Agustín Aoki

Ultrastructural changes in the hypothalamic ventromedial nucleus of ovariectomized rats after estrogen treatment.

The changes produced in the hypothalamic ventromedial nucleus (VMN) of ovariectomized rats after administration of 100 microgram estradiol benzoate/kg body weight were studied using light and electron microscopy. Quantitative morphometric studies included number and size of VMN neurons and nuclei, size and density of terminals and synaptic contacts, spine-to-shaft ratio of postsynaptic elements and relative frequency of two types of synaptic vesicles. Evidence was obtained favoring the concept of heterogeneous composition of the VMN: in ovariectomized animals many cells appeared in a state of quiescence, but other neurons showed no major alterations. Estrogen administration to ovariectomized rats produced evidence of metabolic stimulation such as increase in rough surfaced endoplasmic reticulum, condensation of nucleolar material, enlarged Golgi and presence of pleomorphic mitochondria. The number of neurons in the VMN was not modified by estrogen treatment; however, neuron soma and nuclei were larger. In the ventrolateral division of the VMN terminals and synaptic contacts per unit area were increased after estrogen treatment, but synaptic contact length, terminal size and spine-to-shaft ratio were not modified. The possibility that the differences observed may be consequent to changes in synaptic organization of the VMN related to its estrogen-dependent functions is discussed.

Regulated Expression and Ultrastructural Localization of Galectin-1, a Proapoptotic β-Galactoside-Binding Lectin, During Spermatogenesis in Rat Testis

Abstract Galectin-1, a highly conserved β-galactoside-binding protein, induces apoptosis of activated T cells and suppresses the development of autoimmunity and chronic inflammation. To gain insight regarding the potential role of galectin-1 as a novel mechanism of immune privilege, we investigated expression and ultrastructural localization of galectin-1 in rat testis. Galectin-1 expression was assessed by Western blot analysis and immunocytochemical localization in testes obtained from rats aged from 9 to 60 days. Expression of this carbohydrate-binding protein was developmentally regulated, and its immunolabeling exhibited a stage-specific pattern throughout the spermatogenic process. Immunogold staining using the anti-galectin-1 antibody revealed the typical Sertoli cell profile in the seminiferous epithelium, mainly at stages X–II. During spermiation (stages VI–VIII), a strong labeling was observed at the luminal pole of seminiferous epithelium, localized on apical stalks of Sertoli cells, on heads of mature spermatids, and on bodies of residual cytoplasm. Moreover, spermatozoa released into the lumen showed a strong immunostaining. Following spermiation (stage VIII), galectin-1 expression was restored at the basal portion of Sertoli cells and progressively spread out through the whole cells as differentiation of germinal cells proceeded. Immunoelectron microscopy confirmed distribution of galectin-1 in nuclei and cytoplasmic projections of Sertoli cells and on heads and tails of late spermatids and residual bodies. Surface localization of galectin-1 was evidenced in spermatozoa from caput epididymis. Thus, the regulated expression of galectin-1 during the spermatogenic cycle suggests a novel role for this immunosuppressive lectin in reproductive biology.

Hormonal control of leydig cell differentiation

SummaryThe fine structure of the testicular interstitial cells of the 9-day-old mouse submitted to stimulation with human chorionic gonadotropin (HCG) is reported. As was previously described (Baillie 1964) the interstitial tissue of the prepubertal mouse testis is characterized by the presence of well differentiated epithelioid cells at a quiescent stage. They are characterized by large cytoplasmic depots of lipid droplets and glycogen particles in contrast to poorly developed membranous organelles. These cells are highly sensitive to the action of gonadotropins. Five daily injections of HCG cause their differentiation into cells with active secretory characteristics. The gonadotropin induces a marked depletion of the lipid droplets and glycogen content of the cytoplasm, concurrent with an unusual development of the membranes of the agranular endoplasmic reticulum. Golgi complexes and rough reticulum are prominent. Several changes also appear in the nucleus, especially in the nucleolus.The correlation of the present electron microscopic study of the interstitial cells under HCG stimulation with previous biochemical and physiological findings tentatively suggests that the immature Leydig cells exhibit the basic organization necessary for biosynthesis of steroid hormones.

