Relationships between Leydig cell morphometry and plasma testosterone during postnatal development of the monkey, Macaca fascicularis

In neonates (0 to 3-4 months), the testis contained a mean number of 4.6 X 10(6) Leydig cells representing 4.2 % of its volume; Leydig cell cytoplasm contained 10.2 % of SER. In infants (up to 45 months), Leydig cells regressed but their number increased; their volume density did not change. Leydig cell cytoplasmic volume (454 microns3 ), which was about 2.5-fold less than in neonates (1 119 microns3 ) or adults (1 170 microns3 ), contained only 8.7% of SER. During meiosis stage (38-52 months). Leydig cell numbers and volume density did not vary but the cells reached a maximal size and an amount of SER comparable with that at birth was measured. When spermatogenesis was complete, the Leydig cells represented no more than 0.8% of testis volume, but their number and SER content were significantly increased. Except for a significant decrease when spermatogenesis was completed, Leydig cell lipid content did not change during development, and the volume density of mitochondria did not vary. The mean level of plasma testosterone was 2 ng/ml in neonates and 0.4 ng/ml in infants; it increased to 3 ng/ml during onset of meiosis and reached 10 ng/ml in adults. The profile of testosterone was positively and significantly correlated with the total volume and total number of Leydig cells (P less than 0.01 and P less than 0.02, respectively) and with changes in their cytoplasmic volume (P less than 0.001). Moreover, plasma testosterone levels were positively and significantly correlated with changes in Leydig cell SER content i.e. SER volume density and mean absolute volume per cell (P less than 0.001), total SER in the whole testis (P less than 0.01).     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

Kinetic and autoradiographic studies on binding of hCG to the testicular LH receptors of neonatal rats

The properties of hCG binding to LH receptors of the neonatal (5-day-old) rat testis were analysed and compared with those of the adult testis. The equilibrium association constants (Ka) of hCG-binding were similar at both ages, 2-4 X 10(10) M-1. In contrast, kinetic binding studies revealed that the association and dissociation rate constants of hCG binding were more rapid in the neonatal testis. Likewise, it was observed that the progression from loose (easily dissociable) to tight (non-dissociable) binding was less complete in the young than in the adult testis. Autoradiography of 125I-labelled hCG binding to interstitial cell suspensions at the two ages showed that the gonadotrophin binding per Leydig cell was about 50% lower in the neonatal testis. Conversely, since the surface area of adult Leydig cells was about 4-fold larger, the receptor density appeared to be higher in the neonatal Leydig cells. The rapid recovery of LH receptors after hCG stimulation, typical of the neonatal cells, was due to rapid replenishment of binding in the cells initially occupied by the injected hormone, rather than to an hCG-induced increase of Leydig cell number. Finally, in-vivo experiments with cycloheximide revealed that the rapid recovery of LH receptors was dependent on protein synthesis. These differences in the kinetics of neonatal testicular LH receptor turnover may be involved in the unique functional features of the fetal-neonatal growth phase of rat testicular Leydig cells.     

Seasonal variation in the total volume of Leydig cells in stallions is explained by variation in cell number rather than cell size

Stereological methods were employed in two studies with stallions 1) to determine if seasonal variation in the total volume of Leydig cells is a function of cell number or cell size and 2) to characterize the annual cycle of the Leydig cell population. In the first study, numbers of Leydig cells were calculated for 28 adult (4-20 yr) stallions in the breeding or nonbreeding seasons from nuclear volume density (percentage of the decapsulated testicular volume), parenchymal volume (decapsulated testicular volume), and the volume of individual Leydig cell nuclei. The average volume of the individual Leydig cells was calculated as the total Leydig cell volume/testis (volume density of Leydig cells in the parenchymal volume times parenchymal volume) divided by the number of Leydig cells. The average volume of an individual Leydig cell varied within each season, but means were almost identical for the nonbreeding (6.94 +/- 0.61 picoliter) and breeding (6.91 +/- 0.45 picoliter) seasons. However, Leydig cell numbers per testis were 57% higher in the breeding season, which also had a 58% higher total volume of Leydig cells per testis. In the second study, the numbers of Leydig cells were determined for 43-48 adult horses in each 3-mo period for 12 mo. The number of Leydig cells per testis in May-July was higher (p less than 0.05) than in August-October or February-April, and higher (p less than 0.01) than in November-January. Thus, seasonal fluctuations in the total volume of Leydig cells in adult stallions is a function of the number of Leydig cells that cycle annually.     

