There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation.

The relationship between thyroid function and testicular development in the rat was investigated. Hypothyroidism was induced during fetal or post-natal life by adding methimazole (MMI) to the drinking water of pregnant or lactating mothers. A group of newborn rats was treated with MMI and i.p. injections of L-tri-iodothyronine (L-T3). Hypothyroidism was shown by the reduced serum levels of total T3 and of total thyroxine (T4) in pregnant mothers and in pubertal rats. Testes were studied using light microscopy at 18 and 21 days post coitum or during puberty (21, 35 and 50 days after birth); serum levels of gonadotrophins were also evaluated in pubertal rats. Hypothyroidism had no effect on testicular development during fetal life and when induced in newborn rats it was associated at puberty with reduced serum levels of FSH and LH and with delayed maturation of the testis compared with control rats. The delay in maturation consisted of a reduction in the diameter of seminiferous tubules, and a reduction in the number of germ cells per tubule; this was associated with increased degeneration and arrested maturation of germ cells. In addition, Sertoli cells demonstrated retarded development, as indicated by a delay in the appearance of cytoplasmic lipids and in the development of a tubule lumen. Hormonal and morphological abnormalities were absent in rats treated with MMI plus L-T3. In conclusion, hypothyroidism occurring soon after birth caused reduced levels of gonadotrophins in the serum and a delay in pubertal spermatogenesis, possibly due to retarded differentiation of the Sertoli cells.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10−7M and 10−8M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. −90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.

The relationship between thyroid activity and Sertoli cell function has been investigated in prepubertal rats. Male 28-day-old Wistar rats were used to prepare Sertoli cells by sequential enzyme digestion of the testes. Hypothyroidism, induced by oral administration of methimazole from the day of birth, was characterized by a severe retardation of body and testis growth and a net inhibition of the increase in Sertoli cell gamma-glutamyl transpeptidase (GGT) activity as well as in androgen-binding protein (ABP) and lactate production, which normally occur during postnatal development of Sertoli cells. The functional parameters of Sertoli cells from hypothyroid 28-day-old rats approximated to those of cells from euthyroid 15-day-old animals. These results are consistent with the impairment of protein synthesis in Sertoli cells from hypothyroid rats compared with controls. Body and testis growth were improved and Sertoli cell functions were restored with 3,3',5-tri-iodothyronine (T3) replacement therapy. An excess of T3 in the serum, induced by daily i.p. injections of T3 (100 micrograms/kg body wt) during the last week before the rats were killed, failed to induce changes in body and testis growth or in the activity of GGT and lactate dehydrogenase of Sertoli cells. Cells from hyperthyroid rats exhibited a specific decrease in ABP production. These results indicate that thyroid hormone is necessary for the postnatal maturation of Sertoli cell function and suggest a regulatory role of the hormone on gametogenic development in the prepubertal rat.

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The addition of physiological concentrations (1 nM) of tri-iodothyronine (T3) to the culture medium of Sertoli cells from prepubertal (8-day-old) rats stimulated both protein synthesis (+55%) and lactate (+50%) production, while it inhibited DNA synthesis (-30/35%) and aromatase activity (-45/50%); insignificant T3-dependent effects were observed in cultured Sertoli cells from midpubertal (28-day-old) rats. These data suggest an age-dependent role for thyroid hormone in promoting and maintaining Sertoli cell differentiation at puberty; more-over, the hormone is involved in the regulation of Sertoli cell proliferation. The present study validates the role of Sertoli cells as a specific target for T3 action at the testis level; it also demonstrates the existence of an early and critical direct influence of thyroid hormone on Sertoli cell proliferation and functional maturation.

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