;③TEM:A组大鼠睾的生精小管基膜厚薄不均,基膜组织疏松增厚,成波浪式皱褶,可见基膜断裂,精原细胞与支持细胞及生精小管的基膜之间出现较多空泡,生精小管、生精上皮、生精细胞及支持细胞与相邻细胞之间的间隙扩大。结论:BTB的完整性是其起保护功能的重要基础,乙醇可以破坏其完整性,会使全部生精细胞易受HBV的感染。     

Localization of follicle-stimulating hormone (FSH) immunoreactivity and hormone receptor mRNA in testicular tissue of infertile men

Testicular biopsies from 82 oligo-or azoospermic male patients were subjected to immunostaining using anti-human FSH antibodies. Histological evaluation showed normal spermatogenesis (nspg) in 7 (FSH: 2.7±0.7), mixed atrophy (ma) in 63 (FSH:5.3±0.5), and bilateral or unilateral Sertoli Cell Only syndrome (SCO) in 12 (FSH:21.7±3.5) patients. For the relationship between FSH values and testicular histology, see Bergmann et al. (1994). FSH immunoreactivity was found exclusively in Sertoli cells and in some interstitial cells. Seminiferous epithelium showing normal or impaired spermatogenesis displayed only weak immunoreactivity compared to intense immunoreaction, i.e. large and numerous vesicles in Sertoli cells of SCO tubules in biopsies showing mixed atrophy or SCO. In addition, h-FSH receptor mRNA was demonstrated by in situ hydridization using biotinylated cDNA antisense oligonucleotides. Hybridization signals were found within the seminiferous epithelium exclusively in Sertoli cell cytoplasm associated with normal spermatogenesis and in epithelia showing different signs of impairment, including SCO. It is concluded that: (1) Sertoli cells are the only cells within the seminiferous epithelium expressing FSH receptors; (2) the accumulation of FSH immunoreactivity in Sertoli cells of SCO tubules appears to be a sign of impaired Sertoli cell function.     

Focal disruption of spermatogenesis in the testis of adult rats after a single administration of human chorionic gonadotrophin

Summary The effect of a single injection of 100 i.u. human chorionic gonadotrophin (hCG) upon the morphology of the rat testis was studied by light and electron-microscopy from 12–48 h after treatment. Twelve hours after injection of hCG, emigration of leukocytes occurred across the intertubular blood vessels and, both at this time and at 24 h, infiltrations of leukocytes were observed within the extracellular tissue spaces. Furthermore, 12 h after hCG, the seminiferous epithelium showed focal disruption of spermatogenesis involving germ cell degeneration and pyknosis. Focal damage to the seminiferous epithelium persisted at 24 h and 48 h after injection of hCG, the affected seminiferous tubules showing failure of spermiation, accumulation of extracellular vacuoles and degeneration or partial loss of spermatogonia and primary spermatocytes. The observations show that, after stimulation of the Leydig cells with hCG, the intertubular tissue exhibits an inflammatory-type response and this is associated with focal tissue destruction in the seminiferous tubules. It is concluded that a high dose of hCG exerts anti-spermatogenic effects upon the testis and raises the possibility of unexpected interference with tests of normal Leydig cell function in both laboratory and clinical investigations.     

Passive immunization with anti-laminin immunoglobulin G modifies the integrity of the seminiferous epithelium and induces arrest of spermatogenesis in the guinea pig

