Spermatogenese

Spermatogenesis takes place within the testicular seminiferous tubules which consist of the peritubular lamina propria and the seminiferous epithelium. The latter is composed of germ cells and somatic Sertoli cells. Sertoli cells trigger germ cell development by mediating follicle-stimulating hormone and androgen hormonal stimuli. Spermatogenesis comprises proliferation of spermatogonia, meiosis of spermatocytes, and differentiation of spermatids into spermatozoa (spermiogenesis). There are six distinct and specific germ cell associations (I–VI). These “stages of spermatogenesis” occur sequentially along the length of a tubule. Different defects in spermatogenesis occur in adjacent seminiferous tubules (mixed atrophy) and are associated with deficits in differentiation of Sertoli cells. Biopsy specimens should be fixed in Bouin’s solution. Diagnosis of preinvasive carcinoma in situ is based on the immunohistochemical demonstration of placental-like alkaline phosphatase (PLAP), which is expressed exclusively in carcinoma in situ cells. Histological evaluation should be performed using a score count system, and the use of histological techniques for protein and mRNA expression. Testicular biopsy should only be performed in accordance with strict indication criteria, and histological evaluation should be carried out in specialist centres, i.e. as recommended by the European Academy of Andrology (EAA).     

大鼠曲细精管生殖细胞体外共培养和精子发生过程的观察

目的建立体外长期共培养体系和观察方法,为精子发生过程的研究提供细胞模型。方法采用大鼠曲细精管生殖细胞及支持细胞共培养的方法,对睾丸生精细胞作显微镜观察。结果共培养的支持细胞和生精细胞在体外存活超过6个月。在共培养期间,观察到精母细胞、圆形精子细胞和长形精子细胞。结论在不添加任何细胞因子和生长因子的情况下,大鼠曲细精管生殖细胞长期增生分化,不断产生精子细胞。这一结果暗示组织块和共生的支持细胞可为生殖细胞的增生和分化提供必需的细胞因子。该方法为体外研究精子发生过程提供了实验依据。     

Êxpression of inducible nitric oxide synthase (iNOS) in the azoospermic human testis

Azoospermia, which is the absence of spermatozoa in the ejaculate, is not a rare cause of male infertility. Inducible nitric oxide synthase (iNOS) is a calcium-independent NOS, which is present in the testis and involved in spermatogenesis, and apoptosis of Sertoli and germ cells. Twenty idiopathic infertile men presenting nonobstructive azoospermia were enrolled in this study, and testicular sperm extraction procedures were performed. Tissue extracts were dissected, and the fluid samples were investigated to determine the presence of spermatozoa. Histologic evaluation of the spermatozoa-present samples revealed that seminiferous tubules were normal and were lined by Sertoli cells and spermatogenic cells. However, in the spermatozoa-absent samples, the diameter of the seminiferous tubules was small, and Sertoli-cell-only syndrome was determined in most of the tubules. iNOS expression was very weak in Sertoli cells, germ cells, and in Leydig cells in the spermatozoa-present group. In the spermatozoa-absent group, the immunostaining was very intense in Sertoli and Leydig cells. Electron microscopy findings were supported the histologic results. In conclusion, complete germ cell loss and intense expression of iNOS in the Sertoli and Leydig cells in the spermatozoa-absent groups of azoospermic human testis suggest an essential role of iNOS in spermatogenesis.     

Bilateral prepubertal testicular biopsies predict significance of cryptorchidism-associated mixed testicular atrophy, and allow assessment of fertility

