Hyperandrogenemia elevates expression of apelin and apelin receptor protein in the mice pituitary.

Hyperandrogenemia is associated with polycystic ovarian syndrome (PCOS) and imbalances in the pituitary hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. Apelin and its receptor, APJ (class A, rhodopsin-like G- protein-coupled receptor), belongs to adipokines, and its expression has been shown in the pituitary. It is also well known that, hyperandrogenism and PCOS have deregulation of different adipokines. Whether hyperandrogenism also deregulates the apelin system in the pituitary has yet to be investigated. Thus, we have investigated the expression and localization of apelin and its receptor, APJ, in the letrozole-induced hyperandrogenised pituitary of female mice. Our results showed that the apelin, APJ and androgen receptor (AR) expression were upregulated in the anterior pituitary. Furthermore, the immunostaining of LH exhibited increased abundance than FSH. The circulating LH was also found to be elevated compared to FSH levels. The increased LH synthesis and secretion coincides with elevated apelin system in the pituitary of hyperandrogenised mice. Recently, a direct role of apelin has also been reported in the female pituitary, where apelin inhibits LH secretion. Thus, apelin could be one of the factors for deregulated gonadotropin secretion in hyperandrogenised conditions. However, more research is needed to fully understand the complex interactions between apelin and androgen regarding gonadotropin secretion in hyperandrogenised conditions.

Hormonal dependent expression of apelin and apelin receptor in the ovary and uterus of mice.

Apelin and APJ have been shown to regulate female reproductive functions. However, its uterine expression during the oestrous cycle and its regulation by ovarian steroids, along with gonadotropin regulation in the ovary, has not been investigated. This study aimed to analyze the steroid-dependent uterine expression of apelin/APJ in the uterus along with the oestrous cycle. Furthermore, it also aimed to investigate gonadotropin-dependent ovarian expression of apelin and APJ. To investigate the uterine expression of apelin and APJ during estrous cycle in mice, uterus at different estrous stage were collected. To explore the ovarian steroids dependent expression of apelin system in the uterus, ovariectomized mice were treated with only estrogen at dose of 30 ng/g, only progesterone at dose of 150 µg/g and combined doses. To study the effect of gonadotropin on ovarian expression of apelin system, immature mice were injected with 2.5 IU of pregnant mare serum gonadotropin (PMSG) alone and both PMSG plus 2.5 IU of chorionic gonadotropin (hCG). Apelin and APJ protein expression are modulated by estrous phases in the uterus. The uterine apelin and APJ expression are up-regulated by estrogen and down-regulated by progesterone. The expression and localization of APJ showed increased abundance in the follicles of PMSG treated mice, however, the PMSG plus HCG treatment showed formation of corpus luteum with increased abundance of APJ and progesterone secretion. The expression of apelin and APJ are regulated by pituitary gonadotropin in the ovary and uterine apelin system by ovarian steroid hormone.

Apelin receptor modulation mitigates letrozole-induced polycystic ovarian pathogenesis in mice.

AIMS: Polycystic ovarian syndrome (PCOS) is one of the most common (about 5-20%) reproductive disorders in women of reproductive age; it is characterized by polycystic ovaries, hyperandrogenism, and oligo/ anovulation. The levels and expression of ovarian adipokines are deregulated in the PCOS. Apelin is an adipokine that acts through its receptor (APJ) and is known to express in the various tissues including the ovary. It has also been suggested that apelin and APJ could be targeted as therapeutic adjuncts for the management of PCOS. However, no study has been conducted on the management of PCOS by targeting the apelin system. Thus, we aimed to evaluate its impact on combating PCOS-associated ovarian pathogenesis. METHODS: The current work employed a letrozole-induced-hyperandrogenism PCOS-like mice model to investigate the effects of apelin13 and APJ, antagonist ML221. The PCOS model was induced by oral administration of letrozole (1 mg/kg) for 21 days. A total of four experimental groups were made, control, PCOS control, PCOS + aplein13, and PCOS + ML221. The treatment of apelin13 and ML221 was given from day 22 for two weeks. KEY FINDINGS: The letrozole-induced PCOS-like features such as hyperandrogenism, cystic follicle, decreased corpus luteum, elevated levels of LH/FSH ratio, and up-regulation of ovarian AR expression were ameliorated by apelin13 and ML221 treatment. However, the PCOS-augmented oxidative stress and apoptosis were suppressed by apelin 13 treatments only. ML221 treatment still showed elevated oxidative stress and stimulated apoptosis as reflected by decreased antioxidant enzymes and increased active caspase3 and Bax expression. The expression of ERs was elevated in all groups except control. Furthermore, the PCOS model showed elevated expression of APJ and apelin13 treatment down-regulated its own receptor. Overall, observing the ovarian histology, corpus luteum formation, and decreased androgen levels by both apelin13 and ML221 showed ameliorative effects on the cystic ovary. SIGNIFICANCE: Despite the similar morphological observation of ovarian histology, apelin13 and ML221 exhibited opposite effects on oxidative stress and apoptosis. Therefore, apelin13 (which down-regulates APJ) and ML221 (an APJ antagonist) may have suppressed APJ signalling, which would account for our findings on the mitigation of polycystic ovarian syndrome. In conclusion, both apelin13 and ML221 mediated mitigation have different mechanisms, which need further investigation.

