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.     

Ultrastructure of the seminiferous tubules in oligoasthenoteratozoospermic men associated with varicocele

Varicocele is associated with venous reflux that may cause increased heat and interstitial pressure within the testes, with variable pathological effects on spermatogenesis. This study aimed to study the ultrastructural testicular changes in the seminiferous tubules of 20 infertile severe oligoasthenoteratozoospermia (OAT) men associated with varicocele and five patients with obstructive azoospermia without varicocele as controls. They were subjected to testicular biopsy which was evaluated by transmission electron microscopy. Ultrastructurally, the seminiferous epithelium in the testicular biopsies of infertile severe OAT men associated with varicocele was variably affected in the form of thickening of the peritubular connective tissue, vacuolation of Sertoli cell and germ cell cytoplasm, presence of degenerated and apoptotic cells among the germinal epithelium, altered spermatids and abnormal spermatozoa. It is concluded that varicocele in severe OAT men is associated with ultrastructural changes in the seminiferous tubule.     

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。     

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.     

Mast cells and fibrosis on testicular biopsies in male infertility

Testicular dysfunction correlates with increased testicular mast cells. Mast cells can activate fibroblasts and promote collagen synthesis. The aim of the study was to examine testicular mast cells containing tryptase, and the relationship between mast cells and different fibrosis stages of interstitium and peritubular region of testes. Testicular biopsies obtained from 33 infertile men were assigned to 2 groups: normal spermatogenesis (n = 10) and defective spermatogenesis (n = 23). Total, interstitial, and peritubular mast cells were examined immunohistochemically using antihuman tryptase. The fibrosis stage was evaluated using vimentin and alpha-smooth muscle actin. The ratio of tubules with sclerosis to total tubules was also calculated. In all cases, mast cells were mainly localized in the interstitium. The number of total mast cells was significantly higher in defective spermatogenesis than in normal spermatogenesis (p = .048). In both groups, interstitial mast cells were higher than peritubular mast cells. However, the increase in peritubular region was much higher than the increase in interstitium. Total, peritubular, and interstitial mast cell counts were not different from each other, according to the changing fibrosis stages. Total and interstitial mast cells were significantly higher in the cases with sclerosing seminiferous tubules than in the cases with no sclerosis (p = .04 and p = .024, respectively). The mast cells and the mast cell product tryptase could be involved in the etiology of defective spermatogenesis, especially whenever the last stage (tubular hyalinization and sclerosis) takes place.     

Ontogeny and cellular origin of a rat seminiferous tubule factor(s) that inhibits LH-dependent testosterone production by interstitial cells in vitro

Previous studies have shown the presence of a peptide in spent media from incubated seminiferous tubules (SMST), which inhibits LH stimulation of testosterone production by rat Leydig cells in vitro. The present study has investigated whether the secretion of this inhibitor changes during development in the rat. Seminiferous tubules obtained from rats aged 10, 20, 25, 30, 35, 40, 42, 50 or 60 days were incubated at 32 degrees C for 24 h. Spent media from these incubations were then added to interstitial cells isolated from the testes of rats aged 60 days. Spent media from rats aged 10-30 days had no effect on basal or oLH-stimulated testosterone production by interstitial cells during 3-h incubation. Significant inhibition of LH-stimulated testosterone production was, however, observed with SMST from rats aged 35-60 days. Spent media prepared using tubules from normal, prenatally irradiated (Sertoli cell-enriched) or seminiferous tubules, depleted of peritubular cells, had no effect on basal, but inhibited LH-stimulated, testosterone production. Spent media from peritubular cell cultures had no effect on basal or LH-stimulated testosterone production by interstitial cells. The inhibitory effect of SMST was also dependent on the age of the rats providing the target cells. Interstitial cells from rats aged 10, 20, 50 or 60 days were responsive to the inhibitor while cells from rats aged 30 and 40 days were not. The results of the present study demonstrate that the seminiferous tubule factor(s), which inhibits LH action on interstitial cells, is first secreted at 35 days, a time when the most mature germ cells present are in the early maturation phase. Moreover, interstitial cells are responsive to this factor in both immature (10-20 day-old) and mature (50-60 day-old) rats, but not at ages in between these times. It is suggested that the adult Sertoli cell is the major source of the interstitial cell inhibitor.     

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。     

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).     

Spermatogenesis--physiology and pathophysiology

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).     

Effect of pineal indoles on testicular histology of mice

The effect of late afternoon injections of melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-methoxyindole-3-acetic acid on testicular histology in mice were examined. Melatonin and 5-methoxytryptophol injections caused a reduction in the diameters of seminiferous tubules. The tests of melatonin-treated animals underwent some detectable regressive changes in the seminiferous tubules, whereas administration of 5-methoxytryptamine or 5-methoxytryptophol appeared to cause atrophy in some tubules. The percentage of aspermic tubules in melatonin-treated and methoxytryptamine-treated mice was significantly higher than that of the control. In involuted testes, some seminiferous tubules contained only Sertoli cells together with spermatogonia and spermatocytes, but no discernible spermatids and spermatozoa. Regressing spermatids and cell debris were frequently observed in the tubules. The tested of mice that received daily injections of 5-hydroxytryptophol and 5-methoxyindole-3-acetic acid were indistinguishable from those of the controls.     

