Search

Visfatin/NAMPT (VIS), the hormone exerting a pleiotropic effect, is also perceived as an important factor in the regulation of reproductive processes and pregnancy maintenance. Previous studies confirmed its involvement in the control of porcine pituitary and ovary function. In this study, we hypothesized that VIS may affect the global transcriptome of luteal cells and thus regulate the functioning of the ovaries. Illumina's NovaSeq 6000 RNA sequencing was performed to investigate the differentially expressed genes (DEGs) and long non-coding RNAs (DELs) as well as the occurrence of differential alternative splicing events (DASs) in the porcine luteal cells exposed to VIS (100 ng/mL) during the implantation period. The obtained results revealed 170 DEGs (99 up- and 71 downregulated) assigned to 45 functional annotations. Moreover, we revealed 40 DELs, of which 3 were known and 37 were described for the first time. We identified 169 DASs events. The obtained results confirmed a significant effect of VIS on the transcriptome and spliceosome of luteal cells, including the genes involved in the processes crucial for successful implantation and pregnancy maintenance as angiogenesis, steroidogenesis, inflammation, cell development, migration, and proliferation. [ABSTRACT FROM AUTHOR]

Visfatin (VIS), also known as nicotinamide phosphoribosyltransferase (NAMPT), is the rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Recently, VIS has been also recognized as an adipokine. Our previous study revealed that VIS is produced in the anterior and posterior lobes of the porcine pituitary. Moreover, the expression and secretion of VIS are dependent on the phase of the estrous cycle and/or the stage of early pregnancy. Based on this, we hypothesized that VIS may regulate porcine pituitary function. This study was conducted on anterior pituitary (AP) glands harvested from pigs during specific phases of the estrous cycle. We have shown the modulatory effect of VIS in vitro on LH and FSH secretion by porcine AP cells (determined by ELISA). VIS was also found to stimulate cell proliferation (determined by Alamar Blue) without affecting apoptosis in these cells (determined using flow cytometry technique). Moreover, it was indicated that VIS may act in porcine AP cells through the INSR, AKT/PI3K, MAPK/ERK1/2, and AMPK signaling pathways (determined by ELISA or Western Blot). This observation was further supported by the finding that simultaneous treatment of cells with VIS and inhibitors of these pathways abolished the observed VIS impact on LH and FSH secretion (determined by ELISA). In addition, our research indicated that VIS affected the mentioned processes in a manner that was dependent on the dose of VIS and/or the phase of the estrous cycle. Thus, these findings suggest that VIS may regulate the functioning of the porcine pituitary gland during the estrous cycle. [ABSTRACT FROM AUTHOR]

