Differentiation of Secondary Spermatogonia to Primary Spermatocytes by Mammalian Follicle-Stimulating Hormone in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

In order to elucidate essential factors responsible for the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments, consisting of somatic cells and germ cells almost exclusively secondary spermatogonia, were cultured in control medium for three weeks, most of the testicular cysts still contained only secondary spermatogonia. On the other hand, in the medium supplemented with various kinds of hormones and vitamins primary spermatocytes (zygotene-pachytene) appeared in about 60% of the cysts by the second week. Selective removal of specific hormones and vitamins revealed that follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the differentiation of secondary spermatogonia to primary spermatocytes. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5α-dihydrotestosterone) to the control medium stimulated differentiation. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH. Since our ultrastructural studies revealed a major loss of contact between spermatogonia and Sertoli cells following exposure to FSH, we suggest that FSH triggers differentiation of spermatogonia by acting on Sertoli cells which in turn act on spermatogonia.     

Altered sexual development in male rats after oestrogen administration during the neonatal period.

Male rats given 250 mug oestradiol benzoate by subcutaneous injection on Day 4 of postnatal life showed a marked delay in the onset of the pubertal increase in the weight of the testes and seminal vesicles and in spermatogenesis but not a complete failure of sexual development. The increase in plasma testosterone concentration at puberty was also delayed in oestrogen-treated males but the eventual increase in seminal vesicle weight was closely related in time to the delayed increase in plasma testosterone concentration. Both plasma LH and FSH concentrations were reduced for about 10 days after oestrogen administration as compared to control values. After 22 days of age, plasma LH concentration did not differ significantly from the control values. The plasma FSH concentration of the oestrogen-treated males showed a delayed rise to values equal to or higher than those of controls of the same age. The delayed rise in plasma FSH concentration in the oestrogen treated males preceded the delayed rise in plasma testosterone in these animals. The decrease in plasma FSH concentration from the high prepubertal values to the lower values in adults occurred at different ages in the control and in oestrogen-treated rats but in both groups the decrease occurred as plasma testosterone levels were increasing and the first wave of spermatogenesis was reaching completion. The increase in plasma FSH concentration after castration was reduced in oestrogen-treated males during the period throughout which FSH levels in the intact animals were subnormal but the levels in oestrogen-treated males castrated after the delayed rise in FSH had occurred did not differ from control values. It is suggested that the delayed sexual maturation of male rats treated with high doses of oestrogen in the neonatal period is related principally to abnormalities in the secretion of FSH.     

Regulation of gonadotrophin secretion in rams from birth to sexual maturity. I. Plasma LH, FSH and testosterone levels.

Plasma LH, FSH and testosterone concentrations were measured by radioimmunoassays in male crossbred Merino/Corriedale sheep from birth to 45 weeks of age. FSH levels were 11 and 22 ng/ml at birth, increased to peak levels (mean value of 47 ng/ml) at 5 weeks and fluctuated between 25 and 35 ng/ml for the next 40 weeks. Similarly, LH (less than 0-5 ng/ml) and testosterone (less than 38 ng/100 ml) levels were low at birth and were significantly elevated by 5 weeks of age. LH values varied betwen 0-9 and 3-0 ng/ml for the next 30 weeks and then a secondary rise occurred reaching levels of 2-4 ng/ml by the 41st week after birth. Concentrations of LH subsequently fell to levels observed in adult rams. Testosterone levels rose gradually between the 5th and the 25th week, and then increased rapidly to values of 270-517 ng/100 ml by the 41st week after birth, a time coincident with the peak LH levels. Histological examination of testicular biopsies demonstrated that Sertoli cell maturation occurred 17-21 weeks after birth and was followed by activation of spermatogenesis leading to the presence of spermatozoa in the seminiferous epithelium by 39-42 weeks of age.     