Stereological analysis of the hypothalamic ventromedial nucleus. II, Hormone-induced changes in the synaptogenic pattern

Stereological electron microscopic analysis of the rat hypothalamic ventromedial nucleus (VMN) throughout postnatal development revealed that synaptogenesis takes place up to day 45. Our results disclosed a sexual dimorphism in the synaptic organization of the neuropil of the ventrolateral (VL) subdivision of the VMN. The numerical densities of spine and shaft synapses in the adult male were higher than in the female. A dimorphic pattern in the numerical density of spine synapses occurred as early as day 5, and was present throughout postnatal life, even though in the adult rats both spine and shaft synapses were sexually dimorphic. Neonatal treatment of female rats with testosterone increased the numerical density of axodendritic synapses, inducing a pattern similar to the adult male. On the other hand, administration of tamoxifen to newborn male rats significantly reduced the numerical density of spine synapses to levels comparable to normal female rats.

Toll-Like Receptor 4 in Rat Prostate: Modulation by Testosterone and Acute Bacterial Infection in Epithelial and Stromal Cells

Abstract The prostate gland is the most inflammation-prone organ in the male reproductive tract. However, little information is available regarding the immunobiology of this gland. Toll-like receptor 4 (TLR4) is considered to be a major sensor of danger signals and a key trigger of the innate immune responses. TLRs have also been implicated in the development of different inflammatory diseases in organs in which epithelial-stromal interactions are critical for homeostasis. The purpose of this work was to evaluate the presence and regulation of TLR4 in the rat prostate. Western blot and immunocytochemical studies revealed that constitutive expression of TLR4 in the rat ventral prostate was localized in the epithelial cells, mainly associated with the rough endoplasmic reticulum, as well as in smooth muscle cells in the stroma. In addition, increased concentrations of TLR4 were found in castrated rats, predominantly in hypertrophied smooth muscle cells. On the other hand, using a bacterial prostatitis model, we observed an increment in the TLR4 cytoplasmic content and migration of this receptor to the apical plasmatic membranes of epithelial cells at 24 h and 48 h post-infection. These findings suggest that the prostate gland is able to recognize pathogens and to initiate immune responses. In addition, TLR4 appears to be implicated in the vital stromal-epithelial interactions that maintain prostate homeostasis during prostatitis, as well as following androgen deprivation.

Different behavior of lactotroph cell subpopulations in response to angiotensin II and thyrotrophin-releasing hormone.

Abstract1. In the present investigation we have extended the study of lactotroph subpopulations in primary pituitary cell cultures. Male rats with or without previous estrogenization followed by A-II or TRH treatments were selected as experimental models.2. The TRH increased up to 50% the PRL released in both whole and ORQX + EB rats (P < 0.05). In contrast, A-II treatment introduced no changes in PRL secretion from cell cultures derived from whole male rats but attained a significant augmentation (about 75%) of PRL secreted by ORQX + EB pituitary cells.3. The addition of TRH and A-II to cultures of ORQX + EB-derived lactotrophs induced cytological changes compatible with a high secretory activity. In estrogen-treated rats the prevailing lactotroph subpopulation is type I. In cell cultures from control and A-II treated whole male pituitaries, the majority of lactotrophs consists of atypical subpopulations of II and III cells, with smaller secretory granules (between 150 and 300 nm in diameter).4. Morphometry of immunostained lactotrophs performed on light microscopic preparations revealed that about 30–36% of the total cell count were lactotrophs. This percentage was fixed and did not change significantly after TRH and A-II treatments.5. The present results confirm the presence of morphological and functional subtypes of lactotroph cells in rat pituitary. Typical PRL cell population shows the highest responsiveness to angiotensin II and TRH action. This functional heterogeneity of lactotroph subtypes may reflect an important and scarcely explored factor in the regulatory process of prolactin secretion.

Postnatal development of the hypothalamic ventromedial nucleus: neurons and synapses

Summary1.In this report the postnatal differentiation of the hypothalamic ventromedial nucleus (VMN) was studied. The main maturational changes detected at the fine structural level occurred between 10 and 20 days of postnatal life.2.In 5-day-old rats the majority of neurons was undifferentiated, with rudimentary cytoplasmic organelles. Dendritic profiles presented an empty appearance due to an electron-lucent matrix and scarce content of organelles.3.At 10 days there was a significant proliferation of cytoplasmic organelles in the perikaryon, mainly of those involved in protein biosynthesis as the rough endoplasmic reticulum (RER) and the Golgi complex.4.After 20 days of age the VMN neurons acquired the cytological appearance of adult neurons, with well-organized RER, Golgi complexes, and pleomorphic mitochondria. Concurrent with these changes, there was a marked development of other organelles in the neuropil, which was accompanied by an increase in synaptic density and differentiation of their subsynaptic structures.

Heterogeneity of pituitary lactotrophs: immunocytochemical identification of functional subtypes

The existence of functional lactotroph subpopulations was confirmed in primary pituitary cell cultures of female rats submitted to estrogen treatment and stimulation with thyrotrophin releasing hormone (TRH) and angiotensin II (A-II). In cell cultures of pituitary tissue, prolactin (PRL) producing cells represent about 50% of the total cell count, most of which (90%) correspond to a typical lactotroph subpopulation characterized by large secretory granules, 500-900 nm in diameter, and well developed rough endoplasmic reticulum (RER) and Golgi complex. Few atypical lactotrophs were detected with a quiescent appearance and containing smaller secretory granules, often indistinguishable from granular content of other pituitary cells. Depletion of endogenous estrogen caused by ovariectomy (OVX) decreased the pituitary lactotroph population about 34%, with a relative increase of atypical forms (56%). Replacement therapy with benzoate estradiol (EB) to OVX rats did not reverse the proportion of typical and atypical lactotrophs gauged in control pituitary glands. The predominant lactotroph population of OVX rat was an atypical PRL producing cell which displayed a quiescent appearance compatible with a reduced secretory activity. By contrast, estrogen administration to OVX rats caused a striking development of the RER, a hypertrophy of the Golgi complex and an increased storage of mature and immature secretory granules in the majority of lactotrophs. These features are compatible with a reactivated protein synthesis. Estrogen also enhanced significantly (p < 0.05) the responsiveness of lactotrophs to A-II and the PRL secretion in both intact and OVX + EB treated rats increased by 40% and 30% respectively. By contrast, A-II did not produce any statistically significant response of lactotrophs from OVX female rats. At variance to this observation, in all models tested TRH increased significantly the PRL secretion (p < 0.05). The correlation of PRL secretion and morphology of different lactotroph subtypes authenticates the existence of a lactotroph subpopulation unresponsive to A-II in pituitary cell cultures from rats depleted of estrogen.

The mammosomatotroph: a transitional cell between growth hormone and prolactin producing cells? An immunocytochemical study

In this report the occurrence of mammosomatotroph (MS) cells was correlated with changes in the somatotroph population of adult rat pituitary gland submitted to various experimental conditions (ovariectomized, orchidectomized and intact males, and after treatment with oestradiol benzoate). Cell and volume density of somatotrophs were assessed in sections stained with the immunogold-silver enhancement technique. Mammosomatotrophs were identified by double immunogold labelling at the electron microscopic level. Colocalization of prolactin (PRL) and growth hormone (GH) in the same cell was rarely observed. Only a few MS cells (0.1–0.2% of all parenchymal cells) were found in some experimental models. Oestrogen treatment decreased both cell and volume density of somatotrophs in ovariectomized rats. In this model, serum GH increased significantly but no changes in the pituitary content of the hormone were observed. Our results demonstrate that MS cells are an uncommon cell type in the pituitary of adult ovariectomized, orchidectomized and intact male rats. The oestrogen treatment, which is well known to induce proliferation of lactotrophs, has no effects on the MS population. Data presented in this report do not support the suggested role for mammosomatotrophs as transitional cells in the presumptive interconversion of PRL and GH producing cells.

Hormone-induced differentiation of agranular endoplasmic reticulum in the interstitial cells of the mouse testis

SummaryThe administration of chorionic gonadotrophin to prepuberal mice results in precocious maturation of the testicular interstitial cells. The cytoplasm of the nine-day-old cells is characterized by abundant lipid droplets, large numbers of glycogen particles and mitochondria. By contrast, the membranous organelles are poorly developed.Human chorionic gonadotrophin brings about mobilization of lipid droplets and glycogen particles, and differentiation of large areas of agranular endoplasmic reticulum.The present observations are in agreement with the reports that human chorionic gonadotrophin increases the secretion of testosterone and that the agranular endoplasmic reticulum is the site of storage of steroid and of the enzymes involved in the biosynthesis of androgens.