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder with disrupted uterus structure and function. A positive effect of vitamin D3 (VD3) in female reproduction was observed. Chemerin (RARRES2) and adiponectin (ADIPOQ) are the main adipokines whose levels are altered in PCOS patients. Therefore, the aim of this study was to investigate the impact of VD3 supplementation on RARRES2 and ADIPOQ levels in the uterus of PCOS rats. Methods: We analyzed the plasma levels and uterine transcript and protein expression of RARRES2 and ADIPOQ and their receptors (CCRL2, CMKLR1, GPR1, and ADIPOR1 and ADIPOR2, respectively) in rats with letrozole-induced PCOS. Results: In control animals, VD3 did not change plasma levels of both adipokines, while in PCOS rats supplemented with VD3, they returned to control levels. The expression of RARRES2 and all investigated receptors increased in the uterus of VD3-treated rats; however, the levels of Rarres2 and Gpr1 genes remained unchanged. VD3 supplementation decreased RARRES2, CMKLR1, and GPR1 but increased CCRL2 level to the control value. In the uterus of VD3-treated rats, the transcript and protein levels of ADIPOQ and both receptors ADIPOR1 increased. At the same time, VD3 supplementation induced an increase in Adipoq, Adipor1, and Adipor2 gene expression and restored protein levels to control level values. Conclusions: our findings indicate a new mechanism of VD3 action in the uterine physiology of PCOS rats. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a hematological malignancy characterized by excessive proliferation of abnormal myeloid precursors accompanied by a differentiation block and inhibition of apoptosis. Increased expression of an anti-apoptotic MCL-1 protein was shown to be critical for the sustained survival and expansion of AML cells. Therefore, herein, we examined the pro-apoptotic and pro-differentiating effects of S63845, a specific inhibitor of MCL-1, in a single-agent treatment and in combination with BCL-2/BCL-XL inhibitor, ABT-737, in two AML cell lines: HL-60 and ML-1. Additionally, we determined whether inhibition of the MAPK pathway had an impact on the sensitivity of AML cells to S63845. To assess AML cells' apoptosis and differentiation, in vitro studies were performed using PrestoBlue assay, Coulter electrical impedance method, flow cytometry, light microscopy and Western blot techniques. S63845 caused a concentration-dependent decrease in the viability of HL-60 and ML-1 cells and increased the percentage of apoptotic cells. Combined treatment with S63845 and ABT-737 or MAPK pathway inhibitor enhanced apoptosis but also induced differentiation of tested cells, as well as altering the expression of the MCL-1 protein. Taken together, our data provide the rationale for further studies regarding the use of MCL-1 inhibitor in combination with other pro-survival protein inhibitors. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are one of the most prevalent classes of environmental pollutants. Some evidence shows that PAHs could be involved in human obesity. However, little is known about the distribution patterns of PAHs in human adipose tissue (AT) and the role of PAHs on adipogenesis/lipogenesis. The aims of this pilot study were to determine concentrations of 16 PAHs defined as high-priority pollutants in the plasma and adipose tissue of French and Polish bariatric patients, as well as their correlation with body mass index (BMI), plasma and AT adipokines expression levels. We finally investigated the role of naphthalene on cell proliferation, viability, and differentiation in 3T3-L1 preadipocytes. The concentration of most PAHs was similar in the three types of AT and it was significantly higher in AT as compared to plasma, suggesting bioaccumulation. Polish patients had higher PAH levels in AT than French ones. Only the concentration of naphthalene in AT was positively correlated with the BMI and serum or adipose chemerin, adiponectin and resistin expression, in French but not in Polish patients, who had significantly higher BMIs. Moreover, naphthalene exposure increased the cell proliferation of 3T3-L1 preadipocytes and lipogenesis, and increased the expression of genes involved in adipogenesis after cell differentiation. Taken together, PAHs and more particularly naphthalene could be an obesogenic molecule and increase the risk of obesity. [ABSTRACT FROM AUTHOR]

The apelinergic system comprises two peptide ligands, apelin and ELABELA, and their cognate G-protein-coupled receptor, the apelin receptor APJ. Apelin is a peptide that was isolated from bovine stomach extracts; the distribution of the four main active forms, apelin-36, -17, -13, and pyr-apelin-13 differs between tissues. The mature form of ELABELA-32 can be transformed into forms called ELABELA-11 or -21. The biological function of the apelinergic system is multifaceted, and includes the regulation of angiogenesis, body fluid homeostasis, energy metabolism, and functioning of the cardiovascular, nervous, respiratory, digestive, and reproductive systems. This review summarises the mechanism of the apelinergic system in cell apoptosis. Depending on the cell/tissue, the apelinergic system modulates cell apoptosis by activating various signalling pathways, including phosphoinositide 3-kinase (PI3K), extracellular signal-regulated protein kinase (ERK1/2), protein kinase B (AKT), 5'AMP-activated protein kinase(AMPK), and protein kinase A (PKA). Apoptosis is critically important during various developmental processes, and any dysfunction leads to pathological conditions such as cancer, autoimmune diseases, and developmental defects. The purpose of this review is to present data that suggest a significant role of the apelinergic system as a potential agent in various therapies. [ABSTRACT FROM AUTHOR]