In the testis, the base of the Sertoli cells is in contact with the basement membrane matrix, in which the laminins constitute the major noncollagenous components. We have previously demonstrated that antibodies against a preparation enriched in basement membranes of seminiferous tubules (STBM) or a noncollagenous fraction of STBM passively transferred induced modifications to the basement membranes and focal sloughing of the seminiferous epithelium in the rat. In the present report, we tested the effect of passive immunization with anti-laminin IgG on the limiting membrane of the seminiferous tubules, spermatogenesis, and maintenance of the blood-testis barrier in the adult guinea pig. Rabbit antibodies to laminin 1 (IgG fraction) were injected in adult male guinea pigs (GP). Nonimmunized GP and GP immunized with normal rabbit serum IgG were used as controls. Measurements of variations in the diameter and lumen of the tubules and in the size of individual components of the tubular limiting membrane showed that the highest percentage of tubules with reduced lumen occurred 30 days after passive immunization with anti-laminin, when the limiting membrane was thickest and lesions to the seminiferous epithelium were most severe. The lesions included thickening of the limiting membrane, infolding in the basal lamina, deposits of immune complexes coincident with sloughing of pachytene spermatocytes and spermatids, and vacuolization of the Sertoli cells. Mononuclear cell infiltration of the tubules was rare. Permeability tracer studies revealed that Sertoli cell tight junctions remained impermeable. Fifty and 80 days after treatment, the basement membrane of the tubules and the progression of the spermatogenesis were normal. Passive immunization with anti-laminin IgG provided a valuable experimental model for the in vivo study of the influence of the basement membrane on the issue of spermatogenesis and the integrity of the seminiferous epithelium.     

Developmental study of the different effects on the hybrid sterility of Kit and KitW-v alleles paired with Kit from Mus spretus

The combination of the KitW or KitW-n mutant alleles and KitS from Mus spretus results in male hybrid sterility with small testes. In the present study, reproduction of the combination between KitW-v and KitS alleles was examined. The KitW-v/KitS male was fertile and the histologic structure was normal; the seminiferous tubules showed all of the normal stages of spermatogenesis. The postnatal development of the testis at 8, 12, 16 and 20 days was also studied in the fertile +Kit/+Kit and KitW-v/KitS males and the sterile KitW/KitS. The results showed that at 8 days there was no noticeable difference among the three genotype combinations, while from 12 to 20 days spermatogenesis in the KitW/KitS male nearly stopped before the meiosis stage. The expression of Kit receptor protein from the KitS allele in the sterile testis of the KitW/KitS male was confirmed using western blot analysis. The Kit ligand derived from M. spretus showed two amino acid changes in the extracellular domain compared with that from C57BL and it appears that the ligand-receptor interaction between C57BL and SPR may influence the male hybrid sterility of KitW/KitS.     

The novel dominant mutation Dspd leads to a severe spermiogenesis defect in mice

Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.     

Successful intra- and interspecific male germ cell transplantation in the rat

The lumen of the seminiferous tubules has hitherto been regarded as an immunologically privileged site. We report here the birth of young following transplantation of stem spermatogonia from Long-Evans rats to the seminiferous tubules of Sprague-Dawley rats after treatment with the immunosuppressive agent cyclosporin. Follicle-stimulating hormone was also given to stimulate Sertoli cell proliferation, and testosterone to stimulate the recovery of spermatogenesis. Donor germ cells underwent normal spermatogenesis, and progeny were repeatedly produced from the donor germ cells as demonstrated by microsatellite paternity analysis. In addition, donor germ cells from the cryptorchid testes of LacZ mice were also able to colonize the seminiferous tubules of Sprague-Dawley rats using this protocol. Morphologically normal rat and mouse spermatozoa were present in the epididymis and vas deferens of the recipient rats. This highlights the potential for transplantation of male germ cells between different species.     

Dysplastic development of seminiferous tubules and interstitial tissue in rat hypogonadic (hgn/hgn) testes

The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.     

Demonstration of a stage-specific expression of the ZFY protein in fetal mouse testis using anti-peptide antibodies.

