Development of bovine fetal testis tissue after ectopic xenografting in mice

Testis tissue xenografting represents a versatile model to study testis biology, and to preserve fertility in immature animals. To evaluate whether bovine fetal testes can mature when grafted into mouse hosts, small fragments of testes from midgestation (125 to 145 days of gestation) bovine fetuses were grafted ectopically into immunodeficient castrated male mice. At grafting, donor tissue displayed the typical seminiferous cords composed of gonocytes and primitive Sertoli cells. At 5 or 10 months after grafting, weight of the seminal vesicles in recipient mice was indicative of production of bioactive testosterone by xenografts. Xenografts showed similar development regardless of donor age. At 5 months, tubule formation occurred but germ cell differentiation had not proceeded beyond the spermatogonia stage. At 10 months, an increase in tubule size was evident and pachytene spermatocytes were observed as the most advanced type of germ cells in the xenografts of 2 donors. The number of tubules with germ cells was reduced in xenografts compared to donor tissue, but at 10 months the number of germ cells per tubule was higher than in donors. Germ cell proliferation was similar in donor tissue and xenografts. However, Sertoli cells showed a higher proliferation rate in xenografts collected at 5 months than in donor fetal testes and xenografts collected at 10 months. Sertoli cells in xenografts showed a progressive but incomplete loss of expression of Müllerian inhibiting substance and weak androgen receptor expression, indicating an incomplete Sertoli cell maturation. In conclusion, fetal testis tissue developed partially, qualitatively similar to pubertal testes in situ.     

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.     

Electron microscopic evidence for deep invaginations of the lamina propria towards the seminiferous tubule lumen in a patient with varicocele

Ultrastructural studies on biopsy tissue from the right testis of a 39-year old patient with varicocele revealed 2.5-5 microns thick invaginations of the lamina propria towards the lumen of the seminiferous tubules. These invaginations were of various lengths. The presence of invaginations was confirmed through examination of serial semi-thin sections. In some seminiferous tubules two neighbouring deep invaginations were joined together thus completely encircling and thereby separating the basal compartment, and in some cases even the adluminal compartment, of the seminiferous tubule. The invaginations were surrounded continuously by the basement membrane and contained collagen fibres, cell processes of myoid cells and in some cases also their nuclei.     

Effects of luteinizing hormone, follicle-stimulating hormone, and epidermal growth factor on expression and kinase activity of cyclin-dependent kinase 5 in Leydig TM3 and Sertoli TM4 cell lines

We examined the effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and epidermal growth factor (EGF) on the expression and kinase activity of cyclin-dependent kinase 5 (Cdk5) in Leydig TM3 and Sertoli TM4 cell lines. Hormonal regulation of the expression and activity of Cdk5 by using normal and hypophysectomized rat testes was also investigated to elucidate its role. Cdk5 levels and kinase activity were significantly elevated in TM3 cells that were grown in the presence of 7.5% serum, EGF, or LH and were associated with an increase in testosterone production compared with controls. These increases were accompanied by an increase in proliferation of TM3 cells after treatment with serum or EGF but not with LH suggest that Cdk5 may be involved in cellular differentiation that is induced with LH treatment. In contrast, the presence of neither serum, EGF, nor FSH had a significant effect on Cdk5 activity levels in the Sertoli TM4 cell line, and there was no correlation with proliferative activity or transferrin levels. A significant decrease in Cdk5 expression and activity were noted in rat testis after hypophysectomy compared with normal rat testis and is associated with a simultaneous decrease in testosterone and transferrin levels. Immunohistochemical analysis revealed that Cdk5 was strongly expressed in the nuclei and cytoplasm of Leydig cells, Sertoli cells, spermatogonia, and peritubular cells of normal adult rat testis. After hypophysectomy, the pattern of Cdk5 staining differed markedly from that in normal rat testis and a profound reduction in staining of Cdk5 was observed in each tubule. Our results suggest that LH and EGF influence and modulate Cdk5 expression and activity in Leydig TM3 cells and may, conceivably, be involved in signal transduction cascades that are initiated by hormones or growth factors. Cdk5 in Sertoli TM4 cells is likely to possess some constitutive functions that are not affected by the cells' proliferation state. Moreover, Cdk5 is probably involved in the constitutive and hormonally stimulated activities of the rat testis, in addition to its involvement in cell proliferation.     