INTRODUCTION: Mixed atrophy of the testis (MAT), a frequent finding in biopsies of formerly cryptorchid and/or infertile patients, is defined as the synchronous occurrence of both seminiferous tubules containing germ cells and Sertoli cell only-tubules in variable proportions. In tubules containing germ cells, different types of abnormalities in spermatogenesis may be seen. The presence of adult spermatids in the biopsy, even in small numbers, correlates with successful spermatozoa retrieval for "in vitro" fertilization techniques. Currently, it is unknown whether precursor lesions of MAT can be identified in cryptorchid patients during childhood. MATERIAL AND METHODS: Eighteen formerly cryptorchid adults who had undergone testicular biopsies in childhood had a repeat testicular biopsy to evaluate infertility. In prepubertal biopsies, abnormalities of the testicular parenchyma were classified into types I (slight alterations), II (marked germinal hypoplasia), and III (severe germinal hypoplasia). In postpubertal biopsies, the percentage of tubules containing germ cells and Sertoli cell only-tubules were estimated, as well as the presence of complete spermatogenesis. Abnormalities in spermatogenesis were classified into lesions of the adluminal or basal compartments of seminiferous tubules. RESULTS: Comparison between prepubertal and postpubertal biopsies revealed that most specimens developing from type III lesions presented with incomplete spermatogenesis (P<0.0001) and more severe lesions of the germinal epithelium (P=0.049). DISCUSSION: Type III lesions correlated with MAT characteristics that confer a worse prognosis for in vitro fertilization. Thus, MAT characteristics may be predicted in prepubertal cryptorchid patients, allowing a fertility prognosis. The pathogenesis of these lesions, and their possible inclusion into the spectrum of the testicular dysgenesis syndrome, are discussed.     

Expression of endothelial nitric oxide synthase in the Sertoli cells of men with infertility of various causes

OBJECTIVE: To investigate how endothelial nitric oxide (eNOS) expression in the seminiferous tubules might be related to spermatogenesis, by examining eNOS expression in testicular tissue of patients infertile from various causes. PATIENTS AND METHODS: The study included five fertile men with a normal sperm concentration, nine patients with obstructive azoospermia, 20 with varicocele testes and eight with idiopathic azoospermia (Sertoli cell-only syndrome). Testicular biopsy specimens were examined by immunohistochemistry for eNOS protein expression, in addition to a routine pathological assessment. eNOS protein was detected using an eNOS monoclonal antibody. A Sertoli cell staining index (SSI) was defined as the ratio of stained Sertoli cells per total number of Sertoli cells, and was compared among the groups. RESULTS: eNOS was localized to Sertoli cells in the seminiferous tubules and Leydig cells in the interstium; although some degenerating germ cells stained, normal germ cells did not. The SSI was significantly lower in patients with Sertoli cell-only syndrome than in either fertile men or patients with obstructive azoospermia or varicocele. However, the SSI did not correlate significantly with the Johnsen score. CONCLUSION: The expression of eNOS in Sertoli cells may depend on the existence of germ cells and be associated with germ cell development.     

Recognising the Sertoli-cell-only (SCO) syndrome: a case study

Total Sertoli-cell-only (SCO) syndrome is often confused with a focal SCO picture, in which testicular illness caused damage to seminiferous tubules and compromised the Sertoli cell range of maturation and functions, but from which still some spermatozoa can be retrieved for assisted reproductive techniques. Here, a possibly new SCO syndrome phenotype is reported exhibiting complete lack of germ cells despite normal architecture of the seminiferous tubules with presence of mature Sertoli cells and normal Leydig cells in the intertubular tissue. Sertoli cells are immunonegative for the prepubertal differentiation markers cytokeratin-18, anti-Muellerian hormone and M2A antigen, but reveal a positive signal for the gap junctional protein connexin 43 known to be expressed in Sertoli cells with an adult type of differentiation. The complete lack of germ cells in combination with fully differentiated adult-type Sertoli cells in this case is in contradiction with known SCO subtypes and with the current hypothesis of reciprocal regulation of Sertoli and germ cell differentiation.     