The corpus luteum is a small gland of great importance because its proper functioning determines not only the appropriate course of the estrous/menstrual cycle and embryo implantation, but also the subsequent maintenance of pregnancy. Among the well-known regulators of luteal tissue functions, increasing attention is focused on the role of neuropeptides and adipose tissue hormones—adipokines. Growing evidence points to the expression of these factors in the corpus luteum of women and different animal species, and their involvement in corpus luteum formation, endocrine function, angiogenesis, cells proliferation, apoptosis, and finally, regression. In the present review, we summarize the current knowledge about the expression and role of adipokines, such as adiponectin, leptin, apelin, vaspin, visfatin, chemerin, and neuropeptides like ghrelin, orexins, kisspeptin, and phoenixin in the physiological regulation of the corpus luteum function, as well as their potential involvement in pathologies affecting the luteal cells that disrupt the estrous cycle. [ABSTRACT FROM AUTHOR]

The apelinergic system, which includes the apelin receptor (APJ) as well as its two specific ligands, namely apelin and ELABELA (ELA/APELA/Toddler), have been the subject of many recent studies due to their pleiotropic effects in humans and other animals. Expression of these factors has been investigated in numerous tissues and organs—for example, the lungs, heart, uterus, and ovary. Moreover, a number of studies have been devoted to understanding the role of apelin and the entire apelinergic system in the most important processes in the body, starting from early stages of human life with regulation of placental function and the proper course of pregnancy. Disturbances in the balance of placental processes such as proliferation, apoptosis, angiogenesis, or hormone secretion may lead to specific pregnancy pathologies; therefore, there is a great need to search for substances that would help in their early diagnosis or treatment. A number of studies have indicated that compounds of the apelinergic system could serve this purpose. Hence, in this review, we summarized the most important reports about the role of apelin and the entire apelinergic system in the regulation of placental physiology and pregnancy. [ABSTRACT FROM AUTHOR]

Proper functioning of the body depends on hormonal homeostasis. White adipose tissue is now known as an endocrine organ due to the secretion of multiple molecules called adipokines. These proteins exert direct effects on whole body functions, including lipid metabolism, angiogenesis, inflammation, and reproduction, whereas changes in their level are linked with pathological events, such as infertility, diabetes, and increased food intake. Vaspin-visceral adipose tissue-derived serine protease inhibitor, or SERPINA12 according to serpin nomenclature, is an adipokine discovered in 2005 that is connected to the development of insulin resistance, obesity, and inflammation. A significantly higher amount of vaspin was observed in obese patients. The objective of this review was to summarize the latest findings about vaspin expression and action in endocrine tissues, such as the hypothalamus, pituitary gland, adipose tissue, thyroid, ovary, placenta, and testis, as well as discuss the link between vaspin and pathologies connected with hormonal imbalance. [ABSTRACT FROM AUTHOR]

Previously, we demonstrated the expression of apelin and G-protein-coupled receptor APJ in human placenta cell lines as well as its direct action on placenta cell proliferation and endocrinology. The objective of this study was to examine the effect of apelin on placenta apoptosis in BeWo cells and villous explants from the human third trimester of pregnancy. The BeWo cells and villous explants were incubated with apelin (2 and 20 ng/mL) alone or with staurosporine for 24 to 72 h. First, we analysed the dose- and time-dependent effect of apelin on the expression of apoptotic factors on the mRNA level by real-time PCR and on the protein level using Western blot. Next, we checked caspase 3 and 7 activity by Caspase-Glo 3/7, DNA fragmentation by the Cell Death Detection ELISA kit and oxygen consumption by the MitoXpress-Xtra Oxygen Consumption assay. We found that apelin increased the expression of pro-survival and decreased proapoptotic factors on mRNA and protein levels in both BeWo cells and villous explants. Additionally, apelin inhibited caspase 3 and 7 activity and DNA fragmentation in staurosporine-induced apoptosis as also attenuated oxidative stress by increasing extracellular oxygen consumption. The antiapoptotic effect of apelin in BeWo cells was mediated by the APJ receptor and mitogen-activated protein kinase (ERK1/2/MAP3/1) and protein kinase B (AKT). The obtained results showed the antiapoptotic effect of apelin on trophoblast cells, suggesting its participation in the development of the placenta. [ABSTRACT FROM AUTHOR]