Macrophages in the interstitial tissue of the rat testis

Summary Macrophages were identified in the intertubular tissue of the rat testis by loading animals with a particulate vital dye (trypan blue or India ink) and by localizing immunocytochemically a macrophage membrane antigen (MRC W3/25). Leydig cells were identified by the histochemical staining reaction for 3-hydroxysteroid dehydrogenase activity and by a monoclonal antibody. Macrophages were scattered in the interstitial tissue closely attached to and mixed with the Leydig cells. They were never found in the seminiferous tubules. The macrophages comprised about 25% of all the cells in the interstitium. Double staining with a vital dye and a marker antibody showed that all the phagocytosing cells were macrophages and that the Leydig cells did not take up vital dyes. Double staining for the demonstration of the 3-hydroxysteroid dehydrogenase activity and the macrophage antigen likewise revealed two distinctly different cell populations. Crude Leydig cell preparations obtained by collagenase treatment of the testis contained macrophages (12–14%). Macrophages were present throughout the postnatal prepuberal development of the testis. Their density was increased in the cryptorchid and irradiated testis.     

The intertubular compartment morphometry in capybaras (Hydrochoerus hydrochaeris) testis

The aim of the present study was to characterize the intertubule element volume density, individual and total Leydig cells volume, Leydig cell number per testis and per gram of testis, and leydigosomatic index in adult capybaras. Eight capybaras from a commercial abattoir were utilized. The intertubular compartment volume density and the Leydig cells were 45.2 and 31.13%, respectively. The individual and total Leydig cell volumes were 8.51 and 2169.41 x 10(-12) mL, respectively. The Leydig cell number per testis was 3.8 billion and the Leydig cell number per gram of testis was 126 million. The leydigosomatic index was 0.037%. In conclusion, this study shows that capybaras have one of the greatest individual and total Leydig cell volume and Leydig cell volume density, and that the Leydig cell number per gram of testis is at least double the mean for mammals previously investigated in its order.     

Origin of regenerating Leydig cells in the testis of the adult rat

Summary Ethane dimethanesulphonate (EDS) was used as a specific cytotoxin to eliminate the Leydig cell population of the adult rat testis. Ultrastructural, morphometric and serum gonadotrophin and testosterone analysis was used to study the response of the intertubular tissue of the testis from 1 day to 10 weeks after EDS treatment. In control animals, the testis contained approximately 28 million Leydig cells and 8 million macrophages. Three to seven days after EDS treatment, Leydig cells were absent and serum testosterone was undetectable. Macrophage numbers increased three-fold by 3 days and returned to pretreatment values thereafter. At 2 and 3 weeks post-EDS, foetal-type Leydig cells (1–2 million per testis) appeared in proximity to perivascular and peritubular tissues, a feature also observed at 4 weeks when numerous such cells (15 million per testis) formed prominent clusters in perivascular and peritubular locations. Between 6 and 10 weeks after EDS treatment, the foetal-type Leydig cells were transformed morphologically into adult-type Leydig cells, they occupied central intertubular positions and their numbers were restored to pretreatment values. Regeneration of Leydig cells was reflected by elevated serum testosterone levels which returned towards the normal range. The results demonstrate the regenerative capacity of the testicular intertubular tissue and indicate a dual site of origin of Leydig cells which initially resemble foetal-type Leydig cells prior to establishing the adult-type Leydig cell population. The morphological pattern of Leydig cell regeneration suggests that in addition to gonadotrophic stimulation, local testicular factors from the seminiferous tubules may stimulate Leydig cell growth.     