雄性生殖细胞体外分化研究现状

生殖细胞的体外研究已建立了多种组织和器官培养系统、精曲小管培养系统、Sertoli细胞和生殖细胞共同培养系统等。随着对血 睾屏障和睾丸细胞极性在体外培养中重要性的逐步重视 ,已初步建立了模拟睾丸分隔结构的双室培养系统和藻酸钙三维培养系统。现有培养系统已经成为深入了解和认识精子发生过程的一种强有力工具。目前在体外已经获得某些动物精子发生的整个减数分裂过程 ,但对雄性生殖细胞体外减数分裂的详细调控机制还知之甚少。本文主要介绍了在雄性生殖细胞体外分化尤其是减数分裂研究方面的发展现状     

On the Postnatal Development of the Terminal Segment of the Seminiferous Tubules in Normal and Germ Cell Depleted Rat Testes

The development of the terminal segment of the seminiferous tubules was studied in 5 to 50 days old normal rats. At the age of 5, 10, and 15 days the terminal segment contained fewer gonocytes or spermatogonia than did the corresponding seminiferous tubule. The differentiation of the terminal segment was obvious at 20 days of age due to the high number of germ cells in the seminiferous tubules, where the epithelium became stratified at this stage. The blood-testis barrier in the terminal segment was chiefly established between 15 and 20 days of age as revealed by the lanthanum tracer technique.
To study the effect of the germ cells on the differentiation, the germ cell depleted testes of prenatally irradiated rats were also studied. The modified Sertoli cells of the terminal segment were more vacoulated and had fewer lipid droplets and inter-Sertoli cell junctions than did the Sertoli cells of the seminiferous tubules. The ultrastructure of the modified Sertoli cells of the terminal segment was similar in adult normal and adult SCO (Sertoli cell only) rats. The amount of lipid droplets in the Sertoli cells of SCO rats showed considerable variation among different tubular cross-sections within one testis.     

Sertoli cell junctional complexes in gossypol-treated neonatal and adult guinea pigs

The effect of gossypol, an experimental male contraceptive agent, on the development and maintenance of the blood-testis barrier was determined by feeding gossypol daily to prepubertal and adult guinea pigs, and then examining their testes by electron microscopy of thin sections and freeze-fracture replicas. In guinea pigs of 10 to 30 and 10 to 40 days of age that were fed gossypol, impermeable continuous junctional zones did not develop between adjacent Sertoli cells. Compartmentalization of germ cells in the seminiferous epithelium, therefore, was nonexistent. These findings were obtained by use of the sterol-binding polyene, filipin, used as a low molecular weight tracer in combination with freeze-fracture. In general, the seminiferous tubules lacked lumina and spermatogenesis did not progress beyond the pachytene spermatocyte stage. In adult guinea pigs fed gossypol daily for five weeks, continuous zonules at the base of the seminiferous epithelium appeared intact and were impermeable to filipin. Discontinuous zonules found higher in the epithelium showed distensions between interrupted junctional strands and were permeated by filipin. In addition, vacuolated spaces between Sertoli cells and clumps of heterochromatin were conspicuous in some of the Sertoli cell nuclei. Spermatogenesis was disturbed in about 10% of the seminiferous tubules examined. These perturbations included exfoliation of round and elongated spermatids with concomitant formation of multi-nucleated giant cells. Spermatozoa from these adult male guinea pigs were immotile. These findings suggest that, in neonatal animals, gossypol appears to prevent the maturation of Sertoli cells and this effect is expressed as the failure of focal Sertoli cell tight junctional strands to assemble into continuous zonules. In adult animals, gossypol appears to have no effect on the maintenance of the blood-testis barrier.     

Testicular Biopsy and Hormonal Study in a Male with Noonan''s Syndrome

The testicular biopsy study of a 17-year-old male with Noonan's syndrome revealed seminiferous tubules of reduced diameter with hypospermatogenesis. Many spermatocytes underwent degeneration and many spermatids developed abnormal. The Sertoli cells were similar to immature Sertoli cells. Fully differentiated Leydig cells were rare while precursor Leydig cells were numerous. Both gonadotropin and testosterone levels were low, and a lack of response to LH-RH as well as to clomiphene was found. The testicular biopsy performed at 20 years of age revealed a certain maturation of the seminiferous tubules which increased the germ cell number. The abnormalities in the spermatogenesis as well as the immature appearance of Sertoli cells continued. Leydig cells were more numerous and showed a certain development without reaching the normal pattern. Gonadotropin levels were normal while testosterone levels low. The response to LH-RH was increased and the absence of response to clomiphene persisted. These features suggest a delayed puberty.     

