The role of genotype in the formation of Leydig cell hormone function during postnatal ontogeny in diallele crosses of laboratory mice

Production of testosterone by Leydig cells during the postnatal ontogeny in pubescence under in vitro stimulation by chorionic gonadotropin, dibutiryl-cAMP, and pregnenolon was studied in males of four inbred mouse lines (BALB/c, RT, CBA/Lac, and A/He) and their F1 reciprocal hybrids. Highly statistically significant association between the animal genotype and age was revealed for all parameters studied, which indicates the genotype-dependent formation of the Leydig cell hormone function during the postnatal ontogeny. The effect of genotype was characterized by two specific features. First, in each postnatal ontogeny stages examined correlative genetic variability in respect of the cAMP- and substrate-dependent indices of Leydig cell reactivity was observed. Second, during postnatal ontogeny coordinated genetic variability was subjected to substantial ontogenetic rearrangements. Definite pattern of genetic differences in the Leydig cell hormone activity was formed only at the late pubertal--early post- pubertal stage (60th day after birth). This process coincided with the completion of the Leydig cell morphological differentiation and the appearance of mature cells in the population. Thus, formation of the Leydig cell hormone activity during postnatal ontogeny is under coordinated genetic control, which is also subjected to substantial changes during pubertal differentiation.     

Effect of genotype on hormonal function formation of Leydig cells during mouse postnatal development

Changes in in vitro testosterone production by Leydig cells induced by chorionic gonadotropin, dibutyryl-cAMP, and pregnenolone have been studied during postnatal development of four inbred mouse strains BALB/c, PT, CBA/Lac, and A/He, with contrast hormonal activity of testes in sexually mature males. The interlinear differences significantly change with age of the males by all studied indices indicating genotype-dependent formation of hormonal activity of Leydig cells during postnatal development. Coordinated interlinear variability between all indices of Leydig cells reactivity has been established for each studied period of postnatal development. Hence, we have established coordinated interlinear genetic variability of hormonal function of Leydig cells, which was confirmed by considerable changes in it during postnatal development at puberty. Definitive genotypic differences in hormonal activity of Leydig cells appeared by late pubertal and early postpubertal development (day 60) and coincided with termination of morphological differentiation of Leydig cells and appearance of the differentiated cell population.     

Effect of Genotype on Formation of Hormonal Function of Leydig Cells during Mouse Postnatal Development

Changes in in vitro testosterone production by Leydig cells induced by chorionic gonadotropin, dibutyryl-cAMP, and pregnenolone have been studied during postnatal development of four inbred mouse strains BALB/c, PT, CBA/Lac, and A/He, with contrast hormonal activity of testes in sexually mature males. The interlinear differences significantly change with age of the males by all studied indices indicating genotype-dependent formation of hormonal activity of Leydig cells during postnatal development. Coordinated interlinear variability between all indices of Leydig cells reactivity has been established for each studied period of postnatal development. Hence, we have established coordinated interlinear genetic variability of hormonal function of Leydig cells, which was confirmed by considerable changes in it during postnatal development at puberty. Definitive genotypic differences in hormonal activity of Leydig cells appeared by late pubertal and early postpubertal development (day 60) and coincided with termination of morphological differentiation of Leydig cells and appearance of the differentiated cell population.     

Leydig cell development

This review is about the study of the testis Leydig cells formation and development in prenatal and postnatal periods. Leydig cells of testis are the main place of synthesis and secretion of androgens including testosterone--the main male sexual hormone. Testosterone plays an important role in male reproduction regulation. There are two types (two populations) of Leydig cells during ontogenesis. The first type is fetal Leydig cells, which appear and function in the prenatal masculinization period of the male urogenital system. Another type is adult Leydig cells, which originate during sexual maturation postnatally. Fetal and adult Leydig cells pass the same stages both in the prenatal and postnatal periods. They are Leydig cell progenitors, immature Leydig cells and adult Leydig cells.     