The zinc finger Y (Zfy) gene is located on the Y chromosome of all placental mammals. Although it is phylogenetically conserved and is expressed in mouse fetal testis, it is not the sex determining Y (Tdy) gene. To address the possible function of the Zfy gene in mice, the distribution of Zfy protein in fetal mice was investigated by immunocytochemical staining using several specific antisera against synthetic peptides of the mouse Zfy protein. Analysis of various fetal tissues at different embryonic stages demonstrated a specific staining only in fetal testis. In particular, reactive protein was initially observed in male fetal gonads at day 11.5 postcoitum (p.c.). The immuno-staining intensified in fetal testes at day 12 and 12.5 p.c., decreased drastically in those at day 13 and 14 p.c. and became undetectable in those at day 15 p.c. and beyond. The reactive molecules were distributed mostly within the seminiferous tubules of the embryonic testis. The present observations confirm the previous findings with RT-PCR analysis and indicate that Zfy or Zfy-like protein is expressed in stage-specific manner during early testis differentiation. Its location in the seminiferous tubules suggests a possible role in early germ cell development.     

Effect of 17 alpha-cyanomethyl-17 beta-hydroxy-estra-4, 9-dien-3-one on reproductive organs of the male laboratory mouse

The effect of subcutaneous administration (10, 15 and 20 mg/kg body weight/day, for 21 days; and 20 mg/kg body weight/day, for 28 days) of 17 alpha-cyanomethyl-17 beta-hydroxy- estra-4, 9-dien-3-one (STS 557) on the male reproductive organs of the Parkes strain mouse was investigated. The effect of the treatment on the testis was not uniform; both regressed and normal seminiferous tubules were observed in the same section of the organ. Furthermore, the histological changes observed in the seminiferous tubules in testes of STS 557--treated mice were not different in different dosage groups. In general, in moderately affected seminiferous tubules, the germinal epithelium was thin and consisted of Sertoli cells, spermatogonia, spermatocytes and spermatids; such tubules showed presence of many vacuoles in the epithelium. In severe cases, the tubules had collapsed and were lined by mainly Sertoli cells, spermatogonia and spermatocytes. The treatment also caused marked depression in motility and concentration of spermatozoa in cauda epididymidis, weight of accessory sex glands and in the levels of sialic acid and fructose in the epididymis and seminal vesicle, respectively. By 56 days of drug withdrawal, the alterations induced in the reproductive organs returned to control levels, suggesting that STS 557 treatment induces reversible alterations in the male reproductive organs of Parkes strain mouse.     

Cyclic AMP and genetic sterility of a male restricted (H re /+) mutant of Rattus

Restricted (H ^re /+) male rats marked by a coat color pattern have normal testes at birth. By 9 days postpartum, testes of the mutant animals are smaller than normal and by approximately 90 days of age the animals are sterile. The genetically sterile testes are totally devoid of spermatogonial cells, spermatocytes, spermatids, and spermatozoa, with only Sertoli cells remaining in the seminiferous tubules. Cyclic AMP concentrations in the whole testes (and the seminiferous tubules) of the mutant males are approximately 10–35% greater than in testes of control males when tested at intervals from 5 to 120 days of age. The possible role of excess cyclic AMP in reducing the rate of mitotic division of spermatogonial cells while enhancing differentiation of spermatogonial cells into spermatozoa is discussed. Such a change in the respective rates of mitotic and meiotic divisions would ultimately deplete the mutant testes of all spermatogonial cells.     

Sexual characteristics and spermatogenesis in males of the parthenogenetic gecko Lepidodactylus lugubris (Reptilia, Gekkonidae)

