Immunolocalization of PTHrP in prepubertal and pubertal testis of European bison

The present study deals with immunohistochemical localization of PTHrP in prepubertal and pubertal testis of European bison. PTHrP immunoreactivity was observed in germinal cells in the testis of both prepubertal and pubertal animals. In calves, PTHrP was found in germinal cells, in seminiferous tubules lacking the lumen. The reaction was strong and regularly distributed within the cytoplasm. In adult animals, the reaction showed differentiation in spermatogenic cells. Some cells were strongly and diffusely stained, others exhibited weaker reaction of granular pattern. Sertoli cells and Leydig cells were PTHrP-negative in calves and adult animals.     

Immunolocalization of androgen receptors in testicular cells during postnatal development of the bank vole (Clethrionomys glareolus, S.)

Determination of the cellular distribution of the androgen receptors within the testis is of great importance for an understanding of their essential role in mediating of androgen action in the male gonad. In bank voles, which are seasonally breeding rodents, photoperiod is one of the most important factors inducing profound changes in the morphology and hormonal activity of the testes. Immunolocalization of androgen receptors was found in all somatic cells such as Sertoli cells, Leydig cells, and peritubular-myoid cells, however, distribution of the androgen receptors in various cell types depended on age of animals. Intensity of immunoreactivity was noticed as age and photoperiod-dependent. Males reared under different light regimes showed a significant correlation between the length of light and sexual maturation. Therefore, morphology of the testis from young and adult bank voles was also presented.     

Effects of tri-iodothyronine on testicular interstitial cells and androgen secretory capacity of the prepubertal Rat

The main objective of the study was to investigate the effects of hyperthyroidism on the rat testis interstitium during prepuberty, which is not well understood at present. Male Sprague Dawley rats were injected subcutaneously daily with saline (controls) or tri-iodothyronine (T(3), 50 microg/kg body weight; hyperthyroids) from postnatal Day 1. Rats were killed at Days 5, 7, 9, 12, 16, and 21. One testis of each rat was used to determine LH-stimulated (100 ng/ml) testicular androgen secretory capacity in vitro. The other testis was used either for morphometric studies (n = 5) or for immunolocalization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) to identify steroidogenic cells (n = 3) and 11 beta-hydroxysteroid dehydrogenase 1 (11 beta-HSD1) to differentially identify adult Leydig cells. Daily T(3) injections resulted in significant reductions in body and testis weights. Morphometric analysis revealed that lower testis weights in rats treated with T(3) were mainly the result of reductions of total volume of seminiferous cords/tubules. The number of interstitial mesenchymal cells (MCs) was lower (P < 0.05) in T(3) rats compared with age-matched controls. The number of fetal Leydig cells (FLCs) was not different between the two groups; however, FLC hypotrophy was detected in T(3) rats at Day 16 in contrast to Day 21 in control rats. In both groups, morphologically identifiable adult Leydig cells (ALCs) were observed at Day 12 and thereafter; however, the ALC number per testis in T(3) rats was twice as much as those of controls. Positive immunolabeling for 3beta-HSD was first detected in MC/progenitor cells on Day 9 in rats in the T(3) group (cells were still spindle-shaped) and on Day 12 in rats in the control group. Testicular testosterone production in vitro was lower (P < 0.05) in T(3) rats compared with controls at each age tested and further reductions (<0.05) were observed in T(3) rats at Days 16 and 21. Testicular androstenedione production was also lower (P < 0.05) in T(3) rats at Days 5 and 7, but increased (P < 0.05) thereafter, than in control rats. These findings support that there are more newly formed ALCs in T(3) testes than in those of controls. Moreover, these results demonstrate that hyperthyroidism stimulates premature hypotrophy of FLCs and early differentiation of increased numbers of MCs to ALCs in the prepubertal rat testis, further supporting the view that thyroid hormone has a regulatory role in initiating MC differentiation into ALCs in the prepubertal rat testis.     

Immunohistochemical localization of androgen receptors in testicular cells of mice with mosaic mutation

Mice with mosaic mutation could be one of the models of human Menkes disease, which is associated with abnormal cooper metabolism. The aim of the present study was to localize androgen receptors (ARs) in the testes by means of immunohistochemistry. AR expression was observed in the nuclei of all somatic cells such as Leydig cells, Sertoli cells, and peritubular cells in sections from testes of control and mosaic mutant males. In the latter, very strong immunoreactivity for AR as well as higher levels of steroid hormones in homogenates were noticed in comparison to control mice. No positive immunoreaction for ARs was seen in control sections incubated without the primary antibody.     

Role of thyroid on testicular lipids in prepubertal, pubertal and adult rats. I. Hyperthyroidism

The influence of thyroxine-induced hyperthyroidism on testicular neutral and phospholipids was studied in prepubertal, pubertal and adult rats. Thyroxine treatment (25 micrograms/100 g body weight) for 1 month decreased testicular total lipids, total glyceride glycerols, total cholesterol and total phospholipids. Different classes of glyceride glycerol, cholesterol and phospholipid were also diminished due to thyroxine treatment. All classes of lipids returned to the euthyroid level after the withdrawal of thyroxine treatment. The data obtained in the present study suggest that thyroid hormones have a definite influence on testicular lipid metabolism in rats.     

Nycthemeral plasma and testicular androgen contents in guinea pigs at puberty]

A study based upon transversal sampling involving 13 series of 7 animals each does not show a significant circadian rhythm of testosterone and delta4 androstenedione concentrations in the plasma and testis of the guinea-pig at the age of puberty.     

Streptozotocin-diabetes impairs prolactin binding to Leydig cells in prepubertal and pubertal rats.

Prolactin (PRL) binding to Leydig cells in prepubertal and pubertal streptozotocin (STZ)-diabetic and insulin-treated rats was studied. Prepubertal (30-day-old) and pubertal (50-day-old) rats were made diabetic by single injection of STZ (120 and 100 mg/kg b.wt, respectively). After 3 days of STZ administration, a group of rats was given insulin injections subcutaneously (3 U/100 g b.wt/day in 2 equally divided doses). Animals of prepubertal and pubertal groups were killed on postnatal days 51 and 71, respectively. Age-dependent increase in serum testosterone, PRL levels and PRL receptors on Leydig cells were prevented by STZ-diabetes. Insulin administration partly or completely prevented these changes. These results suggest that steroidogenic defects in Leydig cells of prepubertal and pubertal diabetic rats may be associated with decrease in serum PRL levels and its receptors on Leydig cells. Insulin probably has a role in the maintenance of PRL receptor numbers on Leydig cells during pubertal maturation.     

Luteinizing hormone-releasing hormone in prepubertal and pubertal girls.

Estimations of immunoreactive LH-RH and LH in pooled sera of girls, adult women and postmenopausal women have been carried out. The girls were divided into three groups: I--girls aged 2--4 years, II--girls aged 5--8 years and III--girls 9--12 years of age. The estimated concentrations of LH-RH in particular groups were as following: in group I--1.2 +/- 0.2 pg/ml, in group II--2.2 +/- 0.4 pg/ml, in group III 31.0 +/- 4.4 pg/ml, in adult women 6.3 +/- 1.8 pg/ml. and in postmenopausal women 16.6 +/- 2.4 pg/ml. The concentrations of LH in the same groups were 4.3 +/- 0.7; 4.5 +/- 0.8; 11.0 +/- 1.4, 23.3 +/- 2.4; and 120.0 +/- 14.7 mIU/ml, respectively. The authors suggest that the sexual maturation of girls is initiated by the enhanced hypothalamic activity, reflected in higher concentrations of immunoreactive LH-RH in peripheral serum.     

Gender differentiations of cognitive-motor functioning in prepubertal and pubertal children

The aim of this study was to determine cognitive and motor status factors in female and male children aged 10-14, as well as developmental and/or integration functions according to gender. The study included 162 girls and 134 boys aged 10-14, divided into four groups: 84 girls aged 10-12 (mean age 11.26, SD 0.68), 84 boys aged 10-12 (mean age 11.41, SD 0.50), 78 girls aged 13-14 (mean age 13.52, SD 0.63) and 50 boys aged 13-14 (mean age 13.21, SD 0.53). The significance of quantitative differences between boys and girls in the overall system of variables was defined based on the results of canonic discriminant analysis of variance, and within each variable based on the results on univariate analysis of variance (ANOVA). In the younger age group (10-12 years), girls were superior to boys in a test assessing flexibility (Seated straddle stretch), whereas, compared to girls, boys had greater strength of the trunk (Crossed-arm sit-ups), greater explosive strength ofjump and sprint type (Standing broad jump and 20 m dash), and coordination (Obstacle course backwards and Steps laterally). In the older age group (13-14 years) differences in flexibility were even more prominent in favor of girls, whereas the differences in explosive strength increased in favor of boys, especially of the throwing type with better agility (Steps laterally), balance (Board balance) and greater static strength of arms and shoulders (Bent-arm hang). In order to determine qualitative differences between pubertal and prepubertal girls and boys, the matrix of variable inter-correlations was factorized by the procedure of principal components procedure, that were then transformed to promax solution. The results showed that cognitive functioning had a significant role in the motor efficacy of girls and boys aged 10 to 14. In the age group of 10-12 years, in females, cognitive functioning is related to the motor system which integrates the regulation of muscle tone with agility/coordination, whereas in males there is a relation between cognitive abilities and the regulator of speed of upper extremities movement frequency. In the age group of 13-14 years, in females, cognitive functioning is involved in forming the factors for regulation of coordination and the intensity of energy mobilization in lower extremities, and to some degree, in the factor for regulation of intensity of energy mobilization in upper extremities and strength of the trunk, whereas in males the integration of synergetic regulation of movement in terms of balance and agility in terms of speed of direction change is carried out with significant involvement of cognitive abilities.     

Postnatal changes in testicular development and androgen receptors immunolocalization in D'Man ram lambs

The purpose of this study was to characterize testicular development in D'Man ram lambs, focusing primarily on androgen receptors (ARs) immunolocalization in the adenohypophysis and testis that is not still known in the D'Man ram lamb. Lambs (n = 12) were divided into four groups (three lambs per group). Adenohypophysis and testis were fixed and paraffin embedded; cross-section (3 μm) were stained and evaluated with immunohistochemistry. Testis weight increased at a greater rate between two and five months after birth, which was associated with remarkable changes in testicular histology, including significant increases in the diameter of seminiferous tubules. Spermatogenesis started between three and five months after birth; lumen and elongated spermatids were observed for the first time in three and four months-old animals respectively. ARs detected with immunohistochemistry were located in the nuclei and cytoplasm of adenohypophysis cells, and only in nuclei of testis cells (Leydig, Sertoli, peritubular myoid and germ cells).     

Human sympathoadrenal system and adrenal cortex in prepubertal and pubertal periods

A complex study of the functional state of the sympathoadrenal system and adrenal cortex in 10-15-year-old children of both sexes was carried out using the indices of daily excretion of adrenaline, noradrenaline, 17-ketosteroids, and 17-hydroxycorticosteroids. A synchronism in the functional activity of the mediator component of the sympathoadrenal system as well as of the androgenic and glucocorticoid functions of the adrenal cortex was observed with age and during pubertal development of children. At the same time, heterochronic maturation was observed in the sex groups: in girls at the age of 10 and 12 years and in boys at the age of 14-15 years. The changes of different direction and intensity in the excretion of the studied hormones and hormonal metabolites were observed in the sex and age groups. A sharp increase in the daily excretion of glucocorticoid metabolites accompanied by a considerable decrease in the age index of noradrenaline secretion was observed in 14- and 15-year-old boys from beginning to end of school year; in addition, an increase in the daily excretion of sex hormones was observed at the age of 15 years. In girls, these indices varied within the age range, which points to a more sophisticated neuroendocrine control of physiological functions in girls during puberty.     

Human sympathoadrenal system and adrenal cortex in prepubertal and pubertal periods

A complex study of the functional state of the sympathoadrenal system and adrenal cortex in 10–15-year-old children of both sexes was carried out using the indices of daily excretion of adrenaline, noradrenaline, 17-ketosteroids, and 17-hydroxycorticosteroids. A synchronism in the functional activity of the mediator component of the sympathoadrenal system as well as of the androgenic and glucocorticoid functions of the adrenal cortex was observed with age and during pubertal development of children. At the same time, heterochronic maturation was observed in the sex groups: in girls at the age of 10 and 12 years and in boys at the age of 14–15 years. The changes of different direction and intensity in the excretion of the studied hormones and hormonal metabolites were observed in the sex and age groups. A sharp increase in the daily excretion of glucocorticoid metabolites accompanied by a considerable decrease in the age index of noradrenaline secretion was observed in 14-and 15-year-old boys from beginning to end of school year; in addition, an increase in the daily excretion of sex hormones was observed at the age of 15 years. In girls, these indices varied within the age range, which points to a more sophisticated neuroendocrine control of physiological functions in girls during puberty.     

Variations in testicular androgen receptors and histology of the lamb testis from birth to puberty

Changes in testicular androgen receptor numbers were studied in lambs from 25 to 100 days of age. During this period, cytoplasmic receptors increased from 5 to 80 pmol/testis and nuclear receptors from 1 to 12 pmol/testis, while the total volume of Leydig cells increased 7-fold. The total number of Sertoli cells doubled between 25 and 40 days of age. From 40 days onward their number remained constant while their cellular and nuclear sizes increased by a factor of 3 and 1.5 respectively. Cytoplasmic receptor concentration was positively correlated with the number of Sertoli cells per section of seminiferous tubule, and negatively correlated with the number of germinal cells per cross section. One explanation for these results could be that Sertoli cells are the main androgen target cells in lamb seminiferous tubules.     

Localization of androgen receptors in ram epididymal principal cells

Androgen receptor was immunolocalized in the epididymal epithelium of rams and in isolated cells using an antibody against a synthetic polypeptide representing a portion of the androgen receptor. Immunostaining was predominant in the epithelium in tissue sections. Concentrations of androgen receptor were determined in cells from the central caput, distal caput, and central corpus epididymidis enzymically dissociated and elutriated to provide two fractions. On the average (n = 18), Fraction I contained 8% principal cells while Fraction II contained 71% principal cells; the stromal cells in each fraction were primarily smooth muscle and fibroblasts. For each sample, the number of DHT receptors (fmol) per 10(6) total cells was greater in Fraction II than in Fraction I. Few cells in Fraction I were immunostained for androgen receptor, whereas most cells in Fraction II were intensely stained. The numbers of DHT receptors per cell, or per principal cell, were similar for the central caput and distal caput, but lower in the central corpus epididymidis. The results support our hypothesis that most epididymal DHT receptors are localized in principal cells and confirm that the region between the central caput and proximal corpus of the ram epididymis is most dependent on androgen stimulation.     

Direct measurement of androgen receptors in cultured sertoli cells

Cytoplasmic and nuclear forms of macromolecules with the properties of androgen receptors have been demonstrated by direct labeling techniques in cultured Sertoli cells. The cytoplasmic form was excluded from Sephadex G-200 and could be distinguished from androgen binding protein (ABP) on the basis of size, heat stability, relative electrophoretic mobility, and binding complex dissociation rate. When cultured Sertoli cells were incubated with ³H-testosterone, a time- and temperature-dependent accumulation of label into the nuclear fraction was observed, 46% of which crystallized as authentic testosterone. Specific binding was saturable with an apparent association constant of 0.4nM^?1. Approximately 30% of the nuclear bound hormone was extracted within 1 h by 0.4M KCl and 34% of this was associated with macromolecular species as measured by gel filtration. Unlabeled androgens and to some degree progestogens competed with ³H-testosterone for binding sites. These data constitute direct evidence that Sertoli cells contain androgen receptors.     

Human prepubertal testicular cells in culture: Steroidogenic capacity, paracrine and hormone control

The neonatal human Leydig cell undergoes a transient period of activation during the first months of life. The biological significance of this activation is unknown. Furthermore, little is known about the hormonal regulation of this biological process, even though it coincides with an elevation of LH levels in serum. In order to study the function of human prepubertal testicular culture cells, obtained during the neonatal period, a method for maintaining primary culture cells (isolated from testes collected at necropsy) in culture was developed. Within 24 h after death, testes were collected from 1–36-month-old subjects. Subjects were divided into two age groups, based on the presence or absence of fetal Leydig cells: 1–7-month-old infants (group 1) and 12–36-month-old children (group 2). Testes were digested with collagenase, and cells were seeded in multi-well dishes. Cells were grown in serum-free conditioned media supplemented with 5 mg/l vitamin C, 0.2 IU/l vitamin E and 10% fetal bovine serum for 2 days. Cells were then grown for an additional 4 days in serum-free media in the presence or absence of hLH (40 IU/l), hCG (135 IU/l), rh FSH (1.5 IU/l), rhGH (0.12 IU/l) or insulin (0.9 μmol/l). Concentrations of steroids in media were determined by RIA on day 6 of culture. In basal conditions cells of group 1 (n = 11) secreted more testosterone, androstendione, 17-hydroxyprogesterone, progesterone and dehydroepiandrosterone (mean ± SE: 6.76 ± 1.86, 7.37 ± 1.82, 61.9 ± 1.86, 5.75 ± 1.74 and 8.51 ± 3.23 pmol/10⁶ cells/24 h, respectively) than cells of group 2 (n = 5) (2.95 ± 1.15, 1.50 ± 2.75, 1.44 ± 2.75, 0.78 ± 1.74 and 3.23 ± 1.32, respectively). Under hLH stimulation, cells of group 1 increased testosterone, androstendione and 17-hydroxyprogesterone secretions (to 38.2 ± 0.89, 13.5 ± 1.17 and 51.7 ± 3.23), while progesterone secretion remained unchanged (2.82 ± 1.20). Cell response to rhFSH and rhGH was similar to that of hLH. On the other hand, medium collected from cultures of cells isolated from a Sertoli cell tumor was able to stimulate testosterone secretion in subcultures of control testicular cells in a way similar to that of hCG. In conclusion, (1) these prepubertal human testicular cells can be maintained in primary culture for several days keeping their in vivo steroidogenic potential; (2) cells isolated from young infants can respond to hLH in culture; (3) response to rhFSH is probably mediated by a paracrine factor; (4) response to rhGH is observed in the absence of gonadotropins. Therefore, the early postnatal activation of the human testis might be under multiple pituitary hormone control; and, finally, (5) Sertoli cell tumors can secrete paracrine factors that stimulate steroidogenesis.     

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.