Evaluation of the ultrastructural changes in the human Sertoli cell in testicular disorders and the relationship of the changes to the levels of serum FSH

Sertoli cell ultrastructure was compared in men with testicular disorders (hypospermatogenesis; germ cell aplasia) and men with normal testes to determine if any specific cytological change could be correlated with diminished feedback from the testis resulting in elevated serum FSH levels. The normal Sertoli cell contained smooth endoplasmic reticulum, mitochondria and variable numbers of lipid inclusions, lipofuscin, crystals of Charcot-Böttcher and specialised inter-Sertoli cell junctional complexes. The principal abnormalities in Sertoli cells of men with testicular disorders were: 1) dilated vesicles of smooth endoplasmic reticulum and occasional expansions of the intercellular space; 2) increased numbers of cytoplasmic filaments; and 3) with germ cell aplasia inter-Sertoli cell junctions were complexly arranged due to interdigitation of Sertoli cell processes. Occasionally, increased lipid and lipofuscin aggregations were seen and in germ cell aplasia, aggregations of cytoplasmic glycogen were often present. Although these changes were seen more consistently with germ cell aplasia they were observed frequently with hypospermatogenesis where some tubules contained Sertoli cells with normal features. No correlation was found between abnormal Sertoli cell cytology and serum FSH levels.     

Evaluation of the ultrastructural changes in the human sertoli cell in testicular disorders and the relationship of the changes to the levels of serum FSH

Sertoli cell ultrastructure was compared in men with testicular disorders (hypospermatogenesis; germ cell aplasia) and men with normal testes to determine if any specific cytological change could be correlated with diminished feedback from the testis resulting in elevated serum FSH levels. The normal Sertoli cell contained smooth endoplasmic reticulum, mitochondria and variable numbers of lipid inclusions, lipofuscin, crystals of Charcot-B?ttcher and specialised inter-Sertoli cell junctional complexes. The principal abnormalities in Sertoli cells of men with testicular disorders were: 1) dilated vesicles of smooth endoplasmic reticulum and occasional expansions of the intercellular space; 2) increased numbers of cytoplasmic filaments; and 3) with germ cell aplasia inter-Sertoli cell junctions were complexly arranged due to interdigitation of Sertoli cell processes. Occasionally, increased lipid and lipofuscin aggregations were seen and in germ cell aplasia, aggregations of cytoplasmic glycogen were often present. Although these changes were seen more consistently with germ cell aplasia they were observed frequently with hypospermatogenesis where some tubules contained Sertoli cells with normal features. No correlation was found between abnormal Sertoli cell cytology and serum FSH levels.     

Disrupted expression of intermediate filaments in the testis of rhesus monkey after experimental cryptorchidism

Cytoskeletons in Sertoli cell play an important role in process of spermatogenesis. The expression and distribution of the intermediate filaments, vimentin, keratin and desmin, were studied in the Sertoli cells of the cryptorchid testis of rhesus monkey. Vimentin was localized in the perinuclear region of Sertoli cells of the normal testis. An intense increase in vimentin immunoreactivity was observed with appearance of disorganized staining in the Sertoli cells of the cryptorchid testes. Cytokeratin 18, a marker of immature Sertoli cells, re-expressed in the cells of the adult cryptorchid testes. Desmin was also observed in the Sertoli cells in addition to the peritubular myoid cells on 30 days after the cryptorchid operation. These data suggest that Sertoli cells in primate can be affected by the heat stress. The altered changes in intermediate filaments could be possible to induce the Sertoli cell functional changes that would partially contribute to the germ cell apoptosis leading to azoospermia or oligozoospermia.     

Cellular origins of testicular dysgenesis in rats exposed in utero to di(n-butyl) phthalate