Expression and localization of apelin and apelin receptor protein in the oviduct of letrozole-induced hyperandrogenized mice.

Apelin and its receptor (APJ) are expressed in the reproductive organs of some mammalian females. The function of oviduct has also been suggested to be compromised in the hyperandrogenism condition. However, expression of apelin and APJ has not been shown in the oviduct of hyperandrogenized mice. Thus, the present study has investigated the localization and expression of apelin and APJ in the letrozole-induced hyperandrogenized mice oviduct. Histomorphometric analysis showed decreased lumen of oviduct in the hyperandrogenized mice. Our results showed elevated expression of APJ and decreased abundance of apelin in the hyperandrogenized mice oviduct. This finding suggests impaired apelin signaling in the oviduct of hyperandrogenized mice. The expression of androgen receptor was upregulated while estrogen receptors were downregulated in the hyperandrogenized mice. The expression of HSP70 was also downregulated along with increased expression of active caspase 3 and BAX and decreased expression of BCL2 in hyperandrogenized mice. Furthermore, the phosphorylation of phospho-Ser473-Akt and phospho-Thr308-Akt also showed differential levels in the oviduct of hyperandrogenized mice. Whether this differential phosphorylation of Akt was solely due to impaired apelin signaling in the oviduct, remains unclear. Moreover, increased androgen signaling and suppressed estrogen signaling coincides with elevated apoptosis. In conclusion, hyperandrogenized conditions could also impair the gamete transport and fertilization process due to apoptosis in the oviduct. However, further study would be required to unravel the exact role of apelin signaling in the oviduct in relation to apoptosis.

Possible role of apelin on the ovarian steroidogenesis and uterine apoptosis of infantile mice: An in vitro study.

The expression of adipokines is well-known in the ovary and uterus. Recently we have shown that apelin and its receptor, APJ are developmentally regulated in the ovary and uterus of mice with elevation at postnatal day 14 (PND14). However, its role in the ovary and uterus of PND14 has not been investigated. Thus, we aimed to unravel the role of the apelin system (by APJ antagonist, ML221) on ovarian steroid secretion, proliferation, and apoptosis along with its role in uterine apoptosis in PND14 mice by in vitro approaches. The treatment of ML221 decreased estrogen, testosterone, and androstenedione secretion while increasing the progesterone secretion from the infantile ovary. These results suggest that apelin signaling would be important for ovarian estrogen synthesis in infantile mice (PND14). The abundance of 3ß-HSD, 17ß-HSD, aromatase, and active caspase3 increased in the infantile ovary after ML221 treatment. The expression of ERs and BCL2 were also down-regulated by ML221 treatment. The decreased BCL2 and increased active caspase3 by ML221 suggest the suppressive role of apelin on ovarian apoptosis. The APJ antagonist treatment also down-regulated the ER expression in the uterus along with increased active caspase3 and decreased BCL2 expression. In conclusion, apelin signaling inhibits the ovarian and uterine apoptosis via estrogen signaling in the ovary and uterus.

Expression of Apelin and Apelin Receptor Protein in the Hypothalamo-Pituitary-Ovarian Axis during the Estrous Cycle of Mice.

INTRODUCTION: Apelin is an endogenous peptide, whose expression has been shown in the hypothalamus, pituitary, and ovary; furthermore, it is also called a neuropeptide, binding to apelin receptor (APJ) for various functions. It has been suggested that the hypothalamus, pituitary, and ovarian (HPO) axis is tightly regulated and factors and functions of the HPO axis can be modulated during the estrous cycle to influence reproductive status. To the best of our knowledge, the status of apelin and its receptor, APJ has not been investigated in the HPO axis during the estrous cycle. METHODS: To explore the expression of apelin and APJ in the HPO axis of mice during the estrous cycle, mice were divided into four groups: proestrus (Pro), estrus (Est), metestrus (Met), and diestrus (Di), and apelin and APJ were checked. Further, to explore the role of apelin in gonadotropin secretion, an in vitro study of the pituitary was performed at the Pro and Est stages. RESULT: The expression apelin and APJ in the hypothalamus showed elevation during the estrous cycle of postovulatory phases, Met, and Di. The immunolocalization of apelin and APJ in the anterior pituitary showed more abundance in the Est and Di. Our in vitro results showed that gonadotropin-releasing hormone agonist stimulated luteinizing hormone secretion was suppressed by the apelin 13 peptide from the pituitary of Pro and Est phases. This suggests an inhibitory role of apelin on gonadotropin secretion. The ovary also showed conspicuous changes in the presence of apelin and APJ during the estrous cycle. The expression of apelin and APJ coincides with folliculogenesis and corpus luteum formation and the expression of the apelin system in the different cell types of the ovary suggests its cell-specific role. Previous studies also showed that apelin has a stimulatory role in ovarian steroid secretion, proliferation, and corpus luteum. CONCLUSION: Overall our results showed that the apelin system changes along the HPO axis during the estrous cycle and might have an inhibitory at level of hypothalamus and pituitary and a stimulatory role at ovarian level.