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.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

Morphometric studies on hamster testes in gonadally active and inactive states: light microscope findings

This study provides quantitative information on the testes of seasonally breeding golden hamsters during active and regressed states of gonadal activity. Seminiferous tubules occupied 92.5% of testis volume in adult gonadally active animals. Leydig cells constituted 1.4% of the testicular volume. The mean volume of an individual Leydig cell was 1092 microns 3, and each testis contained about 25.4 million Leydig cells. The volume of an average Sertoli cell nucleus during stage VII-VIII of the cycle was 502 microns 3. A gram of hamster testis during the active state of gonadal activity contained 44.5 million Sertoli cells, and the entire testis contained approximately 73.8 million Sertoli cells. Testes of the hamsters exposed to short photoperiods for 12-13 wk displayed a 90% reduction in testis volume that was associated with a decrease in the volume of seminiferous tubules (90.8% reduction), tubular lumena (98.8%), interstitium (72.7%), Leydig cell compartment (79.3%), individual Leydig cells (69.7%), Leydig cell nuclei (50.0%), blood vessels (85.5%), macrophages (68.9%), and Sertoli cell nuclei (34.1%). The diameter (61.1%) and the length (36.8%) of the seminiferous tubules were also decreased. Although the number of Leydig cells per testis was significantly lower (p less than 0.02) after short-photoperiod exposure, the number of Sertoli cells per testis remained unchanged. The individual Sertoli cell in gonadally active hamsters accommodated, on the average, 2.27 pre-leptotene spermatocytes, 2.46 pachytene spermatocytes, and 8.17 round spermatids; the corresponding numbers in the regressed testes were 0.96, 0.20, and 0.04, respectively. The striking differences in the testicular structure between the active and regressed states of gonadal activity follow photoperiod-induced changes in endocrine function and suggest that the golden hamster may be used as a model to study structure-function relationships in the testis.     

A comparison of Leydig cell function after unilateral and bilateral cryptorchidism and efferent-duct-ligation

Surgical induction of cryptorchidism or ligation of the efferent ducts disrupts spermatogenesis. The response of Leydig cells to disrupted gametogenesis was studied in vitro in tissue and collagenase dispersed Leydig cells obtained from the testes of rats that were made unilaterally or bilaterally cryptorchid or had been efferent-duct-ligated. Four wks after surgery, androgen secretion per mg of tissue or per Leydig cell in response to maximal luteinizing hormone (LH) stimulation was greater in tissue from damaged than from sham-operated testes. It was concluded that disruption of spermatogenesis resulted in Leydig cells that were hyperresponsive to LH stimulation in vitro. Unilateral lesions produced different responsiveness of Leydig cells from the testes ipsilateral and contralateral to the lesion, supporting the hypothesis that intragonadal modulation of Leydig cells function occurs when the function of seminiferous tubules is impaired. Stimulated androgen production of Leydig cells from the contralateral nonligated testis did not differ from that of the sham-operated controls. With unilateral cryptorchidism, which is accompanied by an increase in the temperature of the operated testis, Leydig cells from the scrotal testis were also hyperresponsive compared to those from sham-operated controls. This suggests a possible intergonadal influence of aspermatogenesis caused by cryptorchidism.     

三种波段电磁辐射致大鼠睾丸损伤的比较

目的对比性探讨电磁脉冲(EMP)、S带高功率微波(S-HPM)和X带高功率微波(X-HPM)三种波段电磁辐射致睾丸组织受损的近期和远期效应及其相关敏感指标。方法雄性Wistar大鼠192只,随机分为EMP组、S-HPM组、X-HPM组和对照组,于照后不同时间点采集睾丸组织称重,光镜观察睾丸损伤,并用图像分析技术对曲细精管病变进行定量分析。结果三种波段电磁波辐照后睾丸结构和生精细胞形态损伤基本相似:早期睾丸重及睾丸重/体重比值呈下降趋势;曲细精管生精上皮变薄,生精细胞排列紊乱,精原细胞变性坏死并由管壁脱落,精母细胞和精子数量减少并团聚于管腔中央,支持细胞和间质细胞不同程度变性;曲细精管受损百分率显示EMP组最重,S-HPM最轻,生精细胞受损数量与程度显著增加(P0.05)。结论三种波段电磁辐射对睾丸生精细胞的损伤,具有速发性、时相性、分布不均一性特点;损伤程度呈EMPX-HPMS-HPM;睾丸曲细精管受损百分率可定量反映其损伤程度,可望成为评估电磁辐射致睾丸损伤的敏感指标之一。     

