Variations in the adenohypophysis of the expression of proliferating cellular nuclear antigen, oestrogen and androgen receptors in relation to gonadal steroids during pregnancy of viscachas (Lagostomus maximus maximus).

Viscachas are native rodents of South America that present a long pregnancy of ~154 days. In this work, we analysed variations in the expression of proliferating cellular nuclear antigen, oestrogen and androgen receptors (ERα and AR) in pituitary pars distalis (PD) and pars tuberalis (PT) in relation to oestradiol and testosterone serum levels in non-pregnant and pregnant viscachas. In PD, cell proliferation increased with pregnancy and lactotrophs proliferated during mid-pregnancy (MP). ERα nuclear-immunoreactive cells (ERαn-ir) were maximal in late pregnancy and AR expression did not vary during pregnancy. In PT, cell proliferation and AR expression increased during pregnancy, but ERα expression was very scarce. The immunostaining pattern of receptors was different in PD and PT. The peak of serum oestradiol and testosterone occurred during MP. Our results suggest that cell proliferation and gonadal receptors might be differentially regulated in the pituitary by oestradiol and testosterone during viscacha pregnancy.

Interstitial Cells in the Pineal Gland of Pregnant and Nonpregnant Viscachas (Lagostomus maximus maximus): A Morphometric and Biochemical Study.

The pineal gland of mammals undergoes morphological and biochemical changes throughout the gestation period. In viscachas, a seasonal breeding rodent, pregnancy lasts approximately 154 days and 3 stages can be defined, i.e., early, mid, and late pregnancy. The purpose of this study is to analyze morphometric variations in the expression of S-100 protein, glial fibrillary acidic protein (GFAP), and vimentin in the interstitial cells (IC) in pregnant and nonpregnant viscachas by immunohistochemistry (IHC). We also aim to evaluate a probable relation between glandular activity and pregnancy. The immunopositive percentage area (%IA) for the studied proteins and the number of immunoreactive cells against the S-100 protein with a visible nucleus (nº IC-S-100) were analyzed. Estradiol and progesterone serum levels were also determined by RIA. Variations in the expression of the S-100 protein and GFAP, as well as changes in the nº IC-S-100 related to serum hormone levels, were found between pregnant and nonpregnant viscachas. Viscachas in mid pregnancy exhibited the highest values of %IA for the analyzed proteins, followed by females in late and early pregnancy, while the nonpregnant ones showed the lowest values for all of the groups studied. Likewise, the nº IC-S-100 also varied following the same pattern. Thus, these variations seem to indicate a direct relationship between glandular activity and gonadal hormone levels. On these grounds, we may conclude that IC undergo changes in relation to ovarian hormone levels and participate in the regulation of glandular activity during pregnancy. However, further research is necessary to elucidate this relationship.

Role of macrophage secretions on rat polycystic ovary: its effect on apoptosis.

Polycystic ovarian syndrome is the most common endocrine disorder among women of reproductive age. Little is known about its etiology, although the evidence suggests an intrinsic ovarian abnormality in which endocrine, metabolic, neural and immune factors would be involved. In this work, the effects of macrophage (MO) secretion on ovarian apoptosis in a polycystic ovary syndrome rat model (PCO rat) induced by estradiol valerate are studied. Spleen MO secretions were used to stimulate ovaries and ovarian interstitial and granulosa cells from both PCO and control rats. Ovarian hormones and prostaglandin E2 (PGE2) were measured by RIA; ovarian mRNA levels of Bax, Bcl2 and NFkB by RT-PCR; and ovarian inducible nitric oxide synthase (iNOS) by western blot. The number of apoptotic cells was evaluated by TUNEL. In the PCO ovary, the MO secretions from PCO rats increased the Bax and NFkB mRNA expressions and increased TUNEL staining in both granulosa and theca cells. In addition, the PCO MO secretions produced a decrease of nitric oxide release, iNOS protein level and PGE2 content in the PCO ovary, and it also induced an increase of androstenedione production by PCO interstitial cells, in comparison with control MO secretions. Considering these results and knowing that testosterone stimulates tumour necrosis factor-α production by PCO MO modifying ovarian response by increasing androstenedione, it is reasonable to suggest that the increase of androgens stimulated in ovarian cells by PCO MO secretions could in turn stimulate the cytokine production from MO, thus maintaining an apoptotic vicious cycle in the PCO ovary.