一氧化氮合酶同功异构酶在大鼠睾丸中的表达和定位

目的 :了解一氧化氮 ( NO)在睾丸中的作用。方法 :运用免疫组织化学方法观察 3种一氧化氮合酶 ( NOS)同功异构酶在大鼠生后 4、7、1 4、3 0、60 d睾丸中的分布。结果 :( 1 )生后 4、7、1 4d大鼠睾丸未见 3种 NOS免疫阳性反应 ;( 2 )生后 3 0 d少数精母细胞及生后 60 d生精小管腔面精子和间质细胞呈 NOS1阳性 ;( 3 )生后 3 0 d少数精母细胞、支持细胞和管周类肌细胞呈 NOS2阳性 ,而生后 60 d NOS2阳性反应见于睾丸间质细胞、管周类肌细胞、支持细胞、极少数精母细胞和不成熟精子头部 ;( 4)生后 3 0 d睾丸内少数精母细胞和血管壁呈 NOS3阳性 ,生后 60 d NOS3阳性反应仅见于血管壁。结论 :NO可能参与精子发生、睾酮的分泌过程 ,并调节睾丸内的血流。     

Histopathology of testis in acquired immune deficiency syndrome

Histologic sections from the testes of 32 autopsied patients with acquired immune deficiency syndrome (AIDS) were examined. Almost invariably the testes displayed decreased spermatogenesis, and 20 of the 32 cases showed marked hypospermatogenesis with Sertoli cells predominantly lining the tubules. Although the seminiferous tubules were generally of normal size, the tunica propria at the periphery of the tubules was mildly to moderately thickened in 19 cases and markedly thickened in 10. The interstitial cells of Leydig were unaltered in most patients, with only 4 testes showing Leydig cell hyperplasia. The testicular blood vessels were slightly thickened in many patients, but 5 exhibited moderate to marked intimal proliferation with narrowing of the lumen. Mononuclear inflammatory infiltration of the testicular interstitium was slight in 11 cases, moderate in 6. Only 7 of the 28 AIDS patients with opportunistic infections had evidence of direct involvement of the testes by the infectious organisms. We concluded that the extragonadal endocrine balance of AIDS patients may be deranged due to the infectious process and so deserves clinical evaluation.     

Ectopic Leydig Cells in Seminiferous Tubules of an Infertile Human Male with a Chromosomal Aberration

A case of a human male infertility with chromosomal aberration is reported. The patient showed neither mental retardation nor physical abnormalities except that the testes were somewhat small and soft. Plasma follicle stimulating hormone and luteinizing hormone were 49.0 and 19.0 mIU/ml. Plasma testosterone was 2.6 ng/ml. Karyotype was considered to be 46 XY q-, long arms of the Y chromosome being deleted. Histological features of the testis were peculiar. Seminiferous tubules were small and devoid of spermatogenic cells, consisting only of Sertoli cells. Peritubular boundary layer of the tubules showed a marked increase in width due to the increase of collagen fibers. The base of some Sertoli cells was seen to protrude into the thickened peritubular boundary layer or, though rare, into the interstitial space. Unusual cells which had a round vesicular nucleus and abundant, dense cytoplasms also occurred in the boundary layer of most tubules. These cells were identified as Leydig cells because of an extensively developed smooth endoplasmic reticulum in their cytoplasm, although they lacked Reinke's crystals. These ectopic Leydig cells sometimes lay in direct contact with Sertoli cells in the seminiferous tubule.     

Alkaline phosphatase histochemistry discriminates peritubular cells in primary rat testicular cell culture

Histochemical demonstration of alkaline phosphatase activity appears to be useful in identifying rat peritubular cells in primary testicular cell culture. In both frozen sections of rat testis and Mirsky's fixed, methacrylate-embedded rat testis, the reaction product localized primarily in peritubular cells, vascular endothelium and occasionally in interstitial cells, with much smaller amounts of reaction product associated with elongating spermatids in the germinal epithelium. Occasional late-stage tubules (X-XIV) showed weak reactivity in the epithelium, associated with spermatocytes or Sertoli cells. Ultrastructurally, Gomori-method reaction product was localized to peritubular cells, lymphatics, and spermatogonia in stage VII; no staining was found consistently in Sertoli cells. In isolated cell preparations enriched for Sertoli and germ cells, 1 to 8% of the cells demonstrated alkaline phosphatase activity, while greater than 50% of the cells stained positive for alkaline phosphatase activity in peritubular-enriched fractions. The histochemical demonstration of alkaline phosphatase activity can be useful for identifying peritubular cells in primary cultures of testicular cells.     