Comparison of components of the testis interstitium with testosterone secretion in hamster, rat, and guinea pig testes perfused in vitro

Components of the testis and cytoplasmic organelles in Leydig cells were quantified with morphometric techniques in hamster, rat, and guinea pig. Testosterone secretory capacity per gram of testis and per Leydig cell in response to luteinizing hormone (LH) (100 ng/ml) stimulation was determined in these three species from testes perfused in vitro. Numerous correlations were measured among structures, and between structures and testosterone secretion, to provide structural evidence of intratesticular control of Leydig cell function. Testosterone secretion per gm testis and per Leydig cell was significantly different in the three species: highest in the guinea pig, intermediate in the rat, and lowest in the hamster. The volume of seminiferous tubules per gm testis was negatively correlated, and the volumes of interstitium, Leydig cells, and lymphatic space per gm testis were positively correlated with testosterone secretion. No correlations were observed between volumes of blood vessels, elongated spindleshaped cells, or macrophages per gm testes and testosterone secretion. The average volume of a Leydig cell and the volume and surface area of smooth endoplasmic reticulum (SER) and peroxisomes per Leydig cell were positively correlated, and the volume of lysosomes and surface area of inner mitochondrial membrane per Leydig cell were negatively correlated with testosterone secretion. No correlations were observed between volume and surface area of rough endoplasmic reticulum (RER), Golgi apparatus, and lipid, and volume of ribosomes, cytoplasmic matrix, and the nucleus with testosterone secretion per Leydig cell. These results suggest that Leydig cell size is more important than number of Leydig cells in explaining the difference in testosterone-secreting capacity among the three species, and that this increase in average volume of a Leydig cell is associated specifically with increased volume and surface area of SER and peroxisomes. An important unresolved question is what is the role of peroxisomes in Leydig cell steroidogenesis.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

The fate of fetal Leydig cells during the development of the fetal and postnatal rat testis

The ultrastructure and developmental fate of the fetal generation of Leydig cells of the rat testis was studied from the 17th day of fetal life up to 100 days after birth. The number of fetal Leydig cells per testis was determined by light microscopic morphometric analysis of semithin plastic sections. In fetal testes (days 17-22 postconception), Leydig cells exhibited a characteristic ultrastructure, containing smooth endoplasmic reticulum, many lipid inclusions and glycogen. Testes of 17-day-old fetuses contained about 25 x 10(3) fetal Leydig cells, rapidly increasing to 90 x 10(3) per testis in 21-day-old fetuses. After birth, fetal Leydig cells per testis remained relatively constant up to 2 weeks (80-90 x 10(3) per testis) and were identified by light and electron microscopy which showed their numerous lipid inclusions, their tendency for clustering and their association with interstitial tissue fibroblasts which partly encapsulated the fetal Leydig cells. From 21-100 days after birth, fetal Leydig cell numbers were quite variable with a mean of 45-60 x 10(3) per testis. These results are the first to show that the fetal generation of Leydig cells persist in the adult testis and do not undergo early postnatal degeneration or dedifferentiation into other interstitial cells. The simultaneous occurrence of the fetal Leydig cells and the adult population of Leydig cells indicates that these cells are distinct cell generations which are developmentally unrelated.     

The modified method for isolation and culture of highly homogeneous Leydig cell population from rat testes

A modified method for isolation and culture of a pure population of rat Leydig cells is described. For obtaining crude interstitial cell suspension, decapsulated testes were dispersed in 0.02% collagenase solution in Ca2+, Mg2+--free Hanks medium for 1 hour. Then, approx. 5 X 10(7) cells were centrifuged in 10-90% discontinuous, isoosmotic Percoll gradient at 3000 g for 20 min. The cells from eight fractions obtained were collected and cultured in Eagle's MEM for 4 days. Using morphological methods, 1.059-1.070 g/ml density fraction contained 97% and 1.070-1.080 g/ml fraction contained 90% viable Leydig cells. The cells secreted testosterone to the culture medium and responded to LH stimulation with over four-fold increase in hormone secretion.     

Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis

Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.     

Identification of markers for precursor and leydig cell differentiation in the adult rat testis following ethane dimethyl sulphonate administration.

Administration of ethane dimethane sulphonate (EDS) to adult rats results in the destruction of all Leydig cells, followed by a complete regeneration. We investigated this regeneration process in more detail, using different markers for precursor and developing Leydig cells: the LH receptor, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), transforming growth factor alpha (TGFalpha), and a new marker for Leydig cell maturation, relaxin-like factor (RLF). LH receptor immunoreactivity was found in Leydig cell-depleted testes at 3 and 8 days after EDS administration. The positive (precursor) cells had a mesenchymal-like morphology. The number of LH receptor-positive cells 8 days after EDS administration was 15 +/- 4 per 500 Sertoli cell nuclei. Fifteen days after EDS administration, the first new Leydig cells could be observed. These cells stained positively with both the antibodies against the LH receptor and 3beta-HSD, while some cells also stained positively for TGFalpha. After EDS administration, RLF mRNA disappeared from the testis and reappeared again at the time of the appearance of the first Leydig cells. Concomitant with the increase in the number of Leydig cells, the number of RLF-expressing cells increased. The observations of the present study give further support to the hypothesis that Leydig cell development in the prepubertal testis, and in the adult testis following EDS administration, takes place along the same cell lineage and suggest, therefore, that the adult EDS-treated rat can serve as a model for studying the adult-type Leydig cell development that normally occurs in the prepubertal rat testis.     

Presenilin-1 protein specifically expressed in Leydig cells with its expression level increased during rat testis development

Presenilin-1, mutations of which cause the early-onset of Alzheimer's disease, was shown to be abundantly expressed in the testis as well as the brain. In spite of the high expression level of this protein in the testis, no further analysis has been undertaken. We aimed to study the distribution and developmental changes in presenilin-1 protein, and to provide clues so as to elucidate the role of this protein in the rat testis. To evaluate the specificity of the anti presenilin-1 antibody, rat presenilin-1 protein was expressed in COS-7 cells and the recombinant protein was used for western blot analysis. A positive band of approximately 20 kDa corresponding to the C-terminal fragment of proteolyzed presenilin-1 protein was observed. Using testis and brain tissue samples, a 20 kDa band was detected in both tissues suggesting a similar proteolytic process, but the expression level in the testis was higher than that in the brain. The expression level increased significantly during postnatal testis development. By an immunohistochemical analysis of the rat testis, a strong signal was observed in interstitial cells and further study with cultured TM3 murine Leydig cells revealed an abundant expression of presenilin-1 in Leydig cells. Our study suggests that presenilin-1 expression in Leydig cells may play an important role in Leydig cell function and testis development.     

In vitro effects of Celiptium and MR 14504 on mature rat Leydig cell testosterone production

Percoll-purified mature rat Leydig cells have been used to evaluate the testicular toxicity of two highly potent intercalating agents (Celiptium and MR 14505). Testosterone secretion in the absence and in the presence of human chorionic gonadotropin (hCG) was measured to assess Leydig cell function. Celiptium and MR 14504 induce time- and dose-related inhibitory effects on the production of testosterone by Leydig cells, both in the presence and in the absence of hCG, whatever the concentration of hCG used. We have observed that MR 14504 is about 5 times more potent as an inhibitor of rat Leydig cell steroidogenesis than Celiptium without inducing any cell toxicity. The present study indicates that the Leydig cell is an additional potential site for the primary toxic effects of these drugs in the adult rat testis.