Studies on seminiferous tubule fluid production in the adult rat: effect of hypophysectomy and treatment with FSH, LH and testosterone

Seminiferous tubule fluid production in adult rats was studied using the technique of unilateral efferent duct ligation (EDL), the production rate representing the difference in testis weight over the time since ligation. Following EDL, fluid production increases linearly for 6 h and linearly at a slightly slower rate for a further 18 h with a sharp decrease thereafter. No differences in fluid production were noted for rats aged between 90–310 days. Forty-eight hours after hypophysectomy there was a significant fall (26%) in fluid production prior to any significant decrease in testis weight. Fluid production continued to decline with time after hypophysectomy eventually reaching a plateau 16–44 days later at levels approximately 15% of those found in control rats. Treatment of rats hypophysectomized 4 days earlier with ovine FSH for 3 days did not restore fluid production, but treatment with ovine LH, testosterone propionate (TP) or FSH together with TP for a similar duration all restored fluid production to normal. On the other hand, treatment of intact adult rats with ovine LH significantly increased fluid production but the effect of treatment with testosterone alone did not reach significance. The results indicate that in the adult rat, seminiferous tubule fluid production is controlled principally by testosterone secreted by the Leydig cell in response to LH stimulation.     

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.     

Endocrine parameters, hormone receptors, and functions of the testicular interstitium and seminiferous epithelium in estradiol-immunized Ile-de-France rams

The testicular response of Ile-de-France rams actively immunized against estradiol (E2) was evaluated during both the ovine nonbreeding season (spring) and breeding season (autumn). Plasma concentrations of LH, FSH and testosterone were elevated in E2-immunized rams during both spring and autumn when compared with BSA-immunized controls. Testis weights were significantly elevated by E2 immunization and were characterized by greater interstitial cell volume, including Leydig cells, blood and lymph vessels, greater seminiferous tubule length, and greater numbers of leptotene spermatocytes and round spermatids. Neither Sertoli cell number, Sertoli cell nuclear volume nor testicular FSH receptor number were affected by E2 immunization, but testis weight, Sertoli cell nuclear area, FSH receptor number and LH receptor number were significantly greater in autumn than in spring. A positive effect of E2 immunization on testicular LH receptors was evident in spring but not in autumn. Testicular androgen receptors were suppressed by E2 immunization but were not affected by season. It was concluded that E2 immunization results in moderate stimulation of the ovine testis to increase testosterone secretion and to enhance total daily spermatid production. This effect appears to result from a change in E2 negative feedback and increased pituitary gonadotropin secretion.     

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.     

Intratesticular factors and testosterone secretion: The effect of treatment with ethane dimethanesulphonate (EDS) and the induction of seminiferous tubule damage

The role of seminiferous tubule dysfunction in regulating the levels of a factor (or factors) in testicular interstitial fluid (IF) which stimulates Leydig cell testosterone secretion in vitro, was assessed by injecting rats with the Leydig cell toxin, EDS. Within 72 h of treatment EDS destroyed the Leydig cells and concomitantly reduced IF testosterone to undetectable levels. This was associated with nearly a 2-fold increase (P less than 0.001) in levels of the IF-factor(s) as judged by the enhancement of hCG-stimulated testosterone production (= IF bioactivity). By 3 weeks, and thereafter up to 10 weeks post-EDS, Leydig cells regenerated within the testis, and testosterone levels returned to control values, but IF-bioactivity remained significantly increased. The latter was associated with seminiferous tubule dysfunction as indicated initially by testicular morphology, raised serum levels of FSH and reduced testicular weight. For animals with normal testosterone levels, there was a significant negative correlation (r = -0.57, N = 46; P less than 0.001) between testicular weight and IF bioactivity. A similar increase in IF bioactivity in the presence of normal testosterone levels was observed in rats in which patchy severe seminiferous tubule damage had been induced by short-term cryptorchidism. It is concluded that, in addition to testosterone, seminiferous tubule function may dictate the intratesticular levels of the testosterone-stimulating factor(s) in IF.     

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。     

Functional and Structural Aspects of the Rat Seminiferous Tubules after Treatment with Exogenous Cyproterone Acetate, Testosterone, Progesterone and Oestradiol

Cyproterone acetate, testosterone propionate, progesterone and oestradiol were given to adult rats for 30 days, and the effects on the seminiferous tubules were studied. The contractility of the seminiferous tubules was not affected by cyproterone acetate or progesterone, but was totally abolished by testosterone and oestradiol. The effects of these steroids on spermatogenesis were studied histologically and electron microscopically.
Cyproterone acetate was observed to induce a clear-cut increase in the lipid content of the Sertoli cells and mitochondrial changes in all stages of the cycle of the seminiferous epithelium.
The increase of lipid content was smaller with progesterone. Oestradiol caused an accumulation of phagocytosed lipid material in Sertoli cells. These steroids did not change the protein composition of the rete testis fluid.     

An ex vivo analysis of Sertoli cell actin dynamics following gonadotropic hormone withdrawal

The receptors for the steroid hormone testosterone and the peptide hormone follicle-stimulating hormone are localized to the somatic Sertoli cell in the seminiferous epithelium. In the rat, prolonged gonadotrophic hormone withdrawal has been shown to result in substantial germ cell apoptosis. Previous studies have shown that, coincident with the loss of germ cells following hypophysectomy, the actin cytoskeleton of the Sertoli cell becomes disorganized and diffuse throughout the cell's cytoplasm. The molecular mechanisms that govern Sertoli cell actin filament dynamics in response to the loss of gonadotrophic hormones remain undefined. It was therefore hypothesized that hypophysectomy brings about a decrease in the amount of polymerized actin (F-actin) within the Sertoli cell and that this decrease is associated with changes in the expression of genes known to govern Sertoli actin dynamics. To this end, Sertoli cells were isolated from adult control and hypophysectomized rats. Sertoli cells from hypophysectomized rats were found to contain significantly less (72%) F-actin relative to untreated controls, although overall, beta-actin protein and mRNA expression remained constant. The expression levels of genes known to directly influence the amount of F-actin in cells were then examined by Northern blot analysis. Cofilin and profilin I gene expression was unaffected by hypophysectomy, whereas the expression of profilin II and espin both decreased significantly (47% and 42%, respectively). Taken together, these results suggest that, following hypophysectomy, the actin cytoskeleton of the Sertoli cell shifts to a predominantly depolymerized state, perhaps in part because of decreases in profilin II and espin gene products.     

小睾丸组织超微结构变化与性激素相关性研究

目的 :探讨小睾丸组织超微结构变化和性激素改变及其相关性。　方法 :对 8例小睾丸病人和 12例健康成人血清进行性激素测定 ,光镜及电镜观察小睾丸组织的超微结构变化。　结果 :小睾丸病人和正常对照组血清性激素FSH、LH、T分别为 (2 1.0 5± 9.15 )IU/Lvs (6 74± 3 5 2 )IU/L、(2 2 .88± 6 .2 5 )IU/Lvs (6 6 0± 1 4 8)IU/L、(0 .30± 0 .0 4 )nmol/Lvs (17 5 5± 9 2 5 )nmol/L ,两者相比差异有显著性 (P <0 .0 1) ;精曲小管直径和管壁厚度分别为 (37.33± 6 .80 )、(10 .30± 1.82 ) μm ,与正常组的 (198 4 6± 2 9 84 )、(2 95± 0 2 0 ) μm相比 ,差异有极显著性 (P <0 .0 1) ;组织超微结构变化显著。　结论 :小睾丸组织精曲小管、生精上皮、支持细胞、界膜、间质细胞及血管均发生严重的病理改变 ,其形成可能与遗传和免疫反应有关     

Effect of seminiferous tubule size on hCG-induced regeneration of peritubular Leydig cells in hypophysectomized, EDS-treated rats

Following their selective destruction 3 weeks previously by administration of ethane dimethanesulphonate (EDS) the regenerative capacity of Leydig cells was assessed in relation to seminiferous tubule morphology in hypophysectomized adult rats administered 7 daily injections of 100 iu hCG. Total Leydig cell volume per testis in hCG-treated rats (30.2 ±3.2 μl, mean ± SEM) was significantly ( p <0.01) greater than in the testes of rats at 3 and 4 weeks after EDS-treatment (7.6 ± 0.7 and 22.7 ± 1.4 μl, respectively). Regeneration of Leydig cells in hCG-treated rats significantly ( p <0.05) favoured peritubular locations (18.6 ± 2.8 μl/testis) compared to central or perivascular sites of origin (11.6 ± 1.2 μl/testis). Partial restoration of spermatogenesis occurred in hCG-treated rats (tubule diameters usually >250μm) and a significant inverse correlation was found between peritubular Leydig cell percentage, or total volume per testis, and the volumetric proportion of seminiferous tubules ( r =-0.94, p <0.001) or the seminiferous epithelium ( r =-0.73 to -0.79, p <0.05–0.01). No significant ( p >0.4–0.9) correlation existed between centrally-regenerated Leydig cells and these parameters. The results show that in response to hCG stimulation, Leydig cells are more likely to develop around smaller seminiferous tubules, suggesting that hCG alone cannot mimic the expected pattern of Leydig cell regeneration (central and peritubular origins) which occurs during normal sexual maturation or at 3–4 weeks after EDS treatment. It is concluded that other factors, possibly FSH, are required for typical Leydig cell development which in turn may be influenced by local cellular growth factors originating from either the seminiferous tubules or the adjacent intertubular tissue.     

Does ethane 1,2-dimethanesulphonate (EDS) have a direct cytotoxic effect on the seminiferous epithelium of the rat testis?

The authors examined the possibility that ethane 1,2-dimethanesulphonate (EDS) has a cytotoxic effect on spermatogenesis that is not secondary to androgen withdrawal resulting from the well known cytotoxic effect of EDS on Leydig cells. Adult male rats were implanted with polydimethylsiloxane (PDS) capsules containing testosterone (T) and estradiol (E), and were simultaneously injected with EDS. The PDS-TE implants, by inhibiting luteinizing hormone (LH) production, prevented Leydig cells from repopulating the testis and clamped testosterone within the seminiferous tubules at increasing concentrations relative to implant size. In rats that received EDS alone, the number of advanced spermatids per testis was significantly reduced by 2 weeks, but within 8 weeks returned to the numbers maintained in vehicle-injected control rats or in vehicle-injected rats that received testosterone- and estradiol-filled capsules of 24 cm and 0.1 cm, respectively (PDS-24TE). Surprisingly, in rats that received an EDS injection plus PDS-24TE implants, the number of advanced spermatids per testis was significantly reduced at 8 weeks and severe seminiferous tubule atrophy occurred despite the fact that the testosterone concentration was sufficient to quantitatively maintain spermatogenesis in vehicle-injected rats. In rats injected with EDS and implanted with 24 cm testosterone but not estradiol-filled capsules (PDS-24T), the advanced spermatid number per testis was significantly higher than that in the EDS plus PDS-24TE rats, but significantly lower than that in control rats. These results suggest that EDS may have a cytotoxic effect on the seminiferous epithelium that is independent of the elimination of Leydig cells, and the EDS and estradiol act synergistically to exert a profound toxic effect on spermatogenesis.     

Evaluation of the effect of peritubular cell secretions and the testicular paracrine factor P-Mod-S on Leydig cell steroidogenesis and immunoactive inhibin production

Testicular peritubular cells have been shown to produce a paracrine factor, termed P-Mod-S, under androgen control that has dramatic effects on Sertoli cell function and may provide an important mode of androgen action in the testis. Therefore, the current study was designed to investigate the possibility that peritubular cell secretory products could feedback and regulate Leydig cell function. The Leydig cell functional parameters that were examined included testosterone production and inhibin secretion. Purified forms of P-Mod-S (P-Mod-S(A) and P-Mod-S(B) shown to be biologically active on Sertoli cells) had no effect on basal or gonadotrophin-stimulated production of testosterone or inhibin by Leydig cells. A preparation of peritubular cell-secreted proteins (PSP) with molecular weights greater than 3 kDa did not influence testosterone production by Leydig cells. PSP, however, did influence cultured Leydig cell morphology and improved cell viability. PSP also had no effect on the ability of LH to stimulate Leydig cell testosterone production. Whilst determining the effect of PSP on Leydig cell inhibin production, PSP was found to contain endogenous levels of inhibin apparently due to 2% contamination of the peritubular cell cultures with Sertoli cells. When this endogenous inhibin level was considered, PSP was found to have no influence on basal or hormone-stimulated production of inhibin by Leydig cells. Results of the current study indicate that peritubular cell secretory products, including the paracrine factor P-Mod-S, do not appear to play a major role in the regulation of Leydig cell function. Therefore, the regulation of Leydig cell function by the seminiferous tubule will primarily be due to Sertoli cell secretory products.     

7-Alpha-hydroxytestosterone in seminiferous tubules of rat testis

Production of 4-androstene-7 alpha,17 beta-diol-3-one (7 alpha-hydroxytestosterone) from testosterone was measured in seminiferous tubule and interstitial fractions of rat testis. In adults, specific activity of the 7 alpha-hydroxylase was about 10 times higher in interstitial cells than in seminiferous tubules, but tubule production was a significant portion of the total. Seminiferous tubule 7 alpha-hydroxylase was not detectable in weanlings. Also, we confirmed that 7 alpha-hydroxytestosterone inhibits the activity of the 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase active on testosterone and suggest a role in maturational changes.     

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.     

Maturation of the juvenile rat testis after surgical treatment of cryptorchidism

Rats were made bilaterally cryptorchid at 17 days of age and bilateral orchidopexy performed at 34 days of age. The epididymal content of androgen binding protein (ABP), the weight and morphology of the testis, the cross-sectional area of seminiferous tubules and the testicular concentration of testosterone were then studied at 34, 42, 59 and 120 days of age. Cryptorchidism was followed rapidly by progressive inhibition of spermatogenesis and testicular growth as well as by decreased Sertoli cell secretion of ABP. Orchidopexy resulted in a gradual restoration of spermatogenesis, and all impaired parameters seemed to improve at the same, fairly slow rate. Restoration was not complete, but by 120 days of age the morphological appearance of the testis was compatible with recovery of normal fertility.