Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis.

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.

In vitro regulation of an inducible-type NO synthase in the rat seminiferous tubule cells.

Rat Sertoli cells express an inducible nitric oxide synthase isoform (iNOS) in response to the combined addition of the cytokines--interferon gamma (IFNgamma), tumor necrosis factor alpha (TNF alpha), interleukin-1alpha (IL-1alpha)--and lipopolysaccharides (LPS). We demonstrated that the addition of cytokines and lipopolysaccharides (C+L) to cultured peritubular cells resulted in high nitrite and iNOS mRNA levels, indicating the induction of an iNOS isoform. This enzyme was not induced in cultured pachytene spermatocytes or spermatids. Nitrite production in Sertoli cells and peritubular cells required both IFNgamma and TNF alpha and was potentiated by LPS, whereas IL-1alpha was ineffective. The induction of nitrite production and iNOS mRNA by IFNgamma+TNF alpha+LPS could be further enhanced by basic fibroblast growth factor in Sertoli cells but not in peritubular cells. In contrast, transforming growth factor beta markedly reduced this induction in peritubular cells but had no effect on Sertoli cells. FSH positively modulated the C+L-induced iNOS in Sertoli cells. Dibutyryl cAMP had a synergistic effect with C+L on NOS activity in both Sertoli cells and peritubular cells. In contrast, testosterone did not influence basal or induced NOS activity in these two cell types. These data show that NOS activity in the somatic cells of the seminiferous tubules is induced and regulated by multiple factors that act in combination, and suggest that nitric oxide may participate in the endocrine and paracrine control of testicular function.

Vectorial production of interleukin 1 and interleukin 6 by rat Sertoli cells cultured in a dual culture compartment system.

The bidirectional production of interleukin-1 (IL-1) and IL-6 by Sertoli cells and its regulation by inflammatory and physiological stimuli has been studied using a dual compartment culture system allowing the study of Sertoli cell apical and basal secretory activities. Another Sertoli cell activity, the vectorial transferrin production was also studied in all culture conditions. A low constitutive IL-1 production appeared equally distributed between both poles, while IL-6 and transferrin constitutive production was predominantly directed apically. Two activators of macrophages, lipopolysaccharides and zymosan, were found to induce marked increases of IL-1 in the compartment where they had been added: basal if added to the lower compartment and vice versa. In contrast, after a basal stimulation, IL-6 production was mainly increased in the upper compartment that corresponds to a Sertoli cell apical flux. In this system, IL-1 and IL-6 levels were not modified by FSH; they were not also affected by residual bodies and latex beads, probably due to the fact that, in the bicameral system, phagocytosis is restricted to the Sertoli cells situated at the surface of the inner compartment. IL-1beta, but not IL-1alpha, induced IL-6 secretion in the compartment of stimulation. In conclusion, the present study demonstrates that vectorial secretory patterns of IL-1 and IL-6 production greatly differ and that these cytokines are also differently regulated. These results suggest that Sertoli IL-1 and IL-6 have different targets within the testis and that, in normal and pathophysiological conditions, both the tubular and the interstitial compartments may be influenced by the action of these paracrine factors.

Germ cell control of testin production is inverse to that of other Sertoli cell products.

Recent studies have shown that germ cells can regulate testins, two newly identified Sertoli cell proteins that are associated with junctional complexes. To investigate this possibility, several parameters of Sertoli cell function were investigated over 2-120 days post exposure of the rat testes to x-rays (3 Grays). The irradiation-induced loss of spermatogonia resulted in a maturation-depletion process progressively affecting all germ cell classes. Testis weight began to decrease when the most numerous germ cell type (spermatids) began to decline. A complete or near complete recovery of spermatogenesis and of the testis weight had occurred by day 120 post irradiation. There was no significant change in FSH, epididymal androgen-binding protein, and tubule fluid levels during the first weeks after irradiation, when the seminiferious epithelium was depleted of spermatogonia and germ cells up to early spermatids. In contrast, when the number of the more mature forms of spermatids declined (between day 21 and 54), FSH rose and androgen-binding protein as well as fluid production declined. The subsequent recovery of these parameters was also highly correlated with the number of late spermatids. By contrast, testicular testin contents reacted to the depletion of germ cells with a biphasic increase; a doubling occurred when spermatogonia, spermatocytes, and early spermatids were absent (days 4-28), and a 7-fold rise occurred by day 37 when the number of late spermatids had decreased by 50%. By day 54, when the sperm counts had reached a nadir, testin contents had returned to levels corresponding to about four times the control levels; they progressively recovered thereafter. These observations support the postulate that germ cells negatively regulate testins. This possibility was investigated with in vitro experiments showing that addition of germ cell-conditioned medium to Sertoli cell monolayers inhibited testin secretion in a dose-dependent manner. In conclusion this study; 1) highlights the complex interplay between the various germ cell classes in the control of the Sertoli cell function in the adult testis; 2) establishes that germ cell effects may be opposite on different Sertoli cell products; 3) demonstrates that several classes of germ cells negatively control testicular testin contents; and 4) emphasizes the particular role of late spermatids in Sertoli cell regulation.

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.