Rat Corpus Luteum Research Articles

Protective effects of nobiletin against metabolic and reproductive disturbance in letrozole induced polycystic ovarian syndrome in rats

Polycystic ovarian syndrome (PCOS) is the most prevalent metabolic and endocrine disorder that affects women globally. This research was planned to evaluatethe curative effects of nobiletin (NOB) on biochemical, metabolic, androgenic as well as histological parameters of PCOS induced rats. 24 female albino rats were divided into 4 equal groups: Group-I served as control. Group-II was treated with letrozole (1 mg/kg) (dissolved in 0.5 % CMC) for 21 days, for the induction of PCOs. Group III received letrozole (1 mg/kg) and metformin (2 mg/100 g) for 21 days. Group-IV received letrozole 1 mg/kg and NOB 10 mg/kg for 21 days. Testosterone, estradiol, progesterone, triglycerides, antioxidants (CAT, SOD, GSR, GPx, GST, GSH,), reactive oxygen species, thiobarbituric acid reactive substances, glucose, cholesterol level (HDL-C, LDL-C, VLDL-C, Non-HDL-C) as well as histopathological analysis of ovarieswere performed. However, nobiletin treatment demonstrated beneficial effects by lowering testosterone levels, glucose levels, cysts, and improving estradiol and progesterone level as well as number of follicles and corpus luteum in PCOS rats. Furthermore, dysregulated lipid as well as anti-oxidant profiles in PCOS rats were reverted to the normal level. The current investigation demonstrated that NOB shows the ability to mitigate the irregularities associated with PCOS.

Melatonin as a promising therapeutic intervention for restoring ovarian function in letrozole-induced polycystic ovary syndrome rats

Polycystic ovary syndrome (PCOS) is a common hormonal disorder that affects women of reproductive age and is characterized by multiple ovarian cysts, irregular menstrual cycles, and excessive androgen hormone production. The present study aimed to investigate the therapeutic efficacy of melatonin in alleviating PCOS-induced alterations in female Wistar rats. PCOS was induced in female albino rats by administering letrozole at a dose of 1 mg/kg for 21 days. A total of 24 rats were randomly selected and divided into four groups: group I (normal control), group II (melatonin treatment), group III (letrozole treatment), and group IV (melatonin therapy for PCOS rats). Physical parameters (body and uterus weight), hormone profile (LH and FSH), and steroidogenic enzyme activities and an oral glucose test were assessed using standard methods. Histological analysis was performed using hematoxylin and eosin staining. The results demonstrated that exogenous melatonin administration significantly improved PCOS symptoms in rats, including reduced body weight gain, changes in organ weight/body weight index, blood glucose level, percentage diestrus phase, testosterone, estradiol, progesterone, and LH/FSH ratio, as well as 3β-HSD and 17β-HSD enzyme activity. Histopathological findings revealed well-developed follicles, decreased cystic follicles, and increased antral follicles, Graafian follicles, and corpus luteum in PCOS rats treated with melatonin. These positive outcomes suggest that exogenous melatonin may hold promise as a valuable remedy for PCOS conditions in female rats. Further research is warranted to fully elucidate the underlying mechanisms and potential clinical applications of melatonin in the context of PCOS.

The Effect of Administering Forest Honey to Rats Exposed to Physical Stress on Corticosteroid Levels, Folliculogenesis and the Number of Corpus Luteum

