Age determination of pre- and postnatal skeletons with special reference to current methodological aspects]

From the remains of ten fetal and neonatal skeletons the standard methods of age estimation by means of dental and osseous criteria as well as diaphyseal length were applied and discussed. The ratio of long bone lengths and change of proportions during the first and second half of pregnancy is a useful indicator for the stage of maturation.     

Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture.

Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process. In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome. After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml. At 11 days in culture, samples were removed for morphological characterization and determination of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD). Testosterone concentration was determined by RIA in the culture medium at different intervals. Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions. No mature Leydig cells were present. In basal conditions, the percentages of 3 beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%, respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng.10(6) cells.24 h, respectively. In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng.10(6) cells, respectively). Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-secretory patterns of normal and abnormal human placentas with special reference to human chorionic gonadotropin

[35S]Methionine-labeled protein-secretory patterns resolved by two-dimensional polyacrylamide gel electrophoresis in abnormal hydatidiform-mole placentas were compared with those in normal full-term placentas with special reference to human chorionic gonadotropin (hCG) by means of immunoblotting and immunoelectron-microscopic techniques. Although basic protein-secretory patterns of both placentas were similar to each other, four polypeptide spots appeared and one spot disappeared in the hydatidiform-mole samples. Among four newly synthesized and secreted spots, three were immunoreacted with anti-hCG serum by an immunoblotting experiment. Ultrastructural localization of hCG showed that the labeling intensity of anti-hCG serum in hydatidiform-mole placentas was much heavier than that in full-terms ones. Particularly, the Golgi apparatus, middle-sized granules and large bodies were highly immunoreactive. The present study reveals that hydatidiform-mole placentas have different protein-secretory functions especially in hCG synthesis and secretion from those of normal pregnancy.     

Immunolocalization of estrogen receptor alpha, estrogen receptor beta and androgen receptor in the pre-, peri- and post-pubertal stallion testis

In various species, androgens and estrogens regulate the function of testicular Leydig, Sertoli, peritubular myoid, and germ cells by binding to their respective receptors and eliciting a cellular response. Androgen receptor (AR) is expressed in Sertoli cells, peritubular myoid cells, Leydig cells and perivascular smooth muscle cells in the testis depending on the species, but its presence in germ cells remains controversial. Two different estrogen receptors have been identified, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), and their localization and function in testicular cells varies depending on the species, developmental stage of the cell and type of receptor. The localization of AR in an immature and mature stallion has been reported but estrogen receptors have only been reported for the mature stallion. In the present study, the localizations of AR and ERα/ERβ were investigated in pre-pubertal, peri-pubertal and post-pubertal stallions. Testes were collected by routine castration from 21 horses, of light horse breeds (3 months-27 years). Animals were divided into the following age groups: pre-pubertal (3-11 months; n=7), peri-pubertal (12-23 months; n=7) and post-pubertal (2-27 years; n=7). Testicular tissue samples were fixed and embedded, and the presence of AR, ERα and ERβ was investigated by immunohistochemistry (IHC) using procedures previously validated for the horse. Primary antibodies used were rabbit anti-human AR, mouse anti-human ERβ and rabbit anti-mouse ERα. Sections of each region were incubated with normal rabbit serum (NRS; AR and ERα) or mouse IgG (ERβ) instead of primary antibody to generate negative controls. Androgen receptors were localized in Leydig, Sertoli and peritubular myoid cells of all ages. Estrogen receptor alpha was localized in Leydig and germ cells of all ages but only in pre- and peri-pubertal Sertoli cells and post-pubertal peritubular myoid cells. Estrogen receptor beta was localized in Leydig and Sertoli cells of all ages but in only pre-pubertal germ cells and absent in peritubular myoid cells of all ages. Taken together, the data suggest that estrogen regulates steroidogenesis by acting through ERα and ERβ in the Leydig cells and promotes gametogenesis by acting through ERβ in the Sertoli cells and ERα in the germ cells. In contrast androgen receptors are not found in germ cells throughout development and thus are likely to support spermatogenesis by way of a paracrine/autocrine pathway via its receptors in Leydig, Sertoli and peritubular myoid cells.     

