Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis.

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV-VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.

Immunocytochemical localization of albumin, transferrin, angiotensinogen and kininogens during the initial stages of the rat liver differentiation.

Rat albumin, transferrin, angiotensinogen, T kininogen (TKg) and high molecular weight kininogen (HKg) gene expression was examined immunocytochemically in embryonic and fetal livers. All these plasmatic proteins, angiotensinogen excepted, are detected as early as day 11 of gestation in intestine epithelial cells and embryonic hepatocytes. Angiotensinogen becomes expressible only at day 13 of gestation. During the early fetal period, the protein immunostaining increases strikingly in parallel with the hepatocyte differentiation. Albumin and transferrin are highly expressed comparatively to kininogens and angiotensinogen. For the first time, specific HKg is demonstrated in the rat liver.

Laminin ultrastructural immunolocalization in rat testis during ontogenesis.

The synthesis of one of the main glycoproteins of the basement membrane, the laminin, was demonstrated by ultrastructural immunolocalization during rat foetal (16th day to 20th day of gestation) and postnatal development of the testis. The lamina densa, part of seminiferous tubular basement membrane, is labeled uniformly at all studied stages. The lamina lucida is not well defined before the postnatal stages, at which times discrete immunostaining extends from the lamina densa to the adjacent seminiferous epithelial cells (spermatogonia and Sertoli cells). The extracellular matrix around the peritubular cells is not labeled before birth. Intracellular immunostaining was detected as early as the 16th day of gestation in both Sertoli cells and cells around the seminiferous tubules which will transform later into peritubular cells. It was located in rough endoplasmic reticulum (RER) cisternae and secretory vesicles. After 18-20 days of postnatal life, the immunostaining faints progressively. Some positive material is seen in the RER of the gonocytes at all studied stages. Sertoli cells and peritubular cells are the main producing cells of laminin after the 16th of gestation. The laminin secreted by gonocytes may play an important role in adhesion of gonocytes to the lamina densa and adjacent Sertoli cells before their transition from basal compartment to adluminal compartment.

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation.

Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.

The kallikrein-kinin system in the rat hypothalamus. Immunohistochemical localization of high molecular weight kininogen and T kininogen in different neuronal systems.

High molecular weight kininogen (HKg) and T kininogen (TKg) were detected and localized by immunocytochemistry in adult rat hypothalamus. In addition, kininogens were measured by their direct radioimmunoassay (RIA) or by indirect estimation of kinins released after trypsin hydrolysis and high pressure liquid chromatography (HPLC) separation of bradykinin (BK) and T kinin. A specific HKg immunoreactivity demonstrated with antibodies directed against the light chain (LC) of HKg was colocated with SRIF in neurons of hypothalamic periventricular area (PVA) projecting to external zone (ZE) of median eminence (ME). Heavy chain (HC) immunoreactivity which could be related to HKg or to low molecular weight kininogen (LKg) was detected in some other systems: i) parvocellular neurons of suprachiasmatic (SCN) and arcuate nuclei containing SRIF, ii) magnocellular neurons (mostly oxytocinergic) of paraventricular (PVN) and supraoptic (SON) nuclei, iii) neurons of dorsomedian and lateral hypothalamic areas. TKg immunostaining was restricted to magnocellular neurons of PVN, SON, accessory nuclei (mostly vasopressinergic) and to parvocellular neurons of SCN (vasopressinergic). TKg projections are directed towards the internal zone (ZI) of ME, but very few immunoreactive terminals are detectable in neurohypophysis. TKg staining parallels with vasopressin during water deprivation, and is undetectable in homozygous Brattleboro rats. In some magnocellular neurons, TKg and HC (related to HKg or LKg) are coexpressed. TKg, was also detected in hypothalamus and cerebellum extracts by direct RIA, and BK and T kinin were identified after trypsin hydrolysis. HKg and LKg can act as precursor of BK which can play a physiological role as releasing factor, neuromodulator--neurotransmitter,--or modulator of local microcirculation in hypothalamus. The three kininogens are also potent thiolprotease inhibitors which could modulate both the maturation processes of peptidic hormones and their inactivation and catabolism.

Immunolocalization of type IV collagen and laminin during rat gonadal morphogenesis and postnatal development of the testis and epididymis.

The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.

Anionic sites of the basement membrane of rat seminiferous tubules during ontogenesis.

The anionic sites of the basement membrane of rat seminiferous tubules were demonstrated ultrastructurally in the lamina densa by using cationic polyethyleneimine (PEI). The sites were largely digested out after incubation with heparitinase, indicating a large proportion of heparan sulfates. The anionic sites were present as early as day 16 of gestation on the interstitial side of the lamina densa, and after gestation day 20 they were symmetrically organized on both sides of the lamina densa. The number of sites is not modified postnatally. They appear more irregular in density with advancing age. Experimental conditions as cryptorchidism, fetal irradiation, and ligation of the ductuli efferents lead to unspecific alterations in the distribution of the anionic sites that are parallel to the modifications in the basement membrane.

Anionic sites in the basement membrane of the rat epididymal epithelium during ontogenesis and after testicular and gonadal tract lesions.

Anionic binding sites in the lamina densa of the basement membrane of the rat epididymal epithelium were demonstrated ultrastructurally with the use of cationized polyethyleneimine (PEI). Enzyme digestion with heparitinase removed the anionic sites, indicating that they consist largely of heparan sulfates. The anionic sites are present as early as the 16th day of gestation on the interstitial face of the lamina densa; later during gestation they are localized on both faces of the lamina densa without further modification after birth. The distribution of the anionic sites was identical all along the epididymal duct. After castration and ligation of efferent ducts or in the state of cryptorchidism the sites were more numerous and located inside the thicker portion of the lamina densa. These alterations were more prominent in the initial segment compared to the distal segments, suggesting a differential androgen dependence of the reactive sites and their patterns of distribution.