Expression of p50 C-terminal Src kinase (Csk) in mouse testis

C-terminal Src Kinase (Csk) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases to inhibit their activities. To investigate the possible regulation of spermatogenesis by Src-Csk loop, the postnatal changes in the expression of Csk were examined in mouse testes. Semiquantitative RT-PCR analysis revealed that Csk mRNA increased during neonatal development and peaked at 2 weeks of age. Following the decrease during pubertal development, Csk expression re-increased in adult testes. In Western blot, immature testes showed higher expression of Csk protein than the pubertal or adult testes. In immature testis, Csk immunoreactivity was largely found in the Sertoli cell and there was no visible difference in the Csk immunoreactivity among the seminiferous tubules. In adult testis, however, a differential Csk immunoreactivity was found among the seminiferous tubules. Intense signal was found in the adluminal cytoplasm of the Sertoli cells bearing the post-meiotic differentiating germ cells, suggesting that Csk may participate in the remodeling of seminiferous tubule during late phase of spermatogenesis. Csk immunoreactivity was also found in the Leydig cells, suggesting the possible regulation of Leydig cell function. Src-Csk loop may participate in the differentiation of the seminiferous epithelia and Leydig cells in mouse testis.     

Effects of experimental cryptorchidism and subsequent orchidopexy on seminiferous tubule functions in the lamb

The reversibility of damage caused by cryptorchidism to the seminiferous tubules of the lamb was investigated at various ages. Lambs were made bilaterally cryptorchid either at birth or at 2 months of age. Then orchidopexy was performed at either 2 or 4 months of age. In permanently cryptorchid lambs, spermatogenesis stopped completely, and Sertoli cell function, as measured by FSH receptors, androgen receptors and ABP, was much reduced (-96%, -86% and -81%, respectively). Orchidopexy allowed the cryptorchid seminiferous epithelium to grow again, but the more differentiated the germ cells, the less they were capable of restoration. Even in 0- to 2- and 0- to 4-month-old temporarily cryptorchid lambs that had recovered normal Sertoli cell function, 16 to 49% of the tubules still were empty. It was concluded that cryptorchidism irreversibly damages the seminiferous tubules at a level other than the hormone receptors.     

Lack of RBM expression as a marker for carcinoma in situ of prepubertal dysgenetic testis

Individuals with various intersex states who carry Y-chromosome material bear a high risk of developing testicular neoplasia. In order to gain more insight into the pathogenesis of this neoplasia, the current study evaluates the differentiation of the seminiferous epithelium in 46,XY dysgenetic male pseudohermaphroditism. Immunohistochemical evaluation was performed using the germ cell-specific RNA-binding motif (RBM) protein (encoded by the Y-chromosome) to identify normal germ cells, whereas placental alkaline phosphatase (PLAP) was used to detect neoplastic germ cells. Differentiation of somatic Sertoli cells was assessed using cytokeratin-18 (CK-18) and anti-Müllerian hormone (AMH) as markers for immature Sertoli cells. Specimens were taken from surgically removed dysgenetic gonads of five children (46XY karyotype). Intratubular germ cell neoplasia (carcinoma in situ [CIS] of the testis) was detected in all of them. Normal germ cells revealed immunoreactivity for RBM, whereas the PLAP-positive neoplastic germ cells were negative for RBM expression. Sertoli cells revealed an immature phenotype indicated by AMH expression in their cytoplasm. The design of the current study is unique in its assessment of the state of germ cell differentiation in dysgenetic gonads by the use of the RBM protein, which was expressed only in normal germ cells but not in those of CIS. Testicular dysgenesis interrupted the normal differentiation of the germ line and had no effect on the immature phenotype of the prepubertal Sertoli cells. This points toward the germinal component of CIS as the precursor for the promotion of testis cancer.     

Vasectomy impairs spermatogenesis through germ cell apoptosis mediated by the p53-Bax pathway in rats

PURPOSE: The pathophysiology of impaired spermatogenesis after vasectomy has not been completely investigated. We examined the role of p53 protein in cell cycle arrest and apoptosis of germ cells after vasectomy in the rat. MATERIALS AND METHODS: Eight-week old rats underwent bilateral vasectomy and the testes were harvested 1, 4, 8, 12 and 24 weeks after surgery. Germ cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and electrophoresis assay of DNA fragmentation. Western blot analysis and immunohistochemistry were performed to examine the expression of p53, proliferating cell nuclear antigen, Bax, Bcl-2, p21WAF1/Cip1 and Gadd45. To evaluate spermatogenesis, testicular weight and percent of haploid cells flow cytometry was done. RESULTS: Spermatogenesis impairment was associated with increased p53 and decreased proliferating cell nuclear antigen expression at the delayed phase more than 8 weeks after vasectomy. The number of TUNEL positive germ cells was increased at the early 1-week and delayed phases after vasectomy. Bax but not p21WAF1/Cip1 or Gadd45 expression was increased. p53, Bax and TUNEL positive cells were co-localized in the seminiferous tubules. CONCLUSIONS: Spermatogenesis was impaired after vasectomy by apoptosis but not by cell cycle arrest. The p53-Bax pathway effects apoptosis in the delayed phase after vasectomy in some seminiferous tubules.     

抑制素B βB亚单位在不同病理分型的无精子症患者睾丸组织中的表达

目的:探讨抑制素B(INH B)βB亚单位在不同生精功能状态的人睾丸组织中的表达情况。方法:对83例无精子症患者进行睾丸组织病理检查诊断,根据病理形态的不同分为:唯支持细胞综合征型(n=21);生精功能低下型(n=20);生精阻滞型(n=24);生精功能基本正常型(n=18)。选择上述各型结构完整的睾丸组织,分别应用免疫组化法(SP)对血清INH B βB亚单位在不同生精功能状态的睾丸组织,进行定位研究。结果:各型睾丸组织内均存在血清INH B βB的表达,其分布特点为:间质细胞(Leydig cell)和早期生精细胞多为强阳性表达,呈深棕黄色;支持细胞(Sertoli cell)多为阳性表达;而晚期精子细胞和成熟精子未见表达;生精小管管周类肌细胞呈弱阳性表达。结论:INH B可能是睾丸Sertoli细胞和早期生精细胞的一个联合产物。     

Seminiferous tubule degeneration in human cryptorchid testes

Two types of degenerating seminiferous tubules were found in cryptorchid testes with Sertoli cell hyperplasia of children and adults: 1) tubules with central degeneration, and 2) tubules with total degeneration. Central degeneration begins with degenerative changes in germ cells that accumulate in the lumen of the seminiferous tubule. Some Sertoli cells may also be affected. Degenerated cells finally disappear, and the remaining tubule is composed of only a cuboidal epithelium, which consists mainly of Sertoli cells and occasional germ cells surrounding a wide lumen. Total degeneration is principally seen in tubules with severe germinal hypoplasia. All the seminiferous epithelium cells degenerate and lose their characteristic distribution, forming a disorganized Sertoli cell nodule surrounded by a thickened basement membrane. Lastly, Sertoli cells disintegrate, and the seminiferous epithelium disappears. Tubular degeneration might be related to the thickening of the basement membrane, which hinders metabolic interchange between the seminiferous epithelium and the interstitium.     

Quantitative evaluation of guinea pig spermatogenesis: Epon versus paraffin embedding

A quantitative evaluation of spermatogenesis in the guinea pig was made at the light microscope level following improved methods of fixation and plastic embedding. The pattern of germ cell differentiation was studied by counting the average number of germ cell nuclei per Sertoli cell nucleus during the seminiferous epithelial cycle. Appreciable cell loss occurred during spermatogenesis. As a result, only 20.8% of the theoretically possible number of spermatozoa were produced from each spermatogonium. A slightly better yield of germ cells was obtained from Epon sections in comparison to paraffin sections. Epon sections can be effectively used for quantitative analyses of various cell populations in small biopsy specimens of testis tissue.     

Morphological alterations and intratubular lipid inclusions as indicative of spermatogenic damage in cimetidine-treated rats

Doses of cimetidine (50 mg/kg b/w) were administered to adult male Wistar rats over 52 consecutive days. Besides plasma testosterone levels, morphological and morphometric aspects of the seminiferous tubules as well as histochemical analysis of the lipid content by oil red O were emphasized. Abnormal tubules exhibiting disorganization of their cellular association, loss of germ cells, and multinucleated giant spermatids were usually found. Significant reductions of testis weight and tubular diameter at specific stages (VII-IX), as well as lack of contact between Sertoli cells and spermatids in tubules at stage IX, suggest a possible interference of cimetidine on the histoarchitecture of the seminiferous epithelium. The dense concentration of lipid inclusions in tubules at postspermiation stages indicates phagocytosis and degradation of germ cells. Since no change in serum testosterone levels was verified in cimetidine-treated rats, the authors could not exclude the possibility that besides an antiandrogenic effect, other biochemical factors necessary for normal spermatogenesis could be involved in the testicular alterations.     

Expression of beta-catenin in human testes with spermatogenic defects

beta-catenin is a multifunctional molecule that functions in intercellular adhesion and signal transduction during assembly of AJs between Sertoli cells as well as between Sertoli cells and germ cells. To assess changes in the testicular beta-catenin in male infertility conditions, testicular tissues from obstructive azoospermia with normal spermatogenesis, spermatogenic arrest (SA) and Sertoli cell-only syndrome (SCO) patients were examined for immunohistochemical localization of beta-catenin. In normal spermatogenic tissue, expression of beta-catenin was largely found in the Sertoli cell-germ cell (primarily spermatocytes) contact areas. Interestingly, perinuclear localization of beta-catenin was found in spermatocytes and spermatids. In spermatogenic arrest, beta-catenin in cell contact areas between Sertoli cells and germ cells was greatly decreased, but perinuclear beta-catenin in spermatocytes was not. In SCO, weak or negligible immunoreactivity of beta-catenin was found in cell contacts between Sertoli cells. Nuclear localization of beta-catenin was found in myotubular cells in all samples. Taken together, altered expression of beta-catenin in cell contacts within the seminiferous epithelia in spermatogenic arrest and SCO suggests that interactions between Sertoli cells and germ cell are crucial for expression of beta-catenin, and thus functional development of AJs in seminiferous epithelia in human testis. It should be also emphasized that perinuclear beta-catenin in germ cells may play a specific role in spermatogenesis.     

Expression of the integrin subunits alpha 5, alpha 6 and beta 1 in the testes of the common marmoset

Integrin subunits alpha 5, alpha 6 and beta 1 were localized in the testis of pre-pubertal or adult non-human primates (Callithrix jacchus) by immunofluorescence staining and in situ hybridization. In animals of all ages subunits alpha 5 and beta 1 were localized in cells of the lamina propria of the seminiferous epithelium. In prepubertal animals, the integrin subunits alpha 5, alpha 6, as well as beta 1, were distributed all over the plasma membrane of Sertoli cells. In adult animals the integrin subunits were confined to those plasma membrane regions of Sertoli cells which are assigned to the basal compartment, including the basement membrane of the seminiferous tubules. Protein expression of integrin subunits alpha 6 and beta 1 was most pronounced in tubular stages in which elongated spermatids were not yet present in the adluminal compartment of the epithelium, suggesting that these integrin subunits are particularly essential at certain developmental stages of spermatogenesis. Non-radioactive in situ hybridization revealed that the mRNA for integrin subunits alpha 5, alpha 6 and beta 1 was expressed by Sertoli cells. In situ hybridization, together with immunofluorescence data, shows that these integrin subunits were exclusively synthesized in Sertoli cells. As to functional aspects, it is concluded that during primate spermatogenesis. Sertoli cell integrins may be involved in both cell matrix as well as cell-cell interactions, particularly during early spermatogenesis.