Changes in lysosomal enzymes and mannose-6-phosphate receptors related to sexual maturation in bull epididymis.

One of the most striking features of the mammalian epididymis is the secretion of lysosomal enzymes (LE). These LE may play a role in sperm maturation. In the present study we investigated the activity and distribution of four LE (?-galactosidase (?-Gal), N-acetyl-?-D-glucosaminidase (?-NAG), ?-mannosidase (?-Man) and ?-glucuronidase (?-Glu)) in bull epididymis at two different ages (6 months and 4 years) to determine whether these enzymes vary with sexual maturity. In young, sexually immature (SI) bulls we found high LE activity in the epididymal tissue that accounts for a developed and active lysosomal apparatus. In contrast, low LE activity was measured in sexually mature (SM) bulls, and ?-NAG and ?-Gal were mostly secreted into the lumen. We also attempted to correlate LE distribution with the expression and functionality of mannose-6-phosphate receptors (MPRs), which are thought to be involved in proper delivery of LE to lysosomes. The cation-dependent MPR was highly expressed in SI bulls, with expression decreasing during adulthood, whereas the expression of the cation-independent MPR was higher in SM than SI bulls. In addition, the four enzymes recovered from the epididymal lumen interact with both MPRs at each age. We conclude that the activity and distribution of LE in bull epididymis varies with sexual maturity and that the distribution is regulated differently by the two types of MPR. These findings could provide some molecular basis for male infertility.

Histological changes in the retina provoked by lithium treatment in a nocturnal rodent (Lagostomus maximus maximus).

Daily morphological variations have been previously described in the viscacha (Lagostomus maximus maximus) retina. The aim of this work was to determine the effects of lithium administration on the histology of retinas from this nocturnal rodent since lithium is a drug that has been shown to affect different parameters of circadian rhythms. Adult male viscachas were divided into 2 groups, injected daily with lithium chloride or vehicle for 35 days, and sacrificed at 08:00, 16:00, and 24:00 h for light and electron microscopy studies. The following morphometric parameters were analyzed: the thickness of the photoreceptor layer, the rod outer and inner segments, and the outer nuclear layer. The control group displayed a true daily cycle of photoreceptor renewal similar to that previously reported by us for (untreated) viscachas in their normal habitat. In all lithium-treated groups, we did not observe histological changes in the thickness measurement of the retinal layers. In these groups, the retinas presented ultrastructural characteristics similar to those observed in control animals sacrificed at 24:00 h. In conclusion, chronic lithium administration abolished the daily histological rhythm in the viscacha retina, probably via inhibition of the phagocytosis process in pigment epithelial cells.

Effects of castration on androgen receptors and gonadotropins in the pituitary of adult male viscachas.

The aims of the present study were to determine whether castration results in quantitative immunohistochemical changes in androgen receptors (AR), LH-immunoreactive (IR) cells and FSH-IR cells, and to analyse the colocalisation of AR and gonadotropins in the pituitary pars distalis (PD) of viscachas. Pituitaries were processed for light and electron microscopy. AR-IR, LH-IR and FSH-IR cells were detected by immunohistochemistry. In morphometric studies, the percentage of AR-IR, LH-IR, FSH-IR, LH-IR/AR-IR and FSH-IR/AR-IR cells was determined. In intact viscachas, AR were distributed throughout the PD; they were numerous at the caudal end, with intense immunostaining. LH-IR cells and FSH-IR cells were found mainly in the ventral region and at the rostral end of the PD. Approximately 45%-66% of LH-IR cells and 49%-57% of FSH-IR cells expressed AR in the different zones of the PD. In castrated viscachas, there was a significant decrease in the percentage of AR-IR, LH-IR, FSH-IR, and FSH-IR/AR-IR cells. Some pituitary cells from castrated viscachas also exhibited ultrastructural changes. These results provide morphological evidence that gonadal androgens are directly related to the immunolabelling of AR, LH and FSH. Moreover, the colocalisation of AR and FSH is most affected by castration, suggesting the existence of a subpopulation of gonadotrophs with different regulatory mechanisms for hormonal synthesis, storage and secretion.