Effects of testosterone, estradiol, aromatase inhibitor, gonadotropin and prolactin on the response of mouse testes to acute gonadotropin stimulation

These studies determined the local acute responsiveness of the testis to intratesticular administration of human chorionic gonadotropin (hCG) under basal, stimulated (systemic hCG pre-treated), hypogonadotropic (steroid pre-treatment) and hyperprolactinemic conditions in male mice. In addition, testicular testosterone (T) levels were determined after intratesticular administration of the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) or progesterone under basal or hCG-stimulated conditions. Intratesticular administration of 0.025, 0.25, 2.5 or 25 mIU hCG resulted in a dose-dependent (3- to 14-fold) increase in testicular T concentrations in hCG compared to vehicle-injected testes. Systemic (i.p.) pre-treatment with 5 IU hCG 24 h before prevented any further increases in the already elevated (10-fold basal) T levels after direct intratesticular hCG injection. Pretreatment with 250 micrograms testosterone propionate (TP) reduced basal testicular T concentrations, but resulted in increased responsiveness to intratesticular hCG administration. In contrast, estradiol benzoate (EB) pretreatment, which also reduced basal testicular T concentrations, did not affect the testicular responsiveness to hCG. Hyperprolactinemia reduced testicular responsiveness to intratesticular administration of 0.025, 0.25 or 2.5 mIU hCG, but basal levels of testicular T were elevated. One hour after intratesticular injections of an aromatase inhibitor, 4-OHA; (0.25 micrograms) testis, T levels were increased in males pre-treated with 5 IU hCG (i.p.) 24 h earlier. Higher doses of 4-OHA (2.5, 25 or 250 micrograms) resulted in significant, dose-related increases in basal testicular T levels which were attenuated by hCG-pre-treatment. Intratesticular administration of 20 micrograms progesterone increased testicular T concentrations 2.7-fold, but this effect was attenuated (1.5-fold) in hCG-pre-treated mice, suggesting that enzymatic lesions beyond progesterone may be involved in hCG-induced testicular desensitization. These results indicate that testicular responsiveness to hCG depends on the existing levels of gonadotropic stimulation. However, it is evident that estrogens and prolactin also influence the sensitivity of the testis to gonadotropin.     

Streptozotocin diabetes in the rat with special reference to glucose utilization by the musculature]

The glucose utilization by different skeletal muscle tissues of short-term streptozotocin treated Wistar rats was studied both in vivo and in vitro. The findings permit the following statements: The reduced metabolic conversion of glucose is mainly the result of the diminished transport of glucose into the muscle cells. The utilization of the glucose taken up by the muscle cells for synthesis of glycogen is unchanged in the diabetic animals and can be stimulated by insulin correspondingly as in normal rats. The conversion of the glucose metabolized by the cells to lactate and the time course of the specific activity of glycogen and lactate lead to the conclusion that glycogenolysis in the muscles of streptozotocin diabetic rats during the incubation is enhanced.     

Action of gonadotropins on androgen synthesis by embryonic testes of mouse and rat]

Testes from mouse and rat embryos were cultured in vitro in the presence of 14C-sodium acetate and luteinizing hormone or chorionic gonadotrophin. There is an increase in testosterone and androstenedione synthesis from sodium acetate under both treatments. Sensitivity to gonadotrophins exists already at a relatively early stage, when the testis, through testosterone secretion, is building up the genital tract. But the role of the hypophysis in normal androgen secretion will have to be proven.     

Distribution of LHRH in the rat and mouse brain with special reference to the tanycytes

Summary The distribution of luteinizing hormone-releasing hormone (LHRH) was studied in the rat and mouse brain by means of light and electron microscopic immunohistochemistry using the peroxidase-antiperoxidase method. An immunoreactive product to LHRH antiserum was found near the blood vessels of the vascular organ of the lamina terminalis. In the arcuate nucleus-median eminence region, an immunoreactive material occurred bilaterally in the hypothalamic tissue around the tuberoinfundibular sulci. Electron microscopy revealed that immunoreactive fibers observed light microscopically contain numerous granules 100–130 nm in diameter. No immunoreactive product was located in the tanycytes of the median eminence, the perikarya of hypothalamic neurons, and the parenchyma of several circumventricular organs (subfornical organ, subcommissural organ, pineal organ, area postrema).Supported by grants from the Ministry of Education of Japan and the Ford Foundation     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed an increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and beta-N-acetyl-D-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and gamma-glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.     

Amino acid uptake and protein synthesis in rat testes: stimulation by dissociable factors

Low concentration (25 mM) of sodium in the incubation medium produced a decrease in the amino acid uptake by the testis tissue as well as a reduction in the response to FSH. In this experimental condition, the basal protein synthesis and the stimulatory effect of FSH was not modified. The subcutaneous administration of testosterone to 15 day old rats increased the protein synthesis in the testis without any modification in the amino acid uptake. The addition of DBcAMP (1 mM) or glucose (14 mM) to the incubation medium increased the protein synthesis in the testes of immature (12 day-old) or prepubertal (32 day-old) rats respectively. The amino acid uptake was not modified. In immature rat testes, with protein synthesis completely inhibited by cycloheximide, the restoration of the sodium concentration in the incubation medium to normal levels produced an increase in amino acid uptake. The results above seem to indicate that protein synthesis and amino acid uptake in rat testes tissue can be regulated, at least partially, by different factors.