Differentiation of adult Leydig cells

Adult Leydig cells originate within the testis postnatally. Their formation is a continuous process involving gradual transformation of progenitors into the mature cell type. Despite the gradual nature of these changes, studies of proliferation, differentiation and steroidogenic function in the rat Leydig cell led to the recognition of three distinct developmental stages in the adult Leydig cell lineage: Leydig cell progenitors, immature Leydig cells and adult Leydig cells. In the first stage, Leydig cell progenitors arise from active proliferation of mesenchymal-like stem cells in the testicular interstitium during the third week of postnatal life and are recognizable by the presence of Leydig cell markers such as histochemical staining for 3β-hydroxysteroid dehydrogenase (3β-HSD) and the present of luteinizing hormone (LH) receptors. They proliferate actively and by day 28 postpartum differentiate into immature Leydig cells. In the second stage, immature Leydig cells are morphologically recognizable as Leydig cells. They have an abundant smooth endoplasmic reticulum and are steroidogenically active, but primarily produce 5-reduced androgens rather than testosterone. Immature Leydig cells divide only once, giving rise to the total adult Leydig cell population. In the third and final stage, adult Leydig cells are fully differentiated, primarily produce testosterone and rarely divide. LH and androgen act together to stimulate differentiation of Leydig cell progenitors into immature Leydig cells. Preliminary data indicate that insulin like growth factor-1 (IGF-1) acts subsequently in the transformation of immature Leydig cells into adult Leydig cells.     

Key factors in the regulation of fetal and postnatal Leydig cell development

The primary function of testicular Leydig cells is the production of androgens to promote sexual differentiation in the fetus, secondary sexual maturation at puberty, and spermatogenesis in the adult. The fetal and postnatal (adult) populations of Leydig cells differ morphologically and have distinct profiles of gene expression. As postnatal Leydig cells differentiate, they transition through three discrete maturational stages characterized by decreasing proliferative rate and increasing testosterone biosynthetic capacity. In this review, we discuss the development of both fetal and postnatal Leydig cells and review the regulation of this process by some of the key hormones and growth factors.     

Identification and expression of epidermal growth factor gene in mouse testis

INTRODUCTIONEpidermalgrowthfactor(EGF)wasinitiallyisolatedandpurifiedfromthesubmaxillarygland(SMG)ofmalemouse[1].Itisapolypeptidecomposedof53aminoacidresidues[2].Itinfluencescellproliferationanddifferentiationandmodulatesthegrowthanddevelopmentofmammalianorgans[3--7].AnoteworthyfindingisthatextirpationofmouseSMGresultsinamarkedreductionofserumEGFconcentrationassociatedwithanimpairedspermatogenesis[3].ThisfindingsuggeststhatEGFmayregulatespermproductionanddifferentiation.Inhumantest…     

Changes in the concentration and size of testicular macrophages during development

Structural and functional interactions exist between Leydig cells and testicular macrophages of adult rats. Since the function of Leydig cells changes during critical periods of development and postnatal maturation, it is possible that macrophages are in part involved in regulating this process. As a first step towards gaining an understanding of the development of this paracrine phenomenon, I have undertaken a series of studies designed to determine when macrophages first become identifiable in the fetal tests and to determine whether the concentration or size of macrophages changes during important stages of testicular maturation. Macrophages were identified immunohistochemically in frozen sections of testis from rats at various prenatal and postnatal ages using commercially available monoclonal antibodies to proteins specific to rat macrophages. It was found that macrophages positive for these antigens were found only within the interstitial compartment and were commonly associated with clusters of presumptive Leydig cells that were negative for these antigens. Macrophages were first identifiable in the testis at Day 19 of fetal development. The number of macrophages/unit area of interstitium increased 15-fold between Day 20 of gestation and Day 47 postpartum. The cross-sectional area of the macrophages increased 1.7-fold between Days 13 and 47 postpartum. These results demonstrate that the number and size of testicular macrophages changes with age, suggesting a role for these cells during important times of testicular development and maturation.     

Developmental stages of fetal-type Leydig cells in prepubertal rats

Fetal Leydig cells were studied in rats during and after the perinatal-neonatal period by comparing changes in morphology, number and volume with changes in testicular steroids and serum luteinizing hormone (LH) concentration. Stereologic examination indicated regression of fetal Leydig cells in testis by showing that their total volume as well as the average cell volume decreased between prenatal day 20 and postnatal day 3. The total number and total volume of cells both increased between postnatal days 3 and 11 but the average cell volume did not change during the same time period. Determination of serum LH showed a close correlation between an increase in LH concentration and increases in total number and volume of cells. The combined number of fetal- and adult-type Leydig cells on day 20 was more than 20 times the number of fetal cells at 3 days of age. Electron microscopic analysis showed that fetal Leydig cells after birth formed conspicuous clusters, which were surrounded by a layer of envelope cells and extracellular material. Occasional dividing fetal Leydig cells and possible precursors of fetal or adult Leydig cells were observed. Mitoses of spindle-shaped pericordal cells were frequent during the neonatal period. During and after the second postnatal week fetal Leydig cells again showed signs of regression, indicated by disintegration of the cell clusters, a decrease in cell size, accumulation of collagen between the cells and a decrease in steroid content per cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of platelet-derived growth factor-alpha (PDGF-A) protein in cells of Leydig lineage in the postnatal rat testis