Obligately parthenogenetic lizards usually are all-female populations of hybrids producing diploid oocytes by premeiotic endomitosis and quasi-normal meiosis. In an all-female strain of the gekkonid lizard Lepidodactylus lugubris several phenotypic males arose spontaneously. The sexual characteristics of these males were studied using light and electron microscopy and compared with normal males of the bisexual genus Lygodactylus. Emphasis was layed on morphology of seminiferous tubules, occurrence of spermatogenic stages and ultrastructure of spermatozoa. The phenotypic males possessed preanal pores filled with secretions and a sexual nephric segment which were exactly the same as in normal, reproductively active males. In the testes, density and morphology of non-spermatogenic cell types, the Leydig and Sertoli cells, indicate a normal production of testicular testosterone and a normal function of the blood-testis barrier, respectively. Both in the normal and the phenotypic males, all meiotic cell types of spermatogenesis can be recognised in the seminiferous tubules and are apparently identical, indicating a normal meiosis without impairment in the phenotypic males. In contrast, the differentiation process of spermatids is markedly disturbed in the phenotypic males of L. lugubris. In the normal male, spermiogenesis results in mature spermatids and spermatozoa with small elongated nuclei, an acrosomal complex, and a flagellar tail possessing one axoneme. Spermatozoa fill both the lumen of most seminiferous tubules and the lumina of ductus epididymidis and ductus deferens. In the phenotypic male, spermiogenesis results in seemingly normal spermatids and in spermatozoa with large, non-elongated, deformed nuclei and/or irregular tails possessing more than one axoneme. Both the lumen of most seminiferous tubules and the lumina of the ductus epididymidis and the ductus deferens contain relatively few spermatozoa. We suggest that the phenotypic males inherited the ability for a premeiotic endomitosis from their all-female ancestral lineage. While in females this leads to quasi-normal meiosis and diploid oocytes capable of development, the small nuclei of the spermatozoa are unable to contain a diploid set of chromosomes. Because of the high amount of deformed spermatozoa and possibly uncontrolled loss of genetic material in structurally normal, but aneuploid spermatozoa we conclude that these otherwise perfect males are infertile, thus constituting another example of gametic sterility.     

Recipient preparation is critical for spermatogonial transplantation in the rat

Testis cell transplantation from mice or rats into recipient mouse seminiferous tubules results in donor cell-derived spermatogenesis in nearly all host testes. Normal spermatozoa are produced and, in the most successful mouse transplantations, the donor haplotype is transmitted to progeny of the recipient. However, few studies have been performed in other species. In this report, we demonstrate that rat and mouse testis cells will generate donor cell-derived spermatogenesis in recipient rat seminiferous tubules. Depletion of endogenous spermatogenesis before donor cell transplantation was more difficult in rat than reported for mouse recipients. A protocol employing treatment of neonatal rats with busulfan was most effective in preparing recipients and allowed more than 90% of testes to be colonized by donor cells. Transplantation of mouse testis cells into rat seminiferous tubules was most successful in recipients made cryptorchid and treated with busulfan. In the best experiments, about 55% of rat testes were colonized by mouse cells. Both rat and mouse donor cell-derived spermatogenesis were improved by treatment of rat recipients with leuprolide, a gonadotropin-releasing hormone agonist. The studies indicated that recipient preparation for spermatogonial stem cell transplantation was critical in the rat and differs from the mouse. However, modification of currently used techniques should allow male germ line stem cell transplantation in many species.     

Testicular histological examination of spermatogenetic activity in captive gorillas (Gorilla gorilla)

To clarify the reproductive state of male gorillas, we performed histological examinations on the testicles of 10 male gorillas (Gorilla gorilla). The testicular samples were obtained by autopsy, and ordinal histological preparations were made for light microscopy. The poor spermatogenesis of this species was characterized by the following findings: First, spermatogenesis was evident in only four samples. Meiosis progressed in two samples, but they lacked spermatogenesis. In the remaining four specimens, seminiferous tubules hyalinized without any sign of spermatogenesis. Second, seminiferous epithelia were thin even in the males in which spermatogenesis was observed. Third, degenerated seminiferous tubules were found in all specimens. Fourth, abnormally large syncytial cells were found in the tubules. Six stages in the epithelial cycle of the seminiferous tubules were identified. Testosterone staining made it clear that there were many Leydig cells with spherical or fusiform nuclei in an abundance of interstitial tissue. The relevance of the testicular architecture of gorillas to the mating system is discussed.