Mechanistic study of copper nanoparticle (CuNP) toxicity on the mouse uterus via apelin signaling.

Copper nanoparticles (CuNPs) have been widely utilized in various applications. Due to its wider application, humans are at risk of its exposure. It has been reported that the exposure of CuNPs can lead to organ accumulation and affect organ toxicity. Recent study suggested that CuNPs can translocate into the uterus and affect uterine injury in rat, whereas uterine toxicity still remains unclear. The uterus is an important female organ which is required to sustain pregnancy. Thus, uterine structure and physiology are important. Therefore, this study hypothesized that CuNPs might have a toxic effect on the uterine features of mice. In this study, we have investigated the potential effects of CuNPs on the uterus of mice both in vivo and in vitro. In in vivo study, two groups of female mice were exposed to 5 and 50 mg/kg/day via oral exposure. In vivo results showed that CuNP treatment decreases the body weight and uterus weight and changes in antioxidant status with low estrogen and progesterone levels. Furthermore, CuNPs up-regulated the expression of caspase3 and down-regulated the expression of apelin receptor (APJ). Immunolocalization of apelin showed low abundance in the CuNP-treated uterus. These results suggest a poor apelin signaling in the uterus after CuNP treatment. The in vivo findings were further supported by the in vitro studies. Firstly, the uterus was cultured with 5 and 40 µg of CuNPs, and in the second in vitro experiment, the uterus was divided into 4 groups: control, 40 µg of CuNPs, 40 µg of CuNPs with apelin, and 40 µg of CuNPs with apelin receptor antagonist (ML221). In vitro study showed that CuNPs could directly induce the oxidative stress and apoptosis as well as changing antioxidant status in the uterus. The in vitro apelin 13 (APLN 13) treatments alleviated the expression of BCL2 and improved the antioxidant markers in CuNP-treated uterus. These results also provided an evidence of apelin-mediated signaling in the CuNP-treated uterus. In summary, our results present evidence that CuNPs can stimulate apoptotic pathways which may lead to uterine impairment due to weak apelin signaling.

Postnatal developmental expression and localization of apelin and apelin receptor protein in the ovary and uterus of mice.

Postnatal ovarian and uterine development is crucial to accomplished female fertility. Thus, the investigations of factors that present in pre-pubertal stages are important as it might be responsible for the regulation of ovarian and uterine function. Apelin, an adipokine and its receptor (APJ) are present in female reproductive organs. However, no study has reported its postnatal expression in uterus and ovary. Thus, we investigated the postnatal developmental changes in expression and localization of apelin and APJ in the ovary and uterus of mice. Postnatal ovary and uterus were collected from postnatal day (PND) 1, 7, 14, 21, 42, 65 and performed western blot analysis and immunohistochemistry. Uterine APJ is elevated in PND14 and PND65, whereas, ovarian APJ elevated in PND7, PND14, and PND65. Apelin expression in both ovary and uterus showed intense staining at PND65 and PND14. Our results showed that apelin and APJ abundance was lower at PND21 in uterus and ovary. In conclusion, apelin and APJ are developmentally regulated in the ovary and uterus, and its localization in the different compartments of ovary and uterus suggest its distribution specific physiological role in the uterus and ovary.

Effects of metformin on the uterus of d-galactose-induced aging mice: Histomorphometric, immunohistochemical localization (B-cell lymphoma 2, Bcl2-associated X protein, and active capase3), and oxidative stress study.

d-galactose (DG)-induced rodent aging model has widely been used for the study of age-related dysfunctions of various organs, including gonads and uterus. Antidiabetic drug metformin has gained an attention as antiaging drug in model organism and human but its effect on uterus has not been studied in relation to induced aging. Therefore, we investigated the effect of metformin on uterus of DG-induced aging mice model. Mice were randomly divided into three groups, that is, control (CN), DG-induced aging model and aging model treated with metformin. Histomorphometric results showed significantly decreased number of uterine glands, endometrial thickness, and increased luminal epithelium height in aging model. Furthermore, metformin resumed the number of uterine glands, endometrial thickness, and luminal epithelium height up to CN group. Metformin has also significantly decreased the age-associated oxidative stress (malondialdehyde and lipid hydroperoxide). Superoxide dismutase was significantly decreased in both treated groups compared to the CN group. However, catalase and glutathione peroxidase enzymes were significantly increased by metformin compared to the aging model. Immunostaining of active caspase3 and BAX were intense in the endometrium of aging model compare to CN- and metformin-treated groups. Localization of B-cell lymphoma 2 (Bcl2) showed intense immunostaining in the uterus of CN- and metformin-treated groups, with mild immunostaining in aging model. Our observations suggested that metformin treatment might be helpful for management of age-associated uterine dysfunctions. Moreover, it may be concluded that metformin might ameliorate uterine dysfunctions by reducing oxidative stress, suppressing apoptosis, and increasing the survival/antiapoptotic protein Bcl2.