Platelet-derived growth factor-A (PDGF-A) is a locally produced growth factor in the rat testis secreted by both Sertoli cells and Leydig cells. It has been suggested that PDGF-A may be involved in modulation of testosterone production and may be essential to Leydig cell differentiation, however it is not known at what stage of differentiation PDGF-A begins to be expressed in the cells of Leydig lineage in the postnatal rat testis. Therefore, the objectives of this research were to determine at what postnatal age and in which cell type is PDGF-A first expressed in cells of the adult Leydig cell lineage, and does PDGF-A expression coincide with expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), an indicator of steroid hormone synthesis. Male Sprague Dawley rats of postnatal day 1, 7, 9-14, 21, 28, 40, 60, and 90 were used (n=6). Animals were euthanized and their testicles removed, fixed in Bouin's solution, embedded in paraffin, and 5 micrometers sections were prepared. Immunolocalization of PDGF-A and 3beta-HSD was carried out using a peroxidase-streptavidin-biotin method. PDGF-A was first detected in cells of the Leydig cell lineage at postnatal day 10 in progenitor cells, which were surrounding the seminiferous tubules (peritubular). These cells were confirmed to be the progenitor cells and not the mesenchymal or any other spindle-shaped cells in the testis interstitium by immunolocalization of 3beta-HSD and PDGF-A in the cells in adjacent sections of testis tissue from rats of postnatal days 10-14. After postnatal day 10, PDGF-A was continued to be expressed in subsequent cells of the Leydig lineage through day 90 (adult), however, was not present in peritubular mesenchymal precursor cells of the Leydig cell lineage or any other spindle-shaped cells in the testis interstitium at any tested age. These results revealed that PDGF-A first appears in Leydig progenitor cells in the postnatal rat testis at the onset of mesenchymal cell differentiation into progenitor cells at postnatal day 10 and suggest that a functional role(s) of PDGF-A in postnatally differentiated Leydig cells in the rat testis is established at the time of the onset of postnatal Leydig stem cell differentiation. It is suggested that the significance of the first expression of PDGF-A in the Leydig progenitor cells may be associated with inducing cell proliferation and migration of this cell away from the peritubular region during Leydig cell differentiation.     

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells. Neither growth hormone which was reported previously to stimulate hepatic 5 alpha-reductase activity, nor insulin, insulin-like growth factor-I, or epidermal growth factor, which have been reported to modulate Leydig cell function, had any effect on 5 alpha-reductase activity of Band 3 cells. These studies suggest that the major factor directly stimulating 5 alpha-reductase activity in Leydig cells during early maturation is LH. However, it is possible that other factors acting indirectly may modulate the maturational rise in 5 alpha-reductase activity.     

Reorganization of Functional Systems in Ontogeny

This paper presents data that many mechanisms functioning at a certain stage of development are eliminated from the systemic organization at the subsequent stage of ontogeny. A new point of view on the principles of function formation in ontogeny is proposed. It is based on the fact that in the process of development (embryonic period, early postnatal period, transition to an independent existence, etc.) the subjective structure of the external world changes, therefore adaptation at a certain stage of ontogeny implies creation of another system of function regulation, which provides survival of the organism under new conditions. This becomes possible owing to a reorganization of the functional systems of the organism, including formation of new mechanisms and elimination of everything from the behavioral repertoire, which had lost its adaptive importance at the given stage of ontogeny. Such reorganization is comparable with formation of a new species in the process of evolution (a transition to another ecological niche). To characterize principles of the transition from one stage of ontogeny to another and to emphasize certain common features of the evolutionary and ontogenetic processes, it is suggested to use the term ontogenetic niche.     

