Morphometrical and intracellular changes in rat ovaries following chronic administration of ghrelin

The aim of our investigation was to examine the influence of chronic administration of ghrelin on the rat ovarian state. Morphometrical and intracellular changes in the ovary of 35-d female Wistar rats after sc injection of 1 nmol of ghrelin for 10 consecutive days were studied. Control animals (n = 10) were injected with normal saline using similar method. The ovaries were collected on days 1 and 6 after last injection from each group and subjected to light microscopic morphometric and electron microscopic analysis. It was demonstrated that the number of corpora lutea was significantly lower and the number of ovarian follicles was higher in the treated group on days 1 and 6, than in control (P < 0.01). Moreover, the mean diameter of each follicle, corpora lutea, luteal cell, theca layer, oocyte and zona plucida, but not of granulosa layer, as well as the whole ovarian volume were significantly lower in the treated animals at days 1 and 6 (P < 0.05). Electron microscopic analysis also indicated some intracellular changes associated with apoptosis and cell death such as presence of secondary lysosome, apoptotic bodies, nuclear chromatin condensation as well as margination, nuclear segmentation and vacuolization of cytoplasm of granulosa and theca cells. Our observations provides novel evidences for inhibitory influence of ghrelin on rat ovarian structures and, therefore, for the role of ghrelin as suppressor of female reproductive system.     

Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivo quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 consecutive days or vehicle to the control animals. The rats from each group (n=7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohistochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immunoreactive spermatocytes against Bax increased (P<0.01) in the ghrelin-treated group on day 10, while despite of 30% increment in the Bax level of spermatocytes in the treated rats on day 30, however, it was not statistically significant. During the experimental period, only a few spermatogonia represented Bax expression and the changes of Bax immunolabling cells were negligible upon ghrelin treatment. Likewise, there were immunostaining cells against Bcl-2 in each germ cell neither in the control nor in the treated animals. In fact, ghrelin balanced Bax/Bcl-2 ratio toward at increase of Bax level in the spermatocytes and therefore may stimulate apoptosis in these germ cells. In contrast, ghrelin administration significantly suppressed proliferation-associated peptide PCNA in the spermatocytes as well as spermatogonia (P<0.05). Whereas, caspase-3 activity did not show any marked alteration during the experiment in both groups (P>0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observations also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors.     

Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death

To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H₂O₂-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H₂O₂, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak-1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H₂O₂-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 μM H₂O₂ was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 μM H₂O₂. Meanwhile, H₂O₂ induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulation of Bcl-2 (P < 0.05). When lymphocytes were pretreated with Se before treated with H₂O₂, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P < 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulated Bcl-2 (P < 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress.     

The effect of mechanical stretch stress on the differentiation and apoptosis of human growth plate chondrocytes

The study is aimed to investigate the effect of stretch stress with different intensities on the differentiation and apoptosis of human plate chondrocytes. In the present study, the human epiphyseal plate chondrocytes were isolated and cultured in vitro. Toluidine blue staining and type II collagen immunohistochemical staining were used to identify the chondrocytes. Mechanical stretch stresses with different intensities were applied to intervene cells at 0-, 2000-, and 4000-μ strain for 6 h via a four-point bending system. The expression levels of COL2, COL10, Bax, Bcl-2, and PTHrp were detected by quantitative RT-PCR. Under the intervention of 2000-μ strain, the expression levels of COL2, COL10, and PTHrp increased significantly compared with the control group (P < 0.05), and the expression level of PCNA was also increased, but the difference was not statistically significant (P > 0.05). Under 4000-μ strain, however, the expression levels of PCNA, COL2, and PTHrp decreased significantly compared with the control group (P < 0.05), and the expression level of COL10 decreased slightly (P > 0.05). The ratio of Bcl-2/Bax gradually increased with the increase of stimulus intensity; both of the differences were detected to be statistically significant (P < 0.05). In conclusion, the apoptosis of growth plate chondrocytes is regulated by mechanical stretch stress. Appropriate stretch stress can effectively promote the cells’ proliferation and differentiation, while excessive stretch stress inhibits the cells’ proliferation and differentiation, even promotes their apoptosis. PTHrp may play an important role in this process.     

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.     

Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in ovarian follicular cells of dairy cow at different stages of development

Adiponectin is one of the most important, recently discovered adipocytokines that acts at various levels to control male and female fertility through central effects on the hypothalamus-pituitary axis or through peripheral effects on the ovary, uterus, and embryo. We studied simultaneous changes in the gene expression pattern of adiponectin and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) in granulosa and theca cells, cumulus-oocyte complex, and in corpus luteum in healthy bovine (Bos tarus) follicles at different stages of development. The expression levels of adiponectin, AdipoR1, and AdipoR2 mRNA were lower (P < 0.05) in granulosa and cumulus cells in comparison with that in theca cells and oocyte. In contrast with the oocyte, AdipoR1 in granulosa, theca, and luteal cells was expressed (P < 0.05) more than AdipoR2. Adiponectin expression increased (P < 0.05) in granulosa cells and in cumulus-oocyte complex during follicular development from small to large follicles. Opposite results were observed in theca cells. Expression of adiponectin was highest in the late stages of corpus luteum (CL) regression, whereas lower expression was recorded in active CL (P < 0.05). AdipoR1 and AdipoR2 expression increased during the terminal follicular growth in granulosa and theca cells (P < 0.05) and during the luteal phase progress in CL. There was positive correlation between adiponectin mRNA level in granulosa cells from large follicles and follicular fluid estradiol concentration (r = 0.48, P < 0.05) and negative correlation between adiponectin mRNA abundance in theca cells and follicular fluid progesterone concentration (r = -0.44, P < 0.05). In conclusion, we found that the physiologic status of the ovary has significant effects on the natural expression patterns of adiponectin and its receptors in follicular and luteal cells of bovine ovary.     

Ghrelin ovarian cell expression in patients with polycystic ovary syndrome: an immunohistochemical evaluation.

INTRODUCTION: The influence of ghrelin on different organs has been studied recently, e.g. in the regulation of pituitary hormone release, regulation of energy homeostasis, glucose metabolism and insulin secretion, cell proliferation, and reproductive function. However, the etiology of polycystic ovary syndrome has not been fully explained. The aim of our study was to estimate the presence of ghrelin in polycystic ovaries cells and evaluation of the relationship between ghrelin occurrence and cells proliferation. METHODS: In the present work we have compared ten polycystic ovaries with ovaries without pathology as the control group. We used immunohistochemical method to detect ghrelin. The cells proliferation was evaluated by Ki 67 proliferation index. RESULTS: Ghrelin immunostaining was demonstrated in cytoplasm of ovarian secondary interstitial cells and in atretic corpus luteum. The cell nuclei were ghrelin positive in granulosa, theca layers of follicular cyst in both groups as well as in luteal cells of young corpus luteum in healthy ovaries. Ki 67 immunostaining was observed in granulosa and theca layers of follicular cyst in polycystic and healthy ovaries. CONCLUSIONS: It is possible that local ghrelin expression plays an important role in the direct control of ovarian development and function and ghrelin may participate in patomechanism of PCOS.     

Ghrelin regulates Bax and PCNA but not Bcl-2 expressions following scrotal hyperthermia in the rat

More recently, we have reported the beneficial effects of ghrelin in improvement of histopathological features of the rat testis following local heat exposure. However, the exact mechanism and the precise role of apoptosis- and proliferation-specific proteins in this regeneration process remained to be explored. Thus, thirty adult male Wistar rats were allotted for the experiment and subdivided equally into three groups: control-saline (CS), heat-saline (HS) and heat-ghrelin (HG). The scrota of HS and HG groups were immersed once in water bath at 43 °C for 15 min. HG animals received 2 nmol of ghrelin subcutaneously immediately after heating every other day until day 60 and the other groups were given physiological saline using the same method. The testes of all groups were taken after rat killing on days 30 and 60 after heat treatment for immunocytochemical detection of pro-apoptotic factor Bax, anti-apoptotic protein Bcl-2 and proliferation-associated peptide PCNA in the germ cells. Ghrelin could significantly suppress the Bax expression in spermatocytes compared to the HS group at day 30 (P < 0.05). Likewise, the mean percentages of spermatogonia containing Bax substance were lower in ghrelin-exposed animals, however the differences were not statistically significant. There were immunoreactive cells against Bcl-2 in each germ cell neither in the control nor in the heated animals of experimental groups. In contrast, the number of PCNA immunolabeling cells were higher in HG group in compared to HS or CS animals on both experimental days (P < 0.001). Down-regulation of Bax expression concurrent with overexpression of PCNA in HG group indicates the ability of ghrelin in acceleration of testicular germ cells regeneration following heat stress. These findings indicate that ghrelin may be used as a novel and efficient antioxidant agent to induce resumption of spermatogenesis upon environmental heat exposure.     