Livestock that has stress releases glucocorticoids in response to it, and it causes inhibition of the hypothalamus-pituitary-gonadal axis (HPG) signaling pathway so that it reduces reproductive efficiency. Forest honey can reduce corticosteroid levels as a stress response from physical stress induction which is expected to increase reproductive efficiency including folliculogenesis and the formation of the corpus luteum. This study aims to determine the effect of forest honey on rats (Rattus novergicus) exposed to physical stress on corticosteroid levels, folliculogenesis, and the number of corpus luteum. This study is an experimental laboratory one using 32 rats which were divided into 4 treatment groups; control positive (C) treated with physical stress, treatment 1 (T1) treated with physical stress + honey 2 g/rat/day PO, treatment 2 (T2) treated with physical stress + honey 4 g/rat/day PO and treatment 3 (T3) treated with physical stress + honey 6 g/rat/day PO. All treatments were carried out for 14 days. The results showed that T1 had the lowest corticosteroid level compared to all treatment groups and the corticosteroid level of this group was significantly different (p <0.05) compared to that of C and T3. The folliculogenesis profile showed that the number of primary secondary, tertiary, and Graafian follicles of group T1 was significantly different (p<0.05) compared to that of C, T2, and T3. In terms of the number of corpus luteum, it showed that T1 had the highest number of corpus luteum, and the number of corpus luteum in this group was significantly different (p<0.05) from that of C, T2, and T3. It can be concluded that the administration of forest honey at a dose of 2g/rat/day could reduce corticosteroid levels, improve the folliculogenesis profile, and increase the number of corpus luteum in rats exposed to physical stress. The use of forest honey could reduce corticosteroid levels as a stress response from physical stress induction which was expected to increase reproductive efficiency.

Subacute exposure to Roundup® changes steroidogenesis and gene expression of the glutathione-glutaredoxin system in rat ovaries: Implications for ovarian toxicity of this glyphosate-based herbicide

Studies have indicated that glyphosate induces endocrine disruption and may adversely affect the male reproductive system. However, evidence of its effects on ovarian function is poorly understood so far, making further studies necessary on the mechanisms of the glyphosate toxicity in the female reproductive system. The aim of this work was to evaluate the effect of a subacute exposure (28 days) to the glyphosate-based formulation Roundup® (1.05, 10.5 and 105 μg/kg b.w. of glyphosate) on steroidogenesis, oxidative stress, systems involved in cell redox control and histopathological parameters in rat ovaries. Hence we quantify plasma estradiol and progesterone by chemiluminescence; non-protein thiol levels, TBARS, superoxide dismutase and catalase activity by spectrophotometry; gene expression of steroidogenic enzymes and redox systems by real-time PCR; and ovarian follicles by optical microscopy. Our results demonstrated that oral exposure increased progesterone levels and the mRNA expression of 3β-hydroxysteroid dehydrogenase. Histopathological analysis revealed a decrease in the number of primary follicles and an increase in the number of corpus luteum in rats exposed to Roundup®. An imbalance of the oxidative status was also evidenced by decreasing the catalase activity at all groups exposed to the herbicide. Increased lipid peroxidation and gene expression of glutarredoxin and decreased of glutathione reductase were also observed. Our results indicate that Roundup® causes endocrine disruption of hormones related to female fertility and reproduction and changes the oxidative status by altering antioxidant activity, inducing lipid peroxidation, as well as changing the gene expression of the glutathione-glutarredoxin system in rat ovaries.

A metabolomic approach to study triptolide-induced ovarian damage in rats

Triptolide (TP) is the major active ingredient of Tripterygium wilfordii Hook, a traditional Chinese herb that possesses various pharmacological activities and has been used to treat autoimmune and inflammatory diseases for thousands of years. However, the clinical application of TP is limited due to its multiorgan toxicity, and in particular, its negative impact on female fertility. To date, the specific toxic mechanisms on reproduction induced by TP remain unclear. In the current study, an LC-MS/MS-based metabolomic approach was adopted to study TP-induced reproductive toxicity and its mechanism. Histopathological examination of the ovaries showed that TP significantly induced follicular atresia and decreased the numbers of corpus luteum in rats, as well as reducing the gonadal index and destroying the microstructure of the ovary. Immunohistochemical staining revealed that TP significantly induced apoptosis of rat follicle cells. Metabolomics analysis revealed that 67 and 74 small molecule metabolites in the ovaries and serum, respectively (fold-changes > 1.5, p < 0.05), were significantly different in TP-treated rats compared to CON group rats. Target profiling identified the metabolites arachidonic acid, prostaglandin D2, prostaglandin H2 and prostaglandin E2 as potential serum biomarkers for TP-induced ovary damage.

Activation of Ovarian Taste Receptors Inhibits Progesterone Production Potentially via NO/cGMP and Apoptotic Signaling.