Melatonin prevents testicular damage in hyperlipidaemic mice

The damaging effect of hyperlipidaemia on testicular structure was determined, and the influence of melatonin was evaluated in testicular damage related to hyperlipidaemia. Hyperlipidaemia was induced in ApoE-knockout C57BL/6J male mice fed with high-fat diet alone (group A), or with high-fat diet and melatonin (group B). Six ApoE wild-type C57BL/6J male mice were fed with normal diet, served as controls. At the end of the experimental period, ultrastructural observations showed dramatically histopathological alterations in testicular tissues of group A. The basement membranes of seminiferous tubules were partially thickened and wavy-like in testes of mice with hyperlipidaemia, and vacuolar degeneration of mitochondria and dilation of endoplasmic reticulum were identified as well as the number of mitochondria and lipid droplets decreased significantly in Leydig cells and Sertoli cells. Electrondense deposits were observed in cytoplasms of germ cells. The testicular histostructure in group B treated with melatonin was similar to that of control. Apoptosis was determined by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labelling. Apoptotic germ cells were significantly more numerous in group A than in group B and controls. The results suggest that melatonin may be potential to attenuate testicular damage by improving histopathological changes and reducing germ cell apoptosis in hyperlipidaemic mice.     

Ontogeny and cellular origin of an interleukin-1 -like factor in the reproductive tract of the male rat

We have recently isolated an interleukin-1 (IL-1)-like factor from the rat testis, which originates from the seminiferous tubules and is a protein with an MW of 17,000 and a pI of 5-6. This paper reports on the appearance of the IL-1-like factor during postnatal development and investigates its cellular origin further. IL-1 activity was measured by a murine thymocyte proliferation assay. Very low IL-1 activity was present in culture medium conditioned by seminiferous tubules from rats aged 10 or 20 days. From 30 days of age, increasing amounts were detected, reaching a maximum level in adult animals (60-90 days). No IL-1 activity was found in medium conditioned by peritubular cells. Sertoli cell-enriched seminiferous tubules obtained from experimentally cryptorchid or from prenatally irradiated rats produced much higher levels of IL-1 activity than did those obtained from intact testes. IL-1 activity was detected in efferent duct fluid after ligation of the efferent ducts for 24 h, indicating that the IL-1-like factor was secreted into the tubular lumen. Low levels of IL-1 activity were detected in extracts of epididymal tissue and epididymal sperm, whereas ejaculated seminal plasma, seminal vesicle fluid and extracts of seminal vesicles (together with the coagulating glands) and ventral and dorsolateral prostate lacked IL-1 activity. Instead, seminal plasma inhibited testicular IL-1 activity dose-dependently without affecting cell viability in the thymocyte cultures. Although its biological function remains to be defined, our results indicate that the testicular IL-1-like factor is produced by Sertoli cells and that its appearance during development coincides with the initiation of active spermatogenesis in the rat testis.     

Morphological evidence for two types of idiopathic ''Sertoli-cell-only'' syndrome

Biopsies of testicular specimens taken from 41 patients that were diagnosed as having idiopathic Sertoli-cell-only syndrome were classified into two types, A and B, on the basis of histological and immunohistochemical findings. Thirty eight specimens that were classified as type A exhibited seminiferous tubules of small diameter and with tubular wall hyalinization, but containing normal adult type Sertoli cells. The other three specimens that were classified as type B showed no seminiferous tubular wall hyalinization, and their Sertoli cells had vimentin distribution localized in the subnuclear cytoplasm and had a pseudostratified lining, features resembling the appearance of fetal Sertoli cells. In one patient with a seminoma, a comparative study of the same testis prior to and post-irradiation was undertaken. Judging from this, postpubertal depletion of the germ cell population was considered to be responsible for the tubular atrophy observed in type A. Type B testes, though small in number, were characterized by a morphology distinct from the type A, but their pathogenesis remains unknown.     

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.     

Epidermal growth factor receptors (EGFR) localization in human testis.

An indirect immunofluorescence technique was used to detect and localize epidermal growth factor receptors (EGFR) in human testicular biopsies in different testicular diseases. Monolateral biopsies from twelve infertile subjects were studied from qualitative/quantitative points of view and were examined by immunofluorescence study with anti-EGFR monoclonal antibody. Two different patterns of EGFR expression were observed: a very weak presence of EGFR was detectable on Sertoli cells, peritubular basal structures, and interstitial compartment in testicular biopsies showing a normal spermatogenic status; and an important increase in EGFR expression was observed on the cytoplasm of Sertoli cells and on peritubular basal structures in biopsies exhibiting various degrees of hypospermatogenesis and in a case of Sertoli cells only syndrome. Germ cells did not show EGFR immunolocalization. EGFR seems to be present on different testicular target cells. EGFR expression increases on peritubular and Sertoli cells in the presence of significant tubular damage.