Role of microRNA-195 in cardiomyocyte apoptosis induced by myocardial ischaemia–reperfusion injury

This study aims to investigate microRNA-195 (miR-195) expression in myocardial ischaemia–reperfusion (I/R) injury and the roles of miR-195 in cardiomyocyte apoptosis though targeting Bcl-2. A mouse model of I/R injury was established. MiR-195 expression levels were detected by real-time quantitative PCR (qPCR), and the cardiomyocyte apoptosis was detected by TUNEL assay. After cardiomyocytes isolated from neonatal rats and transfected with miR-195 mimic or inhibitor, the hypoxia/reoxygenation (H/R) injury model was established. Cardiomyocyte apoptosis and mitochondrial membrane potential were evaluated using flow cytometry. Bcl-2 and Bax mRNA expressions were detected by RT-PCR. Bcl-2, Bax and cytochrome c (Cyt-c) protein levels were determined by Western blot. Caspase-3 and caspase-9 activities were assessed by luciferase assay. Compared with the sham group, miR-195 expression levels and rate of cardiomyocyte apoptosis increased significantly in I/R group (both P<0.05). Compared to H/R + negative control (NC) group, rate of cardiomyocyte apoptosis increased in H/R + miR-195 mimic group while decreased in H/R + miR-195 inhibitor group (both P<0.05). MiR-195 knockdown alleviated the loss of mitochondrial membrane potential (P<0.05). MiR-195 overexpression decreased Bcl-2 mRNA and protein expression, increased BaxmRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). While, downregulated MiR-195 increased Bcl-2 mRNA and protein expression, decreased Bax mRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). Our study identified that miR-195 expression was upregulated in myocardial I/R injury, and miR-195 overexpression may promote cardiomyocyte apoptosis by targeting Bcl-2 and inducing mitochondrial apoptotic pathway.     

Acylated and des acyl ghrelin in human portal and systemic circulations

Octanoylation of the gastric peptide ghrelin produces an active isoform that regulates appetite and other metabolic functions. Acylated ghrelin is present in the gastrointestinal tract suggesting that octanoylation may occur in these tissues and thereby affect the acylated ghrelin in the systemic circulation. In this study blood samples were collected simultaneously from portal, arterial, peripheral venous and central venous compartments from patients undergoing laparotomy. ELISA and high sensitivity Bioplex was used to measure the concentration of acylated and des acyl ghrelin. We found median (95% confidence interval (CI)) plasma acylated ghrelin (pg/ml) was 35.8 (30.0–59.6) in the portal compartment compared to 51.5 (37.6–74.8; P < 0.05, n = 11) in the arterial, 39.3 (33.3–56.3) in the portal compartment compared to 55.0 (48.5–77.0; P < 0.001, n = 12) in the peripheral venous and 36.0 (33.1–57.4) in the portal compartment compared to 48.9 (43.3–65.6; P < 0.01, n = 15) in the central venous compartment. Median (95% CI) plasma des acyl ghrelin levels (pg/ml) was 173 (125–220) in the portal compartment compared to 136 (99.3–125; P < 0.001, n = 14)in the arterial, 186 (136–233) in the portal compartment compared to 149 (111–190; P < 0.01, n = 15) in the peripheral venous and 171 (140–208) in the portal compartment compared to 152 (119–175; P < 0.01, n = 15) the central venous compartment. We conclude that plasma acylated ghrelin concentration was significantly lower in portal compared with the systemic compartments whilst plasma des acyl ghrelin was significantly higher in portal compared with systemic compartments. These findings suggest that the liver could be involved in the regulation of circulating ghrelin.     

Chicken oviduct—the target tissue for growth hormone action: effect on cell proliferation and apoptosis and on the gene expression of some oviduct-specific proteins

The aim of this study was to examine the in vivo effect of growth hormone (GH) on cell proliferation and apoptosis and on the gene expression of selected proteins in the chicken oviduct before sexual maturity (first oviposition). Ten-week-old Hy-Line Brown chickens were injected three times a week with 200 μg?·?kg^-1 body weight of recombinant chicken GH (cGH) until 16 weeks of age. Control hens received 0.9 % NaCl with 0.05 % bovine serum albumin as a vehicle. Treatment with cGH increased (P?<?0.05) oviduct weight at 16 weeks of age, i.e. 1–2 weeks before onset of egg laying. The highest number of proliferating (determined by proliferating cell nuclear antigen [PCNA] immunocytochemistry) and apoptotic (determined by TUNEL assay) cells in the oviduct was found in the mucosal epithelium, and the lowest in the stroma. Administration of cGH did not increase (P?>?0.05) the number of PCNA-positive cells but it decreased (P?<?0.01) the number of TUNEL-positive cells, thus increasing the proliferating-to-apoptotic cell ratio in the oviduct. Gene expression (determined by real-time polymerase chain reaction) of apoptosis-related caspase-2 in the magnum and caspase-3 in the magnum and isthmus and their activity (determined by fluorometric assay) in the magnum were attenuated (P?<?0.05) in cGH-treated hens. The gene expression of the magnum-specific ovalbumin and the shell-gland-specific ovocalyxins 32 and 36 was increased (P?<?0.05) in cGH-treated chickens. In contrast, the expression of Bcl-2 and of caspases 8 and 9 was not affected by cGH in any of the oviductal segments. The results suggest that GH, via the orchestration of apoptosis and expression of some oviduct-specific proteins, participates in the development and activity of the chicken oviduct prior to the onset of egg laying.     

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Effects of testosterone and oestradiol on [3H]-thymidine incorporation by porcine granulosa and theca cells

Experiments were carried out to investigate the effects of varying physiological concentrations (0, 10, 100, and 1000 ng ml⁻¹) of oestradiol or testosterone on [³H]-thymidine incorporation by porcine granulosa and theca cells in vitro. Granulosa cells only were recovered from small (1–3-mm) follicles and both granulosa and theca cells recovered from large (4–8-mm) porcine follicles. Cells were cultured for 72 h in medium containing 10% foetal calf serum, 24 h in serum-free medium, and finally 40 h in serum-free medium containing [³H]-thymidine and appropriate steroid treatment. Although DNA per well was significantly higher (P < 0.05) at the end of culture in the theca cells than in the granulosa cells, neither steroid treatment had a significant (P > 0.1) effect on DNA concentration in either cell type. Overall, cells from small follicles incorporated significantly (P < 0.01) more [³H]-thymidine than those from medium follicles. Both oestradiol and testosterone significantly (P < 0.01) inhibited thymidine incorporation by cells from both follicle size categories, with a significant (P < 0.05) hormone × dose interaction. Finally, there was a highly significant (P < 0.001) interaction between the response of cells to different hormone concentrations and the follicle size from which they were recovered. These results indicate that both oestradiol and testosterone may act in an autocrine/paracrine manner to inhibit proliferation and encourage differentiation in follicular cells and thus are likely regulators of the later stage of antral follicle development in the pig.     

Kinetics of Apoptosis and Expression of Apoptosis-Related Proteins in Rat CA3 Hippocampus Cells After Experimental Diffuse Brain Injury

The present study examined kinetics of apoptosis and expression of apoptosis-related proteins Bcl-2, Bax, and caspase-3 in the CA3 hippocampus cells after diffuse brain injury (DBI) induced experimentally in rats. Percentage of apoptotic cells and expressions of above proteins were examined by flow cytometry and immunohistochemistry. Substantial neuronal apoptosis was documented in the CA3 hippocampus cells after DBI (22.26 ± 2.97 % at 72 h after DBI vs. 2.92 ± 0.88 % in sham-operated animals). Expression of Bc1-2 decreased, while expression of Bax and caspase-3 increased after DBI, with caspase-3 expression peaking after that of Bax (72 vs. 48 h, respectively). Further, the Bc1-2/Bax expression ratio decreased prior to increase of caspase-3 expression. In conclusion, cell apoptosis and altered expressions of Bcl-2, Bax, and caspase-3 are present in the CA3 region of hippocampus after experimental DBI. Changes in the Bc1-2/Bax expression ratio may facilitate activation of caspase-3 and aggravate neuronal apoptosis after brain injury.     

Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo

A recent report showed that reversine treatment could induce murine myoblasts dedifferentiation into multipotent progenitor cells and inhibit proliferation of some tumors, and other reports showed that apoptosis of lung adenocarcinoma cells could be induced by aspirin. The aim of the present study was to evaluate the synergistic antitumor effects of reversine and aspirin on cervical cancer. The inhibition rate of reversine and aspirin on cervical cancer cell lines’ (HeLa and U14) was determined by MTT method, cell cycle of HeLa and U14 cells was analyzed by FACS, mitochondrial membrane potential of HeLa and U14 was detected using a JC-1 kit. HeLa and U14 colony formation was analyzed by soft agar colony formation assay. The expression of caspase-3, Bcl-2/Bax, cyclin D1 and p21 was detected by qRT-PCR and Western Blotting. Moreover, tumor weight and tumor volume was assessed using a murine model of cervical cancer with U14 cells subcutaneously (s.c.) administered into the neck, separately or combined with drug administration via the intraperitoneal (i.p.) route. The inhibition rate of cells in the combination group (10 μmol/L reversine, 10 mmol/L aspirin) increased significantly in comparison to that when the drugs were used alone (P < 0.05); moreover, this combination could synergistically inhibit the proliferation of five cervical cancer cell lines (HeLa, U14, Siha, Caski and C33A). In the therapeutic mouse model, tumor weight and tumor volume of cervical cancer bearing mice was more reduced when compared with the control agents (P < 0.05) in tumor-bearing mice. The combination of reversine and aspirin exerts synergistic growth inhibition and apoptosis induction on cervical cancers cells.     

Effects of methoxychlor on IGF-I signaling pathway in rat ovary

Follicular development and other ovarian functions are regulated by growth factors that can be affected by exogenous agents. Methoxychlor (MXC) is an organochloride pesticide that causes female infertility. We investigated how MXC affects the distribution of developing ovarian follicles in adult rats after treatment between embryonic day (E) 18 and postnatal day (PND) 7. We also measured insulin-like growth factor-I (IGF-I) and its receptor, IGF-IR, expressions in ovarian follicles and investigated whether MXC changed the levels of IGF-I and IGF-IR in the ovary. Using immunohistochemical (IHC) staining, we detected IGF-I expression in oocytes and granulosa cells of the follicles, luteal cells, interstitial cells, theca externa and theca interna, and the smooth muscle of ovarian vessels. IGF-IR was co-localized with IGF-I in the ovary except for the theca externa. IGF-I expression was decreased in granulosa cells of preantral and antral follicles after treatment with MXC compared to granulosa cells of preantral and antral follicles of the control group. We also observed that oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the MXC treated groups showed increased IGF-IR expression compared to oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the control group. We also detected more secondary and preantral follicles, and fewer primordial and antral follicles after MXC administration compared to controls. Therefore, the IGF signaling pathway may participate in MXC induced ovary dysfunction and female infertility.     

Preliminary findings on the influence of FTO rs9939609 and MC4R rs17782313 polymorphisms on resting energy expenditure,leptin and thyrotropin levels in obese non-morbid premenopausal women

Given that leptin, ghrelin and thyrotropin play a major role in the regulation of resting energy expenditure (REE) and that the FTO rs9939609 and the MC4R rs17782313 polymorphisms have been proposed to affect energy homeostasis, we hypothesized that both polymorphisms are associated with REE and that these relationships can be mediated by leptin, ghrelin and thyrotropin in obesity. Therefore, the present study aimed to examine the relationships between FTO rs9939609 and the MC4R rs17782313 with REE, leptin, ghrelin and thyrotropin levels in obese women. The study comprised 77 obese (body mass index 34.0?±?2.8 kg/m²) women (age 36.7?±?7 years). We measured body composition by dual-energy X-ray absorptiometry and REE by indirect calorimetry. We analysed fasting leptin, ghrelin and thyrotropin levels and the ratio of leptin to fat mass was calculated. Genotype distributions of the polymorphisms did not deviate from Hardy–Weinberg expectations (P values >0.2). Women carrying the A allele of the FTO rs9939609 had lower REE (1,580?±?22 vs. 1,739?±?35 kcal/day, P?<?0.001) and higher leptin to fat mass ratio (1.33?±?0.05 vs. 1.13?±?0.08 ng/ml kg, P?<?0.05) and thyrotropin levels (1.93?±?0.10 vs. 1.53?±?0.16 μU/ml, P?<?0.05) regardless of age and body mass index. We found no significant influence of the MC4R rs17782313 on energy metabolism or biochemical variables. Our findings confirm that the A allele of the FTO rs9939609 is associated with lower REE and increased plasma leptin levels. We also found an association between the FTO rs9939609 and thyrotropin, suggesting the possible influence of FTO in the hypothalamic–pituitary–thyroid axis as a potential mechanism of the increased adiposity.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.