Effect of estrogens on apoptosis in the pituitary of viscachas (Lagostomus maximus maximus).

Estrogens, acting through their receptors (ERα and ERß), regulate cell turnover in the pituitary gland, influencing cell proliferation and apoptosis across various species. However, their role in pituitary processes in seasonally reproducing animals remains poorly understood. This study aims to investigate the influence of estrogens, through the expression of their specific receptors, on the apoptosis of PD cells in relation to sexual maturity, the reproductive cycle, and pregnancy in a seasonal reproductive rodent (Lagostomus maximus maximus). ERα and caspase-3-cleaved (CASP3c) immunoreactive (-ir) cells were identified through immunohistochemistry. Apoptotic cells were detected using the TUNEL technique, with quantitative analysis facilitated by image analysis software, alongside measurement of serum estradiol levels using radioimmunoassay The immunostaining pattern for ERα included nuclear (ERαn) and cytoplasmic (ERαc) staining. In male viscachas, ERα expression significantly increases from immature to adult animals, correlating with the rise in serum estradiol levels and a decrease in the percentage of apoptotic cells. During the gonadal regression period in adult males, a decrease in the number of ER-ir cells and serum levels of estradiol corresponds with an increase in the number of apoptotic cells. In females, serum levels of estradiol peaked during mid-pregnancy, coinciding with a significant decrease in the number of apoptotic cells in the PD. Simultaneously, the percentage of ERαn-ir cells reaches its maximum value during late pregnancy, indicating the need to maintain the protective action of this gonadal hormone throughout the extensive pregnancy in these rodents. Regional ERα receptor expression and apoptotic cells appear to be associated with distinct PD cell populations and their hormonal responses. Finally, elevated estradiol levels coincide with diminished apoptotic cells in the male reproductive cycle and during pregnancy, suggesting an antiapoptotic role of estradiol in this species.

Protective effect of soybeans as protein source in the diet against cadmium-aorta redox and morphological alteration.

We investigated the effects of cadmium exposition on thoracic aorta redox status and morphology, and the putative protective effect of soybeans in the diet. Male Wistar rats were separated into 6 groups: 3 fed with a diet containing casein and 3 containing soybeans, as protein source. Within each protein group, one was given tap water (control) and the other two tap water containing 15 and 100 ppm of Cd(2+), respectively, for two months. In rats fed with casein diet, 15 ppm of Cd induced an increase of thiobarbituric acid-reactive substances (TBARS), and of the catalase (CAT) and glutathione peroxidase (GPx) activities, which were even higher with 100 ppm of Cd(2+), in aorta. Also, 100 ppm Cd(2+) exposure increased superoxide dismutase (CuZnSOD) activity; CAT, GPX, SOD, Nrf2 and metallothioneine II mRNA expressions and CAT, GPx and NOX-2 protein levels, compared with control. Aorta endothelial and cytoplasmic alterations were observed. However, with the soybeans diet, 15 and 100 ppm of Cd(2+) did not modify TBARS levels; CAT, GPX and Nrf2 mRNA expressions; CAT, GPx and NOX-2 protein; and the aorta morphology, compared with control. The soybean diet attenuates the redox changes and protects against morphological alterations induced, in a dose-dependent way, by Cd in aorta.

Superior mesenteric ganglion via ovarian plexus nerve involved in the cross-talk between noradrenaline and GnRH in rat ovaries.