Age-related changes in the function of the pituitary-gonadal axis in a sterile male rat mutant (hd/hd)

Testicular growth is depressed in the genetically sterile male rat (hd/hd) relative to its LE phenotype littermates (by 50% and 73% at 27 and 90 days of age, respectively). Within the hd/hd testis, both the tubular and seminiferous tubule tissues are affected by the mutation. In addition, there is significantly less germ cell production from the primary spermatocyte stage of spermatogenesis onwards and the total number of Sertoli cells observed is less. In the intertubular tissue, the total volume and the total number of Leydig cells per testis is significantly less, but the mean volume of an average Leydig cell is not modified. The serum gonadotropin levels are higher in the hd/hd rat, whereas from 40 days of age onwards the level of testosterone is lower. The FSH and LH binding affinity constants are unchanged by the mutation; however, the total number of FSH binding sites per 10(6) Sertoli cells is lower while that of LH per 10(6) Leydig cells is greater. Indeed, it is likely that the lesser concentration of serum testosterone in the hd/hd rat is a result of a smaller number of Leydig cells since their individual function is not modified. The testicular androgen binding protein (ABP) content and the ABP output towards the epididymis are lower as a consequence of both a lesser number and an altered function of the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of spermatogenesis and spermiogenesis

Normal testicular function is dependent upon hormones acting through endocrine and paracrine pathways both in vivo and in vitro. Sertoli cells provide factors necessary for the successful progression of spermatogonia into spermatozoa. Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone which are the main hormonal regulators of spermatogenesis. Hormones such as testosterone, FSH and luteinizing hormone (LH) are known to influence the germ cell fate. Their removal induces germ cell apoptosis. Proteins of the Bcl-2 family provide one signaling pathway which appears to be essential for male germ cell homeostasis. In addition to paracrine signals, germ cells also depend upon signals derived from Sertoli by direct membrane contact. Somatostatin is a regulatory peptide playing a role in the regulation of the proliferation of the male gametes. Activin A, follistatin and FSH play a role in germ cell maturation during the period when gonocytes resume mitosis to form the spermatogonial stem cells and differentiating germ cell populations. In vitro cultures systems have provided evidence that spermatogonia in advance stage of differentiation have specific regulatory mechanisms that control their fate. This review article provides an overview of the literature concerning the hormonal pathways regulating spermatogenesis.     

Effects of spinal cord injury on spermatogenesis and the expression of messenger ribonucleic acid for Sertoli cell proteins in rat Sertoli cell-enriched testes

The study was an examination of the effects of spinal cord injury (SCI) on spermatogenesis and Sertoli cell functions in adult rats with Sertoli cell-enriched (SCE) testes. The effects of SCI on the seminiferous epithelium were characterized by abnormalities in the remaining spermatogenic cells during the first month after SCI. Three days after SCI, serum testosterone levels were 80% lower, while serum FSH and LH levels were 25% and 50% higher, respectively, than those of sham control SCE rats. At this time, the levels of mRNA for androgen receptor (AR), FSH receptor (FSH-R), and androgen-binding protein (ABP) were normal whereas those for transferrin (Trf) had decreased by 40%. Thereafter, serum testosterone levels increased, but they remained lower than those of the sham control rats 28 days after SCI; and serum FSH and LH levels returned to normal. The levels of mRNA for AR, ABP, and Trf exhibited a biphasic increase 7 days after SCI and remained elevated 28 days after SCI. FSH-R mRNA levels were also elevated 90 days after SCI. Unexpectedly, active spermatogenesis, including qualitatively complete spermatogenesis, persisted in > 40% of the tubules 90 days after SCI. These results suggest that the stem cells and/or undifferentiated spermatogonia in SCE testes are less susceptible to the deleterious effects of SCI than the normal testes and that they were able to proliferate and differentiate after SCI. The presence of elevated levels of mRNA for Sertoli cell FSH-R and AR, as well as of that for the Sertoli cell proteins, in the SCE testes during the chronic stage of SCI suggests a modification of Sertoli cell physiology. Such changes in Sertoli cell functions may provide a beneficial environment for the proliferation of the stem cells and differentiation of postmeiotic cells, thus resulting in the persistence of spermatogenesis in these testes.     

Gonadotropins, their receptors, and the regulation of testicular functions in fish

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.     

Detection of cellular retinol-binding protein messenger RNA in the somatic cells of the rat seminiferous tubules

A cDNA clone coding for Cellular Retinol-Binding Protein (CRBP) was used as a probe to study the expression of the gene in the somatic cells of the seminiferous tubules (Sertoli and peritubular cells). In this paper we demonstrate that these cells are actively involved in the synthesis of the specific mRNA. In Sertoli cells the gene is modulated by the hormones effective in spermatogenesis, such as FSH and testosterone. Moreover, peritubular cells revealed an approximately two times higher concentration of CRBP steady-state mRNA levels when compared with Sertoli cells.     

The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats

Cryptorchidism for 28 or 10 days resulted in a severe disruption of spermatogenesis (assessed histologically or by fertility tests), Sertoli cell function (assessed by seminiferous tubule fluid production after efferent duct ligation, ABP levels, binding of 125I-labelled FSH to testis homogenates and serum FSH levels) and Leydig cell function (assessed by serum LH and testosterone levels, in-vitro testosterone production, binding of 125I-labelled hCG). Orchidopexy after 28 days of cryptorchidism resulted in a poor recovery of spermatogenesis since the majority of tubules were lined by Sertoli cells and a few spermatogonia. No recovery occurred in the indicators of Sertoli and Leydig cell function. Orchidopexy after 10 days of cryptorchidism also resulted in a poor recovery of spermatogenesis, with a few animals showing partial recovery after 6 months. No recovery occurred in seminiferous tubule fluid production but partial recovery occurred in ABP content and production rate. Serum FSH, LH levels and in-vitro testosterone production by the testis remained elevated and did not change from the values found during cryptorchidism. Fertility testing at 6 months revealed a small number of rats in which fertility was restored although the number of embryos was lower than in controls. In this group of animals there was a significant improvement in a number of indicators of Sertoli cell and Leydig cell function. These data provide further evidence to link the changes in Sertoli cell and Leydig cell function to the germ cell complement present in the testis.     

Maintenance of sexual immaturity in male mice and bucks by immunization against N-terminal peptides of the follicle-stimulating hormone receptor

The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18-38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.     

Effect of specific FSH or LH deprivation on testicular function of the adult rat.

While the need for FSH in initiating spermatogenesis in the immature rat is well accepted, its requirement for maintenance of spermatogenesis in adulthood is questioned. In the current study, using gonadotropin antisera to neutralize specifically either endogenous FSH or LH, we have investigated the effect of either FSH or LH deprivation for a 10-day period on (i) testicular macromolecular synthesis in vitro, (ii) the activities of testicular germ cell specific LDH-X and hyaluronidase enzymes, and finally (iii) on the concentration of sulphated glycoprotein (SGP-2), one of the Sertoli cell marker proteins. Both immature (35-day-old) and adult (100-day-old) rats have been used in this study. Since LH deprivation leads to a near total blockade of testosterone production, the ability of exogenous testosterone supplementation to override the effects of LH deficiency has also been evaluated. Deprivation of either of the gonadotropins significantly affected in vitro RNA and protein synthesis by both testicular minces as well as single cell preparations. Fractionation of dispersed testicular cells preincubated with labelled precursors of RNA and protein on Percoll density gradient revealed that FSH deprivation affected specifically the rate of RNA and protein synthesis of germ cell and not Leydig cell fraction. LH but not FSH deprivation inhibited [3H]thymidine incorporation into DNA. The inhibitory effect of LH could mostly be overriden by testosterone supplementation. LDH-X and hyaluronidase activities of testicular homogenates of adult rats showed significant reduction (50%; P less than .05) following either FSH or LH deprivation. Again testosterone supplementation was able to reverse the LH inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on Spermatogenesis and Steroidogenesis in Culture

This paper summarizes the major pertinent findings of previouslypublished work and includes new experimental data. Culture conditionshave been defined for long-term maintenance of basic testicularstructure, primitive type A spermatogonia, and Sertoli cells.The primitive type A spermatogonia grown for 8 weeks in organculture were capable of restoring complete spermatogenesis afterthe cultures were transplanted into the testes of sexually maturehomologous hosts. Since the primitive type A spermatogonia werethe only germinal elements present in the organ cultures atthe time of transplantation, they probably represent the spermatogenicstem cells. Differentiation of gonocytes or spermatogonia tospermatocytes in late pachytene stage required presence of vitaminsA, C, and E or 4 mM concentration of glutamine in the mediumbut was not affected by hormones. The replication of Sertolicells in vivo and in organ culture was shown to be age relatedand hormone independent. The Sertoli cells possess specificFSH binding receptors and respond to FSH with increased synthesisof c-AMP and androgen-binding protein. The biologic functionof FSH in the Sertoli cells remains to be clarified. Other cellularcomponents of the testis, the germinal, or peritubular cellsdo not bind FSH. In organ cultures of testicular tissue or Leydig-cellcultures all enzymes in the steroid biosynthetic pathway, leadingfrom progesterone to testosterone, remain active for severaldays. In older cultures, the conversion of progesterone to testosteronebecomes impaired primarily due to loss of two specific enzymes:17 hydroxylase and 17-20 lyase. Addition of HCG to monolayercultures of Leydig cells stimulated both the synthesis and releaseof testosterone.     

The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

四川成都市郊正常成年男性血清生殖激素水平及其频数分布

本文用放射免疫测定法(RIA),测定了200～290例中国四川成都市郊30～73岁的正常成年男性血清睾酮(T)促黄体激素(LH).卵泡刺激素(FSH)及泌乳激素(PRL)的水平及其分布,进行了不同年龄水平差异显著性的观察,见到睾酮与泌乳激素水平无显著差异,促黄体激素及卵泡刺激素水平,随年龄之增加而升高,差异有显著性,并测得它们在血清中水平的分布:LH、FSH及PRL均为对数正态公布,T既非正态又非对数正态,但其频率分布较对称,接近正态分布。     

Regulation of gonadal androgen secretion

The various mechanisms regulating testicular and ovarian androgen secretion are reviewed. Testicular androgen secretion is controlled by luteinizing hormone (LH) and follicle stimulating hormone (FSH), which influence the Leydig cell response to the LH. The contribution of prolactin, growth hormone and thyroid hormones to the Leydig cell function is discussed. The ovarian androgen secretion is regulated in a very similar fashion as the Leydig cell of testis. Prolactin, however, has an inhibitory effect on androgen secretion in the ovary. The intratesticular action of androgens is linked to spermatogenesis. Sertoli cells, by producing the androgen-binding protein, contribute to the intratubular androgen concentration. Inhibin production of the Sertoli cell is stimulated by androgens. In the ovary, androgens produced by the theca interna are used as precursors for the aromatization of estradiol, which stimulates together with FSH the mitosis of granulosa cells. The feedback control of androgen secretion is complicated, as the direct feedback mechanisms are joined by indirect feedback regulations like the peptide inhibin, which can be stimulated by androgens. Intragonadal mechanisms regulating androgen production are the cybernins for testicles and ovaries. In the testicle, estrogens from the Sertoli cells regulate the Leydig cell testosterone biosynthesis. In the ovary, nonaromatizable androgens are potent inhibitors of the aromatization activity in the granulosa cell. A peptide with a FSH receptor binding inhibiting activity is found in male and female gonads. Finally, LH-RH-like peptides have been found in the testicle, which are capable of inhibiting steroidogenesis. These gonadocrinins are similarly produced in granulosa cells of the ovary.     

Photoperiodic variations of somatic and germ cell populations in the Soay ram testis

Seasonal changes in the Leydig, Sertoli and stem cells (undifferentiated A0 and cyclic A1) spermatogonia and the daily spermatid production were analysed in the testes of adult Soay rams exposed to short days (8L:16D) or long days (16L:8D) for 12 weeks. The total numbers of Leydig, Sertoli and stem cells (A0 + A1) were not affected by the treatments, but the size of the Leydig and the Sertoli cells, the efficiency of spermatogenesis (i.e. the number of male gametes produced by an A1 spermatogonium) and the daily sperm production were all significantly reduced in the rams exposed to long days. There was a positive correlation between the concentration of FSH and testosterone and many of the cytological changes consistent with a causal role for these hormones in mediating the effects of photoperiod on the testicular function in the ram.     

Effect of prenatal treatment with busulfan on the hypothalamo-pituitary axis, genital tract and testicular histology of prepubertal male rats

Female Wistar rats were treated with busulfan or with solvent on Day 20 of pregnancy. Thirty male offspring of each group were killed at 38 days of age. In busulfan-treated rats, compared to controls, hypothalamic LH-RH content was decreased by 52%, whereas pituitary LH and FSH concentrations were increased by 60 and 43% respectively. Plasma LH and FSH were increased by 112 and 275% respectively. Prolactin concentrations were not changed, but plasma testosterone concentration was decreased by 48%. The total number of Leydig cells per testis was decreased by 52%, and LH binding sites per testis were decreased by 70%. The total number of Sertoli cells was decreased by 44%, while FSH binding sites per testis were decreased by 62%. Spermatogenesis was practically absent after prenatal exposure to busulfan. These data demonstrate that on Day 20 of pregnancy all the dividing cells in the fetal testes were depleted by an antimitotic treatment. The stimulation of the hypothalamo-pituitary axis could have been partly induced by the decrease in testosterone production, and by the aplasia of germ cells involving modifications of the remaining Sertoli and Leydig cells.