The histone deacetylase SIRT1 controls male fertility in mice through regulation of hypothalamic-pituitary gonadotropin signaling

Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis development appears normal in Sirt1(-/-) mice. In contrast, the first round of spermatogenesis arrests before the completion of meiosis with abundant apoptosis of pachytene spermatocytes, abnormal Leydig and Sertoli cell maturation, and strongly reduced intratesticular testosterone levels. We show that this phenotype is the consequence of diminished hypothalamic gonadotropin-releasing hormone expression and strongly reduced luteinizing hormone levels. Rather than having an intrinsic effect on male germ cells per se, our results show that SIRT1 regulates spermatogenesis at postnatal stages by controlling hypothalamus-pituitary gonadotropin (HPG) signaling. In addition to its well studied role in control of metabolism and energy homeostasis, our results thus reveal a novel and critical function of SIRT1 in controlling HPG signaling. This phenotype is more severe than those previously described using mice bred on different genetic backgrounds, and highlights the fact that SIRT1 function is strongly modified by other genetic loci.     

Progenitor cells of the testosterone-producing Leydig cells revealed

The cells responsible for production of the male sex hormone testosterone, the Leydig cells of the testis, are post-mitotic cells with neuroendocrine characteristics. Their origin during ontogeny and regeneration processes is still a matter of debate. Here, we show that cells of testicular blood vessels, namely vascular smooth muscle cells and pericytes, are the progenitors of Leydig cells. Resembling stem cells of the nervous system, the Leydig cell progenitors are characterized by the expression of nestin. Using an in vivo model to induce and monitor the synchronized generation of a completely new Leydig cell population in adult rats, we demonstrate specific proliferation of vascular progenitors and their subsequent transdifferentiation into steroidogenic Leydig cells which, in addition, rapidly acquire neuronal and glial properties. These findings, shown to be representative also for ontogenetic Leydig cell formation and for the human testis, provide further evidence that cellular components of blood vessels can act as progenitor cells for organogenesis and repair.     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Leydig cells, thyroid hormones and steroidogenesis

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.     

Changes in ganglioside composition of photoreceptors during postnatal maturation of the rat retina

To examine at which stage the unusual ganglioside composition observed in adult retinal photoreceptor cells was established, and to see whether ganglioside changes could be correlated to distinct maturational events, quantitative and qualitative variations in gangliosides within pure sheets of photoreceptors during postnatal differentiation and aging of retina were studied. Retinas were separated into their component layers, (particularly photoreceptor layers uncontaminated by other neuronal types) by exploiting a technique of mechanical separation by vibratome. We extracted lipids from the cell membranes and analyzed the ganglioside composition by high performance thin layer chromatography. The data show that from the earliest recordable postnatal age (6 days) until late in life (18 months), photoreceptors contain low quantities of lipid-bound N-acetyl neuraminic acid and a simplified ganglioside profile compared to inner retinal neurons. Specific ganglioside changes occur within photoreceptor cells during postnatal maturation and aging, with downregulation of a-pathway GM1 and overlapping upregulation of b- pathway GD1b taking place during the period corresponding to outer segment formation, correlating with the onset of retinal function.      

Detection of anti-Mullerian hormone receptor II protein in the postnatal rat testis from birth to sexual maturity

Anti-Mullerian hormone (AMH) produced by the immature Sertoli cells negatively regulates the postnatal Leydig cell (i.e. adult Leydig cells/ALC) differentiation, however, the mechanism is sparsely understood. AMH negatively regulates the steroidogenic function of fetal Leydig cells (FLC) and ALC. However, when this function is established in the ALC lineage and whether AMH has a function in FLC in the postnatal testis are not known. Therefore, the objectives of this study were to examine the presence of AMH receptor type II (AMHR-II) in FLC and cells in the ALC lineage in the postnatal mammalian testis using the rat model Male Sprague Dawley rats of days 1, 5, 7-21, 28, 40, 60 and 90 were used. AMHR-II in testicular interstitial cells was detected in testis tissue using immunocytochemistry. Findings showed that the mesenchymal and the progenitor cells of the ALC lineage, were negative for AMHR-II. The newly formed ALC were the first cell type of the ALC lineage to show positive labeling for AMHR-II, and the first detection was on postnatal day 13, although they were present in the testis from day 10. From days 13-28, labeling intensity for AMHR-II in the ALC was much weaker than those at days 40-90. FLC were also positive. The time lag between the first detection of the newly formed ALC in the testis and the first detection of AMHR-II in them suggests that the establishment of the negative regulatory role of AMH on ALC steroidogenesis does not take place immediately upon their differentiation; no change in cell size occurs during this period. The absence of AMHR-II in mesenchymal cells suggests that it is unlikely that the negative regulatory effect of AMH on ALC differentiation in the postnatal testis is achieved via a direct action of AMH on mesenchymal cells. The presence of AMHR-II in postnatal FLC suggests a possible role by AMH on FLC, which warrants future investigations.