There is evidence of the existence of an intraovarian gonadotropin-releasing hormone (GnRH) system. There are also reports about the influence of extrinsic ovarian innervation in gonadal function. Therefore, it is interesting to study the relationship between ovarian sympathetic innervation and GnRH to shed light on possible physiological and pathophysiological implications. This work aimed to investigate whether noradrenergic stimulation of the superior mesenteric ganglion (SMG) can modify the levels of ovarian GnRH and cause functional and morphological changes in the gonad through the ovarian plexus nerve (OPN), during estrus and diestrus II in rats. The SMG-OPN-Ovary system and an ovary without extrinsic innervation were removed from Holtzman rats in estrus and diestrus II stages and placed in specially designed cuvettes containing Krebs-Ringer buffer. In the experimental groups, SMGs and denervated ovaries were stimulated with 10-6 M noradrenaline (NA). GnRH and progesterone levels (in the ovarian incubation medium) and the mRNA expression of 3beta-hydroxysteroid dehydrogenase (Hsd3b3), 20alpha-hydroxysteroid dehydrogenase (Akr1c18), Bax, and Bcl2 were analyzed. Histological studies of the ovaries were performed. In estrus, NA decreased GnRH levels in both experimental schemes. Furthermore, progesterone levels increased while the Akr1c18 expression and Bax/Bcl2 ratio decreased, without causing changes in ovarian morphology. In diestrus, the noradrenergic stimulation of the ganglion increased GnRH levels, decreased progesterone levels, and increased Akr1c18 expression and Bax/Bcl2 ratio. Follicles with histoarchitecture alterations and corpus luteum with signs of cell death were observed. In denervated ovaries, NA increased the levels of GnRH and progesterone. Furthermore, NA decreased the Bax/Bcl2 ratio and histological studies revealed signs compatible with a possible atretogenic effect. In conclusion, noradrenergic stimulation of the SMG-OPN pathway regulates ovarian cyclicity. The SMG modulates the cross-talk between NA and ovarian GnRH, protecting the ovary from atretogenic effects and luteal apoptosis during estrus while inducing luteal regression in the diestrus II.

Cadmium exposure modifies lactotrophs activity associated to genomic and morphological changes in rat pituitary anterior lobe.

Cadmium (Cd) is widely used in industrial applications and is an important contaminant of agricultural products. As an endocrine disruptor, Cd modifies the hormone release of pituitary anterior lobe (PAL). This work was undertaken to evaluate a possible association between phospholipase D (PLD) and prolactin mRNA expressions and the activity of lactotrophs and folliculostellate cells (FSC) in PAL of Cd exposed adult male Wistar rats (Cd, 0.133 mM per liter for 2 months). The PALs were submitted to immunohistochemical and morphometric analysis to determine the percentage of lactotrophs (PRL-ir) and FSC (S-100-ir). Cultured PAL cells were stained with Hoechst 33258 to determine the presence of alterations in nuclear morphology consistent with apoptosis. The expressions of PLD and prolactin mRNA were assessed by RT-PCR. Cd treated rats showed a decrease of PLD mRNA levels that can be associated to both high number of apoptotic cells and increase of S-100 protein expression in FSC. Cd decreased prolactin mRNA expression, number of lactotrophs and percentage of PRL-ir suggesting a low availability of prolactin to be secreted from PAL. Cd modifies the lactotrophs activity of pituitary gland through biochemical, genomic and morphological changes and contributes directly or indirectly to the levels of serum prolactin.

Influence of melatonin and sexual hormones on the expression of proliferating cell nuclear antigen in the adrenal cortex of a seasonal breeder (Lagostomus maximus).

The viscacha (Lagostomus maximus) is a rodent of nocturnal habits, whose physiology and behavior vary according to modifications of environmental signals. The objective of this study is to assess the influence of melatonin and sexual hormones on the viscacha adrenal cortex proliferative activity through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) along with hormonal determinations. PCNA expression was studied in male viscachas to assess the effect of melatonin administration, castration, and the annual reproductive cycle. In female viscachas, PCNA was studied in nonpregnant and pregnant viscachas. PCNA expression was observed in adrenocortical cells (PCNA-A) and endothelial cells (PCNA-E). Melatonin-administered animals showed a significantly lower number of PCNA-A compared to the control group. No significant difference could be established in the number of PCNA-A and PCNA-E between castrated and control animals. However, the morphometric analysis showed an increase in the size of the cortex of castrated animals, along with other cytological features. Significant differences in serum testosterone levels were observed during the male viscacha reproductive cycle, with the lowest levels encountered during the regression period (winter). Male viscachas exhibited a significantly high number of PCNA-A during late autumn and a high number of PCNA-E during winter. In females, hormonal determinations showed a peak of progesterone and estrogen during mid-pregnancy, along with a notably high number of PCNA-A and an increase in the number of PCNA-E. Our results suggest that proliferation in the adrenal cortex of the viscacha varies in relation to melatonin, sexual hormones, and environmental conditions.

Pituitary pars intermedia of male viscacha (Lagostomus maximus maximus): a morphometric study of seasonal and age-related changes in immunohistochemistry.

The aim of this work was to study the pituitary pars intermedia in seasonally captured adult male and immature viscachas by immunohistochemistry and image analysis. The pituitary pars intermedia exhibited a well-developed parenchyma with scarce connective tissue and vascularization. It was formed by a close association of melanotrophs and folliculostellate cells. The folliculostellate cells were stellate in shape with cytoplasmic processes, and they originated follicles with PAS-positive colloid inside. The morphometric parameters of melanotrophs, follicular colloid and folliculostellate cells (S-100-ir and GFAP-ir) varied seasonally and in relation to age. These parameters showed minimal values in the adult males captured in winter and in immature animals, and they were maximal in summer. The percentage of vimentin-positive area of the folliculostellate cells was maximal in immature animals, decreased in relation to age and did not vary seasonally in the adult animals. The greatest development of pars intermedia in the adult animals in relation to the immature ones is probably related to the adults' adaptation to the semiarid environment. The expression of the tested proteins suggests a probable neuroectodermic origin for the folliculostellate cells of the viscacha pituitary pars intermedia. In addition, the cytoplasmic processes of folliculostellate cells might originate an intercellular communication network inside the pars intermedia. The decrease in the morphometric parameters melanotrophs, follicular colloid and folliculostellate cells in winter suggests a low endocrine activity of this zone. This fact might be due to the effect of the short photoperiod and high melatonin serum levels.

Androgen receptors in coeliac ganglion in late pregnant rat.

The ovarian function is controlled by endocrine factors and neural influence. In late pregnant rat, androstenedione, from the coeliac ganglion, has a luteotrophic effect in the ex vivo coeliac ganglion-superior ovarian nerve-ovary system. In this work we investigate the presence of androgen receptors in the coeliac ganglion of late pregnant rats by immunohistochemistry. We also explore, from a physiological point of view, the potential participation of these receptors in the androstenedione ganglionic action on progesterone release and metabolism, as well as on nitrites release in the ovary compartment. The coeliac ganglion was isolated after being fixed in situ and immunohistochemistry was performed. In the system, three experimental groups were used with the addition of (a) androstenedione, (b) flutamide, and (c) androstenedione plus flutamide in the ganglion compartment. Progesterone and nitrite concentrations were determined in the ovary compartment at different incubation times. Corpora lutea samples isolated at the end of incubation were used to determine the expressions and activities of the progesterone synthesis (3beta-hydroxysteroid-dehydrogenase, 3beta-HSD) and degradation (20alpha-hydroxysteroid-dehydrogenase, 20alpha-HSD) enzymes. Immunohistochemistry revealed cytoplasmatic androgen receptor immunoreactivity in neural somas in the coeliac ganglion. In the coeliac ganglion-superior ovarian nerve-ovary system, androstenedione addition increased 3beta-HSD and decreased 20alpha-HSD, showed a tendency to decrease 20alpha-HSD expression, and increased nitrites release in relation to control. Androstenedione plus flutamide decreased progesterone and nitrites release in relation to the androstenedione group. This work demonstrates the presence of androgen receptors in neurons of celiac ganglion and provides evidence for the luteotrophic action of androstenedione via a neural pathway that may be mediated by these receptors.

Ganglionic and ovarian action of acetylcholine during diestrous II in rats. Neuroendocrine control of the luteal regression.

Our aim was to investigate if acetylcholine (Ach), added to the celiac ganglion-superior ovarian nerve-ovary system (CG-SON-ovary) or in ovary incubations, modifies the release of progesterone (P4), androstenedione (A2), dopamine (DA), norepinephrine (NE), gonadotropin-releasing hormone (GnRH), and alters the expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 20α-hydroxysteroid dehydrogenase (20α-HSD), and apoptotic genes in ovarian tissue during the diestrous II (DII) in rats. The CG-SON-ovary system or the ovary alone were removed and placed into separate cuvettes both containing Krebs-Ringer solution (control groups). In experimental groups, 10-6 M Ach was added into the ganglion compartment or into the ovary compartment. P4, A2 and GnRH were measured by RIA, mRNA expression by RT-PCR, and catecholamines by HPLC. In addition, a routine histological technique was applied. In ex-vivo system, 10-6 M Ach into the ganglion compartment decreased P4 and NE release, altered 3ß-HSD and 20α-HSD expression, and decreased bax/bcl-2 ratio, while increasing the release of A2 and DA, and bcl-2 expression. In ovary incubations, 10-6 M Ach decreased P4 and GnRH release, decreased 3ß-HSD and bcl-2 expression, increased A2 release, increased 20α-HSD and bax expression, and the bax/bcl-2 ratio, and induced disorganization of the corpus luteum structure. The peripheral nervous system protected the ovary from the apoptotic mechanisms while in the ovary incubation the effect was reversed. Our results indicate that Ach in DII regulates steroidogenesis and apoptosis in the ovary, by modulating the concentration of neurotransmitters. In vivo, an alteration in the extrinsic cholinergic innervation of the ovary could disrupt the endocrine control of the reproductive function.

A soybean-based diet modulates cadmium-induced vascular apoptosis.

Cadmium (Cd) exposure has been associated with an increased risk of cardiovascular diseases. The diet is a modifiable source of protecting or damaging factors that may affect this risk. Herein we tested the hypothesis that a soybean-based diet (SBD) protects the vascular wall of the aorta against Cd-induced pro-inflammatory and pro-apoptotic effects. To test this hypothesis, we fed male Wistar rats for 60 days with a casein-based diet (CBD) or an SBD. These animals were also exposed to tap-water without (CBD-Co/SBD-Co) or with 15(CBD-15Cd/SBD-15Cd) or 100 (CBD-100Cd/SBD-100Cd) ppm of Cd. Inflammatory parameters (mRNAs and/or proteins) were measured in thoracic aorta tissue. These included inducible and endothelial nitric oxide synthases, cyclooxygenase-2, intracellular-adhesion molecule-1, and vascular cell-adhesion molecule-1. As pro-apoptotic parameters, we measured Bax and Bcl-2 mRNA/protein, as well as TUNEL positive cells in the aorta tissue. Compared to CBD-Co, inflammatory and apoptosis markers increased in the aorta with the concentration of Cd in the drinking water. These effects were not observed in either SBD-15Cd or SBD-100Cd, which were similar to CBD-Co. Cd content in serum and in aortas from animals fed CBD-Co/SBD-15Cd or CBD-Co/SBD-100Cd were similar suggesting that, if any, the effect of SBD is not due to changes in Cd bioaccumulation, but due to secondary effects linked to the composition of the dietary soybean flour. Our findings are consistent with a protective effect of an SBD against Cd-induced inflammation and apoptosis in the thoracic aorta in a rat model.

Immunomorphometric variations of sustentacular cells of the male viscacha adrenal medulla during the annual reproductive cycle. Effects of androgens and melatonin.

The adrenal medulla is crucial for the survival of species facing significant environmental changes. The parenchyma is composed mainly of chromaffin cells, ganglion cells and sustentacular cells (SC). The male viscacha exhibits seasonal variations of gonadal activity and other metabolic functions. The aim of this work was to investigate the influence of the reproductive conditions on the morphology of SC of this rodent. In addition, the effects of testosterone and melatonin on these cells were studied. Immunoexpression of S100 protein, GFAP and vimentin were analyzed. Furthermore, the distribution of adrenergic and noradrenergic chromaffin cells subpopulations was studied for the first time in this species. SC present long cytoplasmic processes in contact with chromaffin cells, probably generating an intraglandular communication network. Significant differences (p < 0.05) in the %IA (percentage of immunopositive area) for the S100 protein were observed according to winter (4.21 ±â€¯0.34) and summer (3.51 ±â€¯0.15) values. In castrated animals, the %IA (6.05 ±â€¯0.35) was significantly higher in relation to intact animals (3.95 ±â€¯0.40). In melatonin-treated animals the %IA (3.62 ±â€¯0.23) was significantly higher compared to control animals (2.65 ±â€¯0.26). GFAP immunoexpression was negative and no noradrenergic chromaffin cells were detected suggesting an adrenergic phenotype predominance. Vimentin was observed in SC, endothelial cells and connective tissue. Results indicate that SC exhibit variations along the annual reproductive cycle, along with castration and the melatonin administration. Our results suggest that in this rodent SC are not only support elements, but also participate in the modulation of the activity of the adrenal medulla; probably through paracrine effects.

Sympathetic innervation regulates macrophage activity in rats with polycystic ovary.

Polycystic ovarian syndrome (PCOS) is a low-grade inflammatory disease characterized by hyperandrogenism and ovarian hyperinnervation. The aim of this work is to investigate whether in vivo bilateral superior ovarian nerve (SON) section in adult rats with estradiol valerate-induced PCOS (PCO rats) affects macrophage spleen cells (MФ) and modifies the steroidogenic ability of their secretions. Culture media of MФ from PCO rats and PCO rats with SON section (PCO-SON rats) were used to stimulate in vitro intact ovaries. Compared with macrophages PCO, macrophages from PCO-SON rats released less tumor necrosis factor-α and nitric oxide, expressed lower Bax and Nfkb mRNA and showed reduced TUNEL staining. Also, in PCO rats, the SON section decreased kisspeptin and nerve growth factor mRNA expressions, without changes in Trka receptor mRNA levels. Macrophage secretions from PCO-SON rats decreased androstenedione and stimulated progesterone release in PCO ovaries, compared to macrophage secretions from PCO rats. No changes were observed in ovarian estradiol response. These findings emphasize the importance of the SON in spleen MΦ, since its manipulation leads to secondary modifications of immunological and neural mediators, which might influence ovarian steroidogenesis. In PCO ovaries, the reduction of androstenedione and the improvement of progesterone release induced by PCO-SON MΦ secretion, might be beneficial considering the hormonal anomalies characteristic of PCOS. We present functional evidence that modulation of the immune-endocrine function by peripheral sympathetic nervous system might have implications for understanding the pathophysiology of PCOS.

Pigmented Cells in the Pineal Gland of Female Viscacha (Lagostomus maximus maximus): A Histochemical and Ultrastructural Study.

The presence of pigment has been demonstrated in different nervous structures such as those of retina, substantia nigra, and locus coeruleus. These pigments have also been described in the pineal gland of different mammal species. Histochemical and ultrastructural studies of the pineal gland of female viscacha (Lagostomus maximus maximus) were performed to analyze the presence of pigmented cells under natural conditions and to evaluate a probable relation between pigment content and glandular activity during pregnancy. The following techniques were applied: hematoxylin-eosin, phosphotungstic acid-hematoxylin, Masson-Fontana silver, DOPA histochemistry, Schmorl's reaction and toluidine blue. Estradiol and progesterone serum levels were determined by RIA. The ultrastructural features of the pineal pigment granules were also analyzed. Pigment granules were observed in a random distribution, but the pigmented cells were frequently found near blood vessels. The pineal pigment was histochemically identified as melanin. Differences in the amount of pigmented cells were found between pregnant and nonpregnant viscachas. The ultrastructural analysis revealed the presence of premelanosomes and melanosomes. Estradiol and progesterone levels vary during pregnancy. In conclusion, the changes in the amount of pigment content and hormone levels may indicate that the pineal gland of female viscacha is susceptible to endocrine variations during pregnancy.

A Seasonal and Age-Related Study of Interstitial Cells in the Pineal Gland of Male Viscacha (Lagostomus maximus maximus).

The pineal gland of viscacha exhibits histophysiological variations throughout the year, with periods of maximal activity in winter and minimal activity in summer. The aim of this work is to analyze the interstitial cells (IC) in the pineal gland of male viscachas in relation to season and age. The S-100 protein, glio-fibrillary acidic protein (GFAP), and vimentin were detected in adult and immature animals by immunohistochemistry (IHC). Double-IHC was also performed. The S-100 protein was localized within both, IC nucleus and cytoplasm. GFAP was present only in the cytoplasm. Vimentin was expressed in some IC, besides endothelial cells, and perivascular spaces. In the adult males, the morphometric parameters analyzed for the S-100 protein and GFAP exhibited seasonal variations with higher values of immunopositive area percentage in winter and lower values in summer, whereas the immature ones showed the lowest values for all the adult animals studied. Colocalization of S-100 protein and GFAP was observed. The IC exhibited differential expression for the proteins studied, supporting the hypothesis of the neuroectodermal origin. The IC generate an intraglandular communication network, suggesting its participation in the glandular activity regulation processes. The results of double-IHC might indicate the presence of IC in different functional stages, probably related to the needs of the cellular microenvironment. The morphometric variations in the proteins analyzed between immature and adult viscachas probed to be more salient in the latter, suggesting a direct relationship between the expression of the S-100 protein and GFAP, and animal age. Anat Rec, 2017. © 2017 Wiley Periodicals Inc. Anat Rec, 300:1847-1857, 2017. © 2017 Wiley Periodicals, Inc.

The cholinergic influence on the mesenteric ganglion affects the liberation of ovarian steroids and nitric oxide in oestrus day rats: characterization of an ex vivo system.

The axons that constitute the ovarian nervous plexus originate mostly in the principal neurons of the superior mesenteric ganglion (SMG) that is part of the sympathetic ganglionic chain and exhibits cholinergic receptors. In order to observe the effect of acetylcholine, the main neurotransmitter in the ganglionic transmission, the purpose of the present work was: first, to standardize an integrated ex vivo superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) system in oestrus day rats; secondly, to determine if the ganglionic cholinergic stimulus modifies the release of nitric oxide and steroids in the ovary compartment in the absence of humoral factors; and thirdly, to investigate if there are differences in the responses between the left and right ovaries caused by the neural stimulus. The ex vivo experimental left and right systems were developed and standardized. The systems were incubated in Krebs-Ringer phosphate buffer in a Dubnoff metabolic shaker. The progesterone release was determined to standardize the incubation times, obtaining different responses between the left and right systems, which shows that both systems have their own autonomic tone. Non-specific stimulation with KCl in the ganglion compartment provoked different responses in terms of release of progesterone and oestradiol. Progesterone decreased in the left and right systems. However, oestradiol diminished at short times and increased at 60 and 120 min in the left ovary, whereas it increases at 30 and 60 min in the right ovary. These different responses show the sensitivity and viability of both systems. When acetylcholine was used in the ganglion compartment, the release of nitric oxide, progesterone, androstenedione and oestradiol was evaluated. The liberation of nitrite increased at 15, 30 and 60 min in the left system and decreased in the right system at 120 min. Progesterone showed a decrease in its release at 15, 30 and 120 min and androstenedione at 15 min in the left ovary compartment. In the right ovary, only progesterone decreased in relation to the control at 120 min while androstenedione did not show significant changes. Oestradiol showed an increase in the left ovary compartment at all the studied times, while in the right ovary it did not show any changes. These results indicate that the neural stimulus from the superior mesenteric ganglion through the ovarian nervous plexus is one of the factors modulating the secretory activity of the ovarian steroids and nitric oxide. The system is viable and also shows a different sensitivity of the left ovary in relation to the right one at least in this cycle stage, characterized by marked irrigation and profound structural changes in the ovary.