Influence of testicular secretions on differentiation in the rat epididymis: ultrastructural studies after castration, efferent duct ligation and cryptorchidism.

The differentiation of the rat epididymis was studied in prepubertal castrated, ligated or cryptorchid rats, in order to assess the influences of blood-borne and luminal androgens. The principal cells showed partial differentiation: decrease in cell height, decreased numbers of cytoplasmic organelles implicated in the elaboration phenomena (Golgi apparatus, smooth endoplasmic reticulum), whereas the organelles implicated in the absorptive function remained relatively intact. The lamina densa of the basement membrane underlying the epithelium was irregular, thicker than normal and followed the irregular outline of the basal parts of the epithelial cells. These changes were evident in castrated rats, to a lesser degree in ligated and cryptorchid rats, and were more prominent in the initial part of the duct. On the other hand, the narrow cells and the clear cells followed a normal differentiation pattern in the experimental rats, suggesting that a differential androgen dependence exists among the various type of epididymal cells.

Ultrastructural study of epithelial cells and basement membrane. Differentiation of the rat epididymis after prenatal irradiation.

The effects of prenatal irradiation on the testis are well documented, but less is known about its effects on epididymal differentiation. Pregnant rats were irradiated on the 18th day of gestation. The increase in microfilaments and lipid inclusions in the epithelial cells, in favor of a direct radiation effect, is maximal at birth and disappears thereafter. Narrow cells and clear cells show a normal differentiation pattern. On the other hand, the principal cell maturation is largely altered. The synthesis capacities are decreased based on a reduction in the size of the Golgi apparatus and the smooth endoplasmic reticulum. The aspects of invaginations of the apical plasmalemma, coated vesicles and multivesicular bodies are not modified, suggesting normal absorption functions. The epithelial basement membranes become irregular and thicker than normal, enfolding the basal part of the epithelial cells. The basement membrane proteoglycans, demonstrated by the cationic marker polyethyleneimine, are irregularly distributed in contrast to the normal pattern. These modifications of the principal cells and the basement membrane are more prominent in the proximal epididymis. This suggests a differential maturation dependence of the epithelial cells on the luminal factors, normally secreted by the testis, and likely disturbed by prenatal irradiation which leads to germ cell degeneration, and then to a new balance in the seminiferous epithelium.

[Ultrastructural analysis of anionic sites of the basal membrane of the seminiferous tubules of subjects with severe oligospermia]. / Analyse ultrastructurale des sites anioniques de la lame basale du tube séminifère de sujets présentant une oligospermie sévère.

Several morphological alterations of the basement membrane of the seminiferous tubules have been reported in the patients presenting with a testicular sterility. The proteoglycans are one of the major components of the basal membrane and may be ultrastructurally defined by the use of the cationic marker polyethyleneimine (PEI). In comparaison to the normal distribution of the anionic sites labelled by PEI which is characterized by a regular pattern of distribution along both the epithelial and the interstitial faces of the lamina densa, the pattern of distribution of the sites is largely altered in 6 patients, 27 to 40 years of age, with oligo- or azoospermia. Several types of alterations are reported: parallel thickening of the lamina densa with an abnormal distribution of the anionic sites inside the lamina densa, stratifications of the basal membrane depressing the seminiferous epithelium with anionic sites labelled on both faces of the lamina densa-like layers, massive expansions of the lamina densa with either a regularly circumferential labelling of the sites or a random type of distribution. The digestion of the sites by the enzyme heparitinase is highly suggestive of the presence of proteo-heparan-sulfate. The analysis of the distribution of the anionic sites may represent a usefull tool for studying the pathogenesis of testicular sterility.

Influence of testicular secretions on epididymis oxidative metabolism in the rat.

The oxidative metabolism of the epididymis has been investigated in 40-day-old rats under normal and experimental conditions. No difference in the oxidative metabolism was observed between the initial, middle and terminal segments of the epididymal duct of normal rats. After bilateral or unilateral castration or efferent duct ligation, the oxidative metabolism was found to be exclusively dependent upon plasmatic androgens in the terminal segment in contrast to the proximal segment oxidative metabolism which is dependent of both normal luminal secretions and normal plasmatic androgens.

Differentiation of the rat epididymis after withdrawal of androgen.

The differentiation capacity of the rat epididymis after depletion of androgen was studied in organ culture and in castrated rats. The differentiation of "narrow cells' in 5- and 10-day-old explants and in 10-day-old castrated rats suggests that: (i) the testicular androgens are not essential for their differentiation, (ii) a differential androgen dependence exists among the epididymal cell types, (iii) the undifferentiated epithelial cells are the precursors of the narrow cells.

In vitro development of seminiferous tubules from immature rat testis: ultrastructural and morphometric studies.

Seminiferous tubules from 5 day-old rats maintained in a semi-solid culture system were examined for 4, 8 or 12 days. Morphometric and ultrastructural studies show a significant increase in the seminiferous tubule diameter and normal Sertoli cell differentiation. All the germinal cells degenerate except the spermatogonia.

[Postnatal development of oxidative metabolism of the epididymis in rats]. / Evolution postnatale du métabolisme oxydatif de l'épididyme de rat.

The oxidative metabolism of the epididymis decreases significantly from birth to 12 days and increases gradually after 20 days. Its evolution is contemporary with the development of the interstitial gland and follows the variations of plasmatic and tissular testosterone levels.