Oocyte maturation is a critical stage in embryo production and female reproduction. The aims of this study were to determine: (i) the mRNA and protein expression of vaspin and its receptor 78-kDa glucose-regulated (GRP78) in porcine cumulus–oocyte complexes (COCs) by real-time PCR and Western blot analysis, respectively, and their localisation by immunofluorescence; and (ii) the effects of vaspin on in vitro oocyte maturation (IVM) and the involvement of mitogen ERK1/2 (MAP3/1)- and AMPKα (PRKAA1)-activated kinases in the studied processes. Porcine COCs were matured in vitro for 22 h or 44 h with vaspin at a dose of 1 ng/mL and nuclear maturation assessed by Hoechst 33342 or DAPI staining and the measurement of progesterone (P4) level in the maturation medium. We showed that vaspin and GRP78 protein expression increased in oocytes and cumulus cells after IVM. Moreover, vaspin enhanced significantly porcine oocyte IVM and P4 concentration, as well as MAP3/1 phosphorylation, while decreasing PRKAA1. Using pharmacological inhibitors of MAP3/1 (PD98059) and PRKAA1 (Compound C), we observed that the effect of vaspin was reversed to the control level by all studied parameters. In conclusion, vaspin, by improving in vitro oocyte maturation via MAP3/1 and PRKAA1 kinase pathways, can be a new factor to improve in vitro fertilisation protocols. [ABSTRACT FROM AUTHOR]

Phoenixin (PNX) neuropeptide is a cleaved product of the Smim20 protein. Its most common isoforms are the 14- and 20-amino acid peptides. The biological functions of PNX are mediated via the activation of the GPR173 receptor. PNX plays an important role in the central nervous system (CNS) and in the female reproductive system where it potentiates LH secretion and controls the estrus cycle. Moreover, it stimulates oocyte maturation and increases the number of ovulated oocytes. Nevertheless, PNX not only regulates the reproduction system but also exerts anxiolytic, anti-inflammatory, and cell-protective effects. Furthermore, it is involved in behavior, food intake, sensory perception, memory, and energy metabolism. Outside the CNS, PNX exerts its effects on the heart, ovaries, adipose tissue, and pancreatic islets. This review presents all the currently available studies demonstrating the pleiotropic effects of PNX. [ABSTRACT FROM AUTHOR]

Formation and limited lifespan of corpus luteum (CL) are important for proper ovarian periodicity and fertility. Failed vascularization, imbalance between proliferation and apoptosis leads to luteal phase deficiency and infertility. The aim of this study was to examine the effect of vaspin on angiogenesis, apoptosis and proliferation as well as the involvement of 78-kDa glucose-regulated protein receptor (GRP78) and mitogen-activated kinase (MAP3/1) in these processes. Porcine luteal cells were incubated with vaspin (0.1–10 ng/mL) for 24 h to 72 h and then mRNA and protein expression of angiogenesis: vascular endothelial growth factor (VEGFA), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), VEGFA receptors (VEGFR1, VEGFR2), apoptosis: caspase 3, bcl-2-like protein 4 (BAX), B-cell lymphoma (BCL2), and proliferation: proliferating cells nuclear antigen (PCNA), cyclin A factors as well as secretion of VEGFA, FGF2, ANGT1 were measured by real-time polymerase chain reaction (PCR), immunoblotting and enzyme-linked immunosorbent assay (ELISA), respectively. Moreover, apoptosis was assessed by caspase activity using the Caspase-Glo 3/7 assay, while proliferation was by alamarBlue. We found that vaspin enhanced luteal cell angiogenesis, proliferation, and significantly decreased apoptosis. Additionally, using GRP78 siRNA and the pharmacological inhibitor of MAP3/1 (PD98059), we observed that the effect of vaspin was reversed to the control level in all investigated processes. Taken together, our results suggest that vaspin is a new regulator of female fertility by direct regulation of CL formation and maintenance of luteal cell function. [ABSTRACT FROM AUTHOR]