Taste receptors are not only expressed in the taste buds, but also in other nongustatory tissues, including the reproductive system. Taste receptors can be activated by various tastants, thereby exerting relatively physiologic functions. The aim of this study was to investigate the effects and potential mechanisms underlying ovarian taste receptor activation on progesterone production using saccharin sodium as the receptor agonist in a pseudopregnant rat model. Taste 1 receptor member 2 (TAS1R2) and taste 2 receptor member 31 (TAS2R31) were demonstrated to be abundantly expressed in the corpora lutea of rats, and intraperitoneal injection of saccharin sodium can activate both of them and initiate their downstream signaling cascades. The activation of these ovarian taste receptors promoted nitric oxide (NO) production via endothelial nitric oxide synthase (eNOS). NO production then increased ovarian cyclic guanosine 3',5'-monophosphate (cGMP) levels, which, in turn, decreased ovarian cyclic adenosine 3',5'-monophosphate levels. In addition, the activation of ovarian taste receptors induced apoptosis, possibly through NO and mitogen-activated protein kinase signaling. As a result, the activation of ovarian taste receptors reduced the protein expression of steroidogenesis-related factors, causing the inhibition of ovarian progesterone production. In summary, our data suggest that the activation of ovarian taste receptors inhibits progesterone production in pseudopregnant rats, potentially via NO/cGMP and apoptotic signaling.

Hormonal regulation and function of an RNA helicase, Ddx5 in corpus luteum of adult Wistar rats

Corpus luteum (CL) is an endocrine tissue involved in regulation of reproductive cycle and early pregnancy establishment. In the present study DEAD-box helicase-5 (Ddx5), a member of the DEAD box family of RNA helicases was investigated for its expression, regulation and function in CL of Wistar rats. Ddx5 was expressed in adult rat CL. Primary cell culture from supra-ovulated ovaries were established for in vitro studies. Addition of luteinizing hormone (LH; 100 ng/ml), a luteotrophic factor in primary cell culture, decreased Ddx5 RNA expression (foldchange:0.6 ± 0.075) while prostaglandin alpha (PGF2α; 1μM), a luteolytic factor caused an increase (foldchange:2.4 ± 0.4) compared to control group. Under in vivo conditions, the administration of PGF2α or gonadotropin-releasing hormone antagonist; cetrorelix (CET) caused luteolysis as well as an increase in the protein level of Ddx5 (foldchange:1.9 ± 0.27 and 1.4 ± 0.09 viz.; p < 0.05) in CL of adult rats. LH was administered post CET treatment which suppressed Ddx5 protein expression (foldchange:0.8 ± 0.16; p < 0.05) compared to CET treated group. Further, it was observed that the expression of Ddx5 was upregulated (foldchange:1.5 ± 0.23; p < 0.05) in CL during late pregnancy compared to mid pregnancy concomitant to luteolysis in adult rats. Overall, the results suggest for the first time that Ddx5 is expressed in rat CL and regulated by luteolytic and luteotrophic factors in an inverse fashion. Further, the data significantly correlates ddx5 expression to CL regression suggesting involvement of ddx5 in luteolysis. These results suggest a significant role of Ddx5 in female reproduction biology and warrant in depth examination of the function of Ddx5 in CL.

Effect of Aqueous Extract of Glycyrrhiza glabra on the Biochemical Changes Induced by Cadmium Chloride in Rats.

Twenty four rats were divided into four groups (6 rats/group): 1-control group; 2-licorice (Glycyrrhiza glabra) extract: rats administered with an oral dose of licorice (3mg/ml/kg/day) using stomach tube for 4weeks; 3-cadmium chloride group: rats administered with an oral dose of CdCl2 (10mg/kg/day) using stomach tube for 4weeks; and 4-licorice extract + CdCl2 group: rats administered orally with both licorice (3mg/ml/kg/day) and CdCl2 (10mg/kg/day) using stomach tube for 4weeks. Administration of CdCl2 induced significant increase in thiobarbituric acid reactive substance (TBARS), paraoxonase-1 (proxon-1), caspase-3 (casp-3) activities, and significant decrease in superoxide dismutase (SOD), catalase (CAT) activities, and glutathione (GSH) content in hepatic tissue. Significant increase in TBARS and kidney injury molecule-1 (KIM-1) and significant decrease in SOD, CAT activities, and GSH content in renal tissue were recorded. Significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) activities, urea, creatinine, and significant decrease in butyryl choline esterase (BChE), total triiodothyronine (T3), and total thyroxin (T4) were recorded in serum. Histological investigation of renal cells showed vacuolations of endothelium lining glomerular tuffs and vacuolations of epithelium lining renal tubules. Investigation of ovarian tissue showed dilatation of interstitial blood vessels and congestion of multiple corpus luteum in CdCl2-treated rats. Significant improvements in the biochemical and histological changes were observed in CdCl2 + licorice extract group. It could be concluded that licorice extract alleviates the hazardous effects of cadmium chloride, which may be attributed to its antioxidant properties.

PROTECTIVE EFFECTS OF AJWA ON NICOTINE INDUCED CHANGES IN BLOOD VESSELS AND CORPUS LUTEUM

Introduction: Nicotine adversely affects ovarian functions and Ajwa (Phoenix dactylifera) being an antioxidant prevents nicotine from causing toxicity.&#x0D; Aims &amp; Objectives: To assess the effects of Ajwa on nicotine induced vascular and luteal changes in adult albino rats.&#x0D; Material &amp; Methods: Study was conducted in the Anatomy Department of Federal Postgraduate Medical Institute, Shaikh Zayed Medical Complex Lahore. Four equal groups were made of 40 adult female albino rats. 1ml distilled water was given to the rats of control (A) group by gastric intubation, experimental group B was given nicotine injection intraperitoneally (0.1 mg/kg body weight) for 28 days. The rats of experimental group C were given Ajwa fruit extract 1gm/kg body weight daily by gastric intubation, while rats of experimental group D were given 0.1mg/kg body weight nicotine intraperitoneally plus 1gm/kg body weight Ajwa fruit extract by gastric intubation for 28 days. Results: Vascular congestion was found in one rat of group A &amp; D and in all animals of group B. Corpus luteum of group B revealed congestion and was focally infiltrated by inflammatory cells.&#x0D; Conclusion: Ajwa improves nicotine induced changes in blood vessels and corpus luteum of adult albino rats, so it may be useful to improve infertility or subfertility induced by smoking in women.

Expression patterns of claudin-5 and its related signals during luteal regression in pseudopregnant rats: The enhanced effect of additional PGF treatment

To study the expression patterns of claudin-5 and its related signals during luteal regression in rats, a sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpus luteum (CL). A total of 35 rats were treated with one PGF or two PGF at an interval of 24 h from day 7 of pseudopregnancy to induce CL regression. Serum and ovaries were collected at 0, 2, 4, 8 or 24 h after one PGF injection (1 PGF), 2 or 24 h after two PGF injections (2 PGF). The serum progesterone level was detected by RIA; the ovarian expression of claudin-5, the phosphorylations of STAT3 (p-STAT3), Akt (p-Akt), ERK1/2 (p-ERK) and p38 MAPK (p-p38) were detected by western blot, real-time PCR and IHC. Results showed that serum progesterone (P4) decreased after PGF treatment. Claudin-5 mRNA decreased at 4 h and 8 h after 1 PGF and 2 h after 2 PGF, and claudin-5 protein decreased at 4 h after 1 PGF. p-STAT3 increased at 4 h after 1 PGF and 2 h after 2 PGF. p-ERK increased at 2 h after 2 PGF. The level of p-Akt decreased at 4 h after 1 PGF. PGF treatment did not alter the phosphorylation of p38 MAPK at any time points in this study. IHC results revealed that claudin-5 was expressed in the nuclei and cytoplasm of steroidogenic cells and in the vessels, while PGF induced-p-STAT3 was expressed uniformly in the cytoplasm of luteal steroidogenic cells. In conclusion, PGF treatment decreased the expression of claudin-5 and the additional PGF treatment enhanced the decrease in claudin-5 mRNA expression and the increases in ERK1/2 and STAT3 phosphorylation in the corpus luteum of pseudopregnant rats, which will contribute new information to the further study of molecular mechanism of luteal regression.

Study on the mechanism of Bushen Culuan Chongji treating "kidney deficiency and blood stasis" in ovulatory dysfunctional infertility

Kidney deficiency and blood stasis is the main cause of ovulatory dysfunctional infertility. Kidney deficiency is the main pathological mechanism. Blood stasis is the main pathological manifestation, and it is an important factor throughout. Bushen Culuan Chongji is under the guidance of traditional Chinese medicine(TCM) theory, previous years of clinical experience, combined with the etiology and pathogenesis of anovulatory infertility and modern pharmacological research results, selected, not only maintains the TCM syndrome differentiation and different diseases features, but also reflects the superiority of the combination of disease. In the study of Bushen Culuan Chongji in the treatment of anovulatory infertility, there was no acute toxicity and no LD50 was detected. No adverse effects and side effects were found in the reproductive, genetic, toxicity, teratogenic, and perinatal tests in their high and low dose groups. In pharmacodynamics experiments, it can promote the development and maturation of follicles and the formation of corpus luteum in rats. Taking the kidney deficiency and blood stasis syndrome as the breakthrough point, systematically study the efficacy, safety and mechanism of six ovulatory dysfunctional infertility diseases, including abnormal uterine bleeding-ovulatory disorders, polycystic ovary syndrome, high prolactin, luteinized unruptured follicle syndrome, luteal phase defect and diminished ovarian reserve/premature ovarian failure. It verified the contribution degree of reinforcing kidney and resolving stasis TCM, reflected the characteristics of combination between disease differentiation and syndrome differentiation, interpreted the treatment principles of treating different diseases with the same method, and provided scientific basis for clinical treatment.

Type-dependent differences in Fas expression and phagocytes distribution in rat corpora lutea during natural regression: an immunohistochemical evidence.

Though Fas/Fas ligand (FasL) system-dependent apoptosis is considered to be the primary form of cell death in regressing corpus luteum (CL), the cellular identity and regulation of expression of the ligand and receptor molecules are not fully understood. Here, we focused on immunohistochemical determination of Fas expression during natural regression with comparison of three different types of rat CLs. Detected Fas was in good spatial association with cleaved caspase-3 and FasL proteins and with macrophages and neutrophils. In CLs of the cycle and pseudopregnancy, Fas-positive cell types included large and small luteal (steroidogenic) cells and capillary endothelial cells mainly, and blood-derived immune cells occasionally. Fas signals were abundant at multiple focal inflammatory-like sites. In contrast, Fas signals in CL of pregnancy did not localize in steroidogenic cells, but almost exclusively in endothelial cells and granulocytes. The signals scattered evenly throughout the CL tissue as phagocytes also did. In all CLs types, the numbers of Fas-expressing cells increased transiently after functional inactivation and at the early phase of structural regression. This observation revealed spatio-temporally regulated expression of Fas that was highly associated with apoptotic and phagocytotic systems and type-dependent differences in Fas expression and phagocytes dynamics in naturally regressing CL of rats.

Effect of feeding a high-fat diet independently of caloric intake on reproductive function in diet-induced obese female rats.

IntroductionGlobally, the prevalence of overweight and obesity is increasing, predisposing females to health hazards including compromised reproductive capacity. Our objective was to investigate the effect of ad libitum, isocalorically and hypocalorically restricted high-fat diet (HFD) feeding on reproductive function in diet-induced obese female rats.Material and methodsTwenty female albino Sprague Dawley rats were used; 5 rats were kept on a standard pellet animal diet to serve as a control group (A) and 15 rats were fed a HFD for 9 weeks to induce obesity. The HFD fed animals were equally divided into three groups: an ad libitum HFD group (B), an isocalorically restricted HFD group (C), and a hypocalorically restricted HFD group (D). Estrous cyclicity, hormonal levels, ovarian histopathology and caspase-3 immunoreactivity were evaluated.ResultsThe HFD-fed rats in groups B, C and D had significant irregularity in estrous cyclicity Vs group A (p = 0.001, 0.003 and 0.034 respectively). Groups C and D had significant reduction in serum progesterone level (p = 0.006 and 0.018 Vs A). Isocaloric restriction of HFD feeding significantly increased serum LH. Groups B and C had a significant increase in caspase-3 expression in the ovary (p < 0.001).ConclusionsAd libitum HFD interfered with the normal estrous cycle and enhanced apoptosis of luteal cells in obese female rats. The HFD restriction interfered with the normal estrous cycle and caused functional insufficiency of the corpus luteum in obese female rats. These results suggest that HFD feeding determinately affects female reproductive function independently of caloric intake.

Luteal activity of pregnant rats with hypo-and hyperthyroidism

BackgroundLuteal activity is dependent on the interaction of various growth factors, cytokines and hormones, including the thyroid hormones, being that hypo- and hyperthyroidism alter the gestational period and are also a cause of miscarriage and stillbirth. Because of that, we evaluated the proliferation, apoptosis and expression of angiogenic factors and COX-2 in the corpus luteum of hypo- and hyperthyroid pregnant rats.MethodsSeventy-two adult female rats were equally distributed into three groups: hypothyroid, hyperthyroid and control. Hypo- and hyperthyroidism were induced by the daily administration of propylthiouracil and L-thyroxine, respectively. The administration began five days before becoming pregnant and the animals were sacrificed at days 10, 14, and 19 of gestation. We performed an immunohistochemical analysis to evaluate the expression of CDC-47, VEGF, Flk-1 (VEGF receptor) and COX-2. Apoptosis was evaluated by the TUNEL assay. We assessed the gene expression of VEGF, Flk-1, caspase 3, COX-2 and PGF2α receptor using real time RT-PCR. The data were analyzed by SNK test.ResultsHypothyroidism reduced COX-2 expression on day 10 and 19 (P < 0.05), endothelial/pericyte and luteal cell proliferation on day 10 and 14 (p < 0.05), apoptotic cell numbers on day 19 (p < 0.05) and the expression of Flk-1 and VEGF on day 14 and 19, respectively (p < 0.05). Hyperthyroidism increased the expression of COX-2 on day 19 (P < 0.05) and the proliferative activity of endothelial/pericytes cells on day 14 (p <0.05), as well as the expression of VEGF and Flk-1 on day 19 (P < 0.05).ConclusionsHypothyroidism reduces the proliferation, apoptosis and expression of angiogenic factors and COX-2in the corpus luteum of pregnant rats, contrary to what is observed in hyperthyroid animals, being this effect dependent of the gestational period.

Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum.

Angiogenesis in the developing corpus luteum (CL) is a prerequisite for establishment and maintenance of an early pregnancy. To explore the physiological significance of insulin-like growth factor-binding protein-7 (IGFBP7) in the developing CL, the effects of IGFBP7 on vascular endothelial growth factor (VEGFA)- and luteinizing hormone (LH)-induced in vitro tube formation were tested using isolated luteal microvascular endothelial cells (LECs). Capillary-like tube formation of LECs and their proliferation were stimulated by both VEGFA and LH. IGFBP7 treatment suppressed VEGFA- or LH-induced tube formation. The proliferation and migration of LECs, and phosphorylation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase 1/2 were inhibited by IGFBP7. Furthermore, IGFBP7 attenuated VEGFA-enhanced cyclooxygenase (COX)-2 mRNA expression and prostaglandin E2 secretion. These findings suggest the possibility that luteal IGFBP7 secretion may suppress the stimulatory effect of VEGFA on angiogenesis in the early CL.

Effects of Hypo‐ and Hyperthyroidism on Proliferation, Angiogenesis, Apoptosis and Expression of COX‐2 in the Corpus Luteum of Female Rats

Although thyroid dysfunction occurs frequently in humans and some animal species, the mechanisms by which hypo- and hyperthyroidism affect the corpus luteum have not been thoroughly elucidated. This study evaluated the levels of proliferative activity, angiogenesis, apoptosis and expression of cyclooxygenase-2 in the corpus luteum of female rats with thyroid dysfunction. These processes may be important in understanding the reproductive changes caused by thyroid dysfunction. A total of 18 adult female rats were divided into three groups (control, hypothyroid and hyperthyroid) with six animals per group. Three months after treatment to induce thyroid dysfunction, the rats were euthanized in the dioestrus phase. The ovaries were collected and immunohistochemically analysed for expression of the cell proliferation marker CDC-47, vascular endothelial growth factor (VEGF), VEGF receptor Flk-1 and cyclooxygenase-2 (COX-2). Apoptosis was evaluated using the TUNEL assay. Hypothyroidism reduced the intensity and area of COX-2 expression in the corpus luteum (p<0.05), while hyperthyroidism did not alter COX-2 expression in the dioestrus phase. Hypothyroidism significantly reduced the expression of CDC-47 in endothelial cells and pericytes in the corpus luteum, whereas hyperthyroidism did not induce a detectable change in CDC-47 expression (p>0.05). Hypothyroidism reduced the level of apoptosis in luteal cells (p<0.05) and increased VEGF expression in the corpus luteum. In contrast, hyperthyroidism increased the level of apoptosis in the corpus luteum (p<0.05). In conclusion, thyroid dysfunction differentially affects the levels of proliferative activity, angiogenesis and apoptosis and COX-2 expression in the corpus luteum of female rats.

Differential Morphological Effects in Rat Corpora Lutea among Ethylene Glycol Monomethyl Ether, Atrazine, and Bromocriptine

Ethylene glycol monomethyl ether (EGME) or atrazine induces luteal cell hypertrophy in rats. Our previous study suggested that EGME stimulates both new and old corpora lutea (CL), while atrazine stimulates new CL. Bromocriptine (BRC) is known to suppress the luteolysis in rats. This study investigated the light- and electron-microscopic luteal changes induced by EGME, atrazine, or BRC. Female rats were treated with EGME (300 mg/kg/day), BRC (2 mg/kg/day), EGME and BRC (EGME + BRC), or atrazine (300 mg/kg/day) for 7 days. Luteal cell hypertrophy induced by EGME, EGME + BRC, and atrazine was subclassified into the following two types: CL hypertrophy, vacuolated type (CL-V) characterized by intracytoplasmic fine vacuoles, and CL hypertrophy, eosinophilic type (CL-E) characterized by eosinophilic and abundant cytoplasm. The proportions of CL-V and CL-E were different among the treatments. BRC-treated old CL showed lower proportion of endothelial cells and fibroblasts than normal old CL. Ultrastructural observation revealed that the luteal cells of CL-V contained abundant lipid droplets, whereas those of CL-E in EGME and EGME + BRC groups showed uniformly well-developed smooth endoplasmic reticulum. No clear ultrastructural difference was observed between the control CL and atrazine-treated CL-E. These results indicate that EGME, atrazine, and BRC have differential luteal morphological effects.

DNA Methylation and Histone Modifications Are Associated with Repression of the Inhibin α Promoter in the Rat Corpus Luteum

The transition from follicle to corpus luteum after ovulation is associated with profound morphological and functional changes and is accompanied by corresponding changes in gene expression. The gene encoding the α subunit of the dimeric reproductive hormone inhibin is maximally expressed in the granulosa cells of the preovulatory follicle, is rapidly repressed by the ovulatory LH surge, and is expressed at only very low levels in the corpus luteum. Although previous studies have identified transient repressors of inhibin α gene transcription, little is known about how this repression is maintained in the corpus luteum. This study examines the role of epigenetic changes, including DNA methylation and histone modification, in silencing of inhibin α gene expression. Bisulfite sequencing reveals that methylation of the inhibin α proximal promoter is low in preovulatory and ovulatory follicles but is elevated in the corpus luteum. Increased methylation during luteinization is observed within the cAMP response element in the promoter, and EMSA demonstrate that methylation of this site inhibits cAMP response element binding protein binding in vitro. Chromatin immunoprecipitation reveals that repressive histone marks H3K9 and H3K27 trimethylation are increased on the inhibin α promoter in primary luteal cells, whereas the activation mark H3K4 trimethylation is decreased. The changes in histone modification precede the alterations in DNA methylation, suggesting that they facilitate the recruitment of DNA methyltransferases. We show that the DNA methyltransferase DNMT3a is present in the ovary and in luteal cells when the inhibin α promoter becomes methylated and observe recruitment of DNMT3a to the inhibin promoter during luteinization.

A Regulatory Mechanism Between Progesterone and Notch Signaling Pathway Maintains the Functionality of the Corpus Luteum from Pregnant Rats.

The Notch signaling pathway includes a family of transmembrane receptors (Notch1-4) that interact with specific ligands (Delta-like family, Jagged1 and Jagged2). This system regulates cell fate decisions, including proliferation, differentiation and apoptosis. The transmembrane receptors are cleaved upon binding of their ligands, leading to the release of the intracellular domain (NICD), which translocates to the nucleus where it functions as a transcriptional coactivator of regulatory genes of cellular fate. This processing requires the activity of two proteases, namely tumour necrosis factor a-converting enzyme and presenilin/gamma-secretase. We have previously shown that Notch1-4 receptors and the ligand Dll4 are expressed in the corpus luteum (CL) of pregnant rats and that the administration of prostaglandin F2 alpha decreases the levels of mRNA and protein expression of these proteins. Furthermore, blocking ovarian Notch system with the gamma-secretase inhibitor DAPT decreases serum levels of progesterone (P4) and increases pro-apoptotic protein expression in the CL. However, the role of P4 in regulating the Notch pathway in the CL of pregnant rats remains unclear. In this study CL′s were isolated from ovaries of rats on day 16 of gestation and cultured for 4 hours in the presence of aminoglutethimide (inhibitor of cytochrome P450scc 0.15 mM), DAPT (gamma secretase inhibitor, 20 uM), both inhibitors and the inhibitors with the addition of P4 (500ng/ml). Luteal function was assessed by measuring P4 levels in the medium by RIA. The CL levels of Notch1-4 and members of the ERK and PI3K/AKT pathways were measured by Western blot. The treatment of CL′s with each inhibitor significantly decreased P4 levels (P < 0.05, n = 5) in the culture medium, effect which was similar in the presence of both compounds. Furthermore, these inhibitors decreased protein levels of Notch1 intracellular fragment (NICD) compared to the control group (P < 0.05, n = 5). However, no significant difference was observed for NICD of Notch4. In addition, ERK and AKT phosphorylation decreased with the addition of aminoglutethimide or/and DAPT in the CL culture medium. These results suggest the existence of a regulatory mechanism between progesterone and the Notch signaling pathway to maintain the functionality of the CL in pregnancy. Supported by: CONICET (PIP1223), Universidad de Buenos Aires (X-218) and ANPCYT (PICT, 2010-0248).

Allopregnanolone Alters the Luteinizing Hormone, Prolactin, and Progesterone Serum Levels Interfering with the Regression and Apoptosis in Rat Corpus Luteum

Steroids synthesized in the central nervous system are termed "neurosteroids". They are synthesized and metabolized in several brain areas. The objective of this work was to determine if 1 intracerebroventricular allopregnanolone injection in rats can interfere in luteal regression in a close relationship with modifications in LH, progesterone, and prolactin serum concentrations. Allopregnanolone was injected during proestrus morning and the animals were sacrificed on oestrous morning. Ovulation test and histological analysis were performed in the oestrus morning with light and electron microscopy. Serum prolactin, LH, and progesterone levels were measured by radioimmunoassay. The allopregnanolone injection significantly decreased luteinizing hormone serum level and the number of oocytes on oestrus. Progesterone and prolactin serum levels were increased after this injection. The inhibition of apoptotic figures due to allopregnanolone administration was detected in the already formed corpora lutea belonging to the previous ovary cycle and it was significantly lower than in vehicle group (control). When the GABA(A) antagonist (bicuculline) was administered alone or previously to allopregnanolone, no effect on the ovulation rate was observed. No changes in the apoptotic cell numbers were observed with respect to those of vehicle group. These results show that the effect of centrally injected allopreganolone over reproductive function could be due to a centrally originated LH mediated effect over ovarian function that affects luteal regression, through the inhibition of apoptosis and stimulation of progesterone and prolactin release.