Foetal exposure of male rats to di(n-butyl) phthalate (DBP) induces testicular changes similar to testicular dysgenesis syndrome in humans, including the formation of focal 'dysgenetic areas' within post-natal testes, surrounded by otherwise normal tubules exhibiting complete spermatogenesis. We hypothesize that these dysgenetic areas form when Sertoli (and other) cells are 'trapped' during the abnormal formation of large Leydig cell (LC) clusters in foetal life and by post-natal day (d) 4 these groups of intermingled cells attempt to form seminiferous tubules. It is likely that the malformed tubules resulting correspond to the dysgenetic areas evident in later life. This also provides a plausible explanation for the occurrence of LCs within seminiferous cords/tubules in or bordering the dysgenetic areas. In our previous studies intratubular LCs (ITLCs) were identified by immunostaining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), the definitive LC cytoplasmic marker. However, the possibility remained that the 'presumptive' ITLCs were in fact Sertoli cells that had aberrantly gained the ability to express 3beta-HSD. Therefore, the aim of the present study was to fully characterize the ITLCs induced by in utero DBP exposure in d25 rats using a number of LC- (3beta-HSD, P450 side-chain cleavage enzyme, insulin-like factor 3, oestrogen receptor alpha) and Sertoli cell- (vimentin, Wilm's tumour-1) specific markers. Our results show that ITLCs express all four LC-specific markers but do not express either of the Sertoli cell markers. It is therefore concluded that the ITLCs are bona fide LCs that are abnormally located within the seminiferous tubules of DBP-exposed rats in post-natal life.     

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.     

Ultrastructural changes in Sertoli cells in ageing humans

Ultrastructural study of seminiferous tubules in ageing men revealed a varying degree of spermatogenetic arrest associated with changes in the Sertoli cells. Approximately half of the Sertoli cells showed a normal mature nuclear appearance although the cytoplasm was altered morphologically. These cells were classified as containing abundant lipid droplets (30%), containing large cytoplasmic vacuoles filled with an amorphous material similar to that in the tubule lumen and surrounded by junctional specializations (8%), multinucleated Sertoli cells (4%), or Sertoli cells with numerous mitochondria displaying tubular cristae (2%). The remaining 7% of Sertoli cells had an immature nuclear appearance and sparse development of the cytoplasmic organelles; these cells probably represent dedifferentiated Sertoli cells. Although individual differences were marked, a correlation between the increase in gonadotrophin levels and changes in both germ cell development and Sertoli cell structure was observed.     

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.     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.     

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。     

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.     

Immunohistochemical distribution of S-100 protein and subunits (S100-alpha and S100-beta) in the swamp-type water buffalo (Bubalus bubalis) testis

The distribution and localization of S-100 protein (S-100) and its subunits (S100-alpha and S100-beta) in the testis of swamp-type water buffalo were investigated using immunohistochemistry. S-100 was detected in the Sertoli cells in the convoluted seminiferous tubules, modified Sertoli cells lining the terminal segment of the seminiferous tubules and in the intratesticular excurrent ducts (straight tubules and rete testis). S100-beta showed the same distribution and localization with that of S-100. However, the cytoplasmic extension of the Sertoli cells in S100-beta staining showed less staining intensity compared with that of S-100. S100-alpha showed a positive staining only in the modified Sertoli cells of the terminal segment of the seminiferous tubule. Endothelial cells of blood vessels were also positive with the proteins while the Leydig and spermatogenic cells showed a negative reaction. The localization of S-100 in the testis of the water buffalo was in parallel with that of other artiodactyls which supports the hypothesis that this protein is a multifunctional protein. S100-beta in the Sertoli cells suggests that this protein is involved in establishing blood-testis barrier. Its presence in the modified Sertoli cells and in the epithelium of the excurrent ducts suggest secretory and absorptive function, respectively. Meanwhile, S100-alpha, which was detected only in the modified Sertoli cells, is involved in the secretory activity of these cells that are related to exocrine function.     

S-100 Protein immunoreactivity in bovine testis

The present study deals with immunohistochemical localization of S-100 protein in the bovine testis. Immunoreactivity for the protein was seen in Sertoli cells of the seminiferous tubules and as a particularly intense staining in the terminal tubular segment (transitional region, middle portion, and terminal plug), which is mainly composed of modified Sertoli cells. Immunoreactivity was also found in epithelial cells of the straight testicular tubules and rete testis, and in the endothelium of capillaries, veins and lymphatic vessels. Although the functional significance of S-100 protein immunoreactivity in the Sertoli cells remains unclear, the present results suggest that it may be involved in the microtubule assembly-disassembly system. The specificity of the immunolabelling observed should enable the antigen and/or antibody to S-100 to be used as an investigative and diagnostic tool in the study of bovine Sertoli cell function.     

Intratubular large cell hyalinizing sertoli cell neoplasia of the testis: a report of 8 cases of a distinctive lesion of the Peutz-Jeghers syndrome

We report the clinical and pathologic features of 8 boys with Peutz-Jeghers syndrome who had distinctive testicular lesions. The patients were 4 to 13 years of age (mean, 6.5 y), and all had gynecomastia, which was the presenting feature in 7. Physical examination demonstrated bilateral testicular enlargement in the absence of a discrete mass. Advanced bone age and elevated serum estradiol were demonstrated in 3 and 4 cases, respectively. Testicular biopsy, performed in all cases, usually showed no gross abnormality, but on microscopic examination there were patchily distributed clusters of expanded seminiferous tubules that contained large Sertoli cells with vacuolated to eosinophilic cytoplasm admixed with globular deposits of basement membrane that extended from a thickened peritubular basement membrane. Small, focal calcifications occurred in 3 cases; no invasive tumor was present in any of the cases. Follow-up was available in 5 patients after biopsy, and none showed evidence of progression at 10 months to 5 years (median, 4 y). Review of the previously reported cases of testicular lesions in Peutz-Jeghers patients verified a low frequency of invasive tumors (27%) and no known case with metastasis. The testicular lesions seen in patients with Peutz-Jeghers syndrome mostly represent multifocal intratubular neoplasia of large Sertoli cells with unique morphology distinct from other lesions such as the large cell calcifying Sertoli cell tumor and sex cord tumor with annular tubules. The process usually remains confined to the tubules for prolonged intervals (years), but it may occasionally progress to invasive large cell Sertoli cell tumors with or without associated calcification. This indolent course justifies management by careful follow-up, including ultrasound examination, rather than orchiectomy in the majority of cases. Orchiectomy is indicated when there is evidence of an invasive tumor and may be necessary to control hormonal manifestations.     

Mechanism of alcoholic testicular damage]

To observe the mechanism of alcoholic testicular damage, in a previous experiment we used weanling male SD rats aged 45 days, weighing about 200 g, and fed a liquid diet (Lieber's) containing 5% ethanol. The latter accounted for 36% of total caloric intake for 7 weeks, but did not result in testicular atrophy. In a later experiment, we used a liquid diet in which ethanol accounted for 46% of the total calorie count. It provided a high-fat, low-protein content which simulated the nutritional background of patients with alcoholic liver diseases. This diet resulted in testicular atrophy. Histological and biochemical changes accompanying this experimental testicular atrophy included the following: 1) The testes of alcohol-fed animals contained smaller seminiferous tubules with reduced numbers of total cells, but no degeneration was seen in the spermatids. 2) In the peritubular wall of the seminiferous tubules, we observed curvature, irregularities, infolding of the basement membrane, and lamellation of the lamina densa, as well as hyperplasia of collagen fibers in the tunica propria. 3) In the cytoplasm of the Sertoli cells, deposits of gigantic fat droplets and stratification of the mitochondria were observed. The permeability of the Sertoli cell tight junction was confirmed using the Lanthanum method. 4) Testosterone levels in both the serum and testes declined. 5) Lactate dehydrogenase-X (LDH-X) activity in the testes declined. 6) Low Km alcohol dehydrogenase (ADH) activity localized in the testicular interstitial tissue was increased. These results indicate that the composition of three major nutritional elements as well as alcohol concentration are important in the mechanism of alcoholic testicular damage, and this damage affects both the testicular interstitial cell and the seminiferous tubules, particularly the Sertoli cells and peritubular wall of the latter. In addition, the findings suggest that ADH is involved in alcohol metabolism in the interstitial cells of the testes.     

睾丸细胞生物学研究进展

睾丸是男性生殖腺,由生精小管和间质构成。生精小管主要由生精细胞和支持细胞组成,是精子发生场所;间质中主要是间质细胞,间质细胞合成与分泌雄激素。本文介绍睾丸3种细胞的发育分化,以及成年期睾丸细胞的结构和生物学研究进展。     

Pattern of Sertoli cell degeneration in cryptorchid prepubertal tests

Seventy-three testicular biopsies from 54 children (aged 2 months-14 years) with undescended testes were examined by light and electron microscopy. The biopsies included abdominal, inguinally fixed, inguinally moveable, and retractile testes. Alterations in Sertoli cell morphology were found in all biopsies. The alterations included dilated elements of rough endoplasmic reticulum, vacuolization of the cytoplasm, mitochondria with poorly preserved cristae, increase in electron density of the matrix, elongation of the nuclei, and irregularities of the nuclear membrane. According to the numerical appearance of these cells and to the extent of lesions in single Sertoli cells, seven phases in the continuous process of tubular alteration were distinguished. The most severe tubular damaged (phase VII) occurred when the seminiferous epithelium consisted exclusively of necrotic cells. All phases of tubular alterations were seen regularly in each of the biopsies investigated. Germ cells occurred only in phases I-IV and were never observed in tubules in phases V-VII. Significant differences became evident between inguinal and retractile testes by morphometric evaluation. It was demonstrated that the number of germ cells per cross-sectioned tubule (S/T value) correlated negatively with the percentage of tubules in phases V-VII. In contrast to inguinal testes, a complete absence of Sertoli cells and an S/T value less than 0.1 were never found in retractile testes and the percentage of tubules in phases V-VII was reduced significantly compared with inguinal testes. Our findings indicate that (i) maldescended testis in patients between 1 and 15 years-of-age is associated with a special pattern of Sertoli cell degeneration; (ii) Sertoli cell degeneration is a continuous process, which can lead eventually to complete dissolution of the seminiferous epithelium; (iii) total degeneration is not related to age but is dependent on testicular position; (iv) a defined phase of degeneration excludes germ cell development, and therefore enhanced Sertoli cell degeneration in cryptorchid testes must also account for the reduction in germ cell number.     

Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptorchidism in the rat

Summary.  In order to elucidate the respective effects of depletion of germ cells and of increase in testicular temperature, rats of the same Wistar strain were rendered experimentally bicryptorchid or sterilized by a busulfan injection in utero and compared to control animals. In both models, germ cells were depleted but numeric evolution and functions of somatic cells differed. The aim of that work was to compare the numeric evolutions of testicular somatic and germ cells to their respective functions in each model before puberty and in adult rats of the same strain. Serum concentrations of FSH, LH and testosterone were compared at 20, 40 and 110 days of age. Histological analyses of Sertoli and germ cells in the seminiferous tubules and of Leydig cells in the intertubular tissue were performed before puberty and at adulthood. Testosterone serum concentrations were depleted in both models starting at 40 days of age and more in busulfan-treated rats. Both FSH and LH levels were increased from 20 days onwards in experimental rats. Additional cryptorchidism in busulfan-treated rats depressed the serum testosterone concentration. At 17 days of age, the cryptorchidism do not modify somatic or germ cell populations while busulfan treatment has induced a decrease of both these populations. Conversely, the cross sectional area of the somatic testicular cells was not affected whatever the treatment. In adult testes of busulfan-treated and cryptorchid rats, the total numbers of Sertoli and Leydig cells and of germ cells per testis were decreased. The cellular size of the perivascular Leydig cells was not modified by any of the treatments whereas the size of the Sertoli cells was reduced.
In conclusion, in both models the absence of germ cells induces a decrease in Sertoli cell function, while the increase in testicular temperature provokes degeneracies of Sertoli and germ cells in the seminiferous tubules of the rat.     

The rat testis produces large amounts of an interleukin-1-like factor

Homogenates of whole testis, isolated seminiferous tubules, testicular cytosol, conditioned media from seminiferous tubules obtained from intact or cryptorchid rats, as well as seminiferous tubules devoid of peritubular cells, showed high concentrations of interleukin-1 (IL-1). Cytosol from spleen showed low IL-1 activity, while no activity was found in cytosol from heart, kidney, prostate, ovary or liver. Interleukin-1 activity was not detected in spent medium from cultures of immature Sertoli cells (10-day-old rats) or from peritubular cells or in homogenates of interstitial cells from adult rats. Ultrogel AcA 44 gel chromatography and HPLC size exclusion chromatography exhibited a single peak of IL-1 activity corresponding to a relative molecular mass of 17,000-20,000 (Mr = 17-20 K). Similarly, chromatofocusing revealed only one peak of activity with an apparent isoelectric point of 5-6. It is concluded that the rat testis contains large amounts of an IL-1 alpha-like factor. The adult Sertoli cell or possibly germ cells are suggested as its primary source. Testicular IL-1-like activity is of particular interest in view of the intense cell proliferation during spermatogenesis, and the tendency to testicular relapse of acute lymphoblastic leukaemia.     

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.