Adipokines influence energy metabolism and have effects on male reproduction, including spermatogenesis and/or Sertoli cell maturation; however, the relationship between these active proteins and androgens in testicular cells is limited. Here, we studied the impact of short-term exposure to flutamide (an anti-androgen that blocks androgen receptors) on the expression of chemerin, apelin, vaspin and their receptors (CCRL2, CMKLR1, GPR1, APLNR, GRP78, respectively) in adult rat testes. Moreover, the levels of expression of lipid metabolism-modulating proteins (PLIN1, perilipin1; TSPO, translocator protein) and intercellular adherens junction proteins (nectin-2 and afadin) were determined in testicular cells. Plasma levels of adipokines, testosterone and cholesterol were also evaluated. Gene expression techniques used included the quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) and immunohistochemistry (IHC). The androgen-mediated effects observed post-flutamide treatment were found at the gonadal level as chemerin, apelin, and vaspin gene expression alterations at mRNA and protein levels were detected, whereas the cellular targets for these adipokines were recognised by localisation of respective receptors in testicular cells. Plasma concentrations of all adipokines were unchanged, whereas plasma cholesterol content and testosterone level increased after flutamide exposure. Differential distribution of adipokine receptors indicates potential para- or autocrine action of the adipokines within the rat testes. Additionally, changes in the expression of PLIN1 and TSPO, involved in the initial step of testosterone synthesis in Leydig cells, suggest that testicular cells represent a target of flutamide action. Increase in the gene expression of PLIN1 and TSPO and higher total plasma cholesterol content indicates enhanced availability of cholesterol in Leydig cells as a result of androgen-mediated effects of flutamide. Alterations in adherens junction protein expression in the testis confirm the flutamide efficacy in disruption of androgen signalling and presumably lead to impaired para- and autocrine communication, important for proper functioning of adipokines. [ABSTRACT FROM AUTHOR]

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis–controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors (n = 20) were obtained from patients (31–45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients (n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy. [ABSTRACT FROM AUTHOR]

Vaspin, a visceral adipose tissue-derived serine protease inhibitor, is expressed in the porcine ovary; it induces the activation of various kinases and steroidogenesis. The aim of this study was to examine the effect of vaspin on granulosa (Gc) proliferation, cell cycle regulation, and apoptosis. Porcine Gc was incubated with vaspin (0.01–10 ng/mL) for 24 to 72 h, proliferation was measured using alamarBlue assay, cell cycle progression was assessed using flow cytometry, and cyclin (D, E, and A) protein expression was measured using immunoblotting. Apoptosis was assessed by measuring caspase activity using Caspase-glo 3/7 assay. Furthermore, histone-associated DNA fragments levels were measured using a cell-death detection ELISA; BAX (bcl-2-like protein 4), BCL2 (B-cell lymphoma 2), caspases (-3, -8, and -9), p53 mRNA, and protein expression were assessed using real time PCR and immunoblotting. We found that vaspin significantly enhanced Gc proliferation and cell cycle progression into the S and G2/M phases and decreased apoptosis. We observed that siRNA silencing of the glucose-regulated protein (GRP78) receptor and pharmacological inhibitors of mitogen-activated kinase (MAP3/1/ERK1/2), Janus kinase (STAT3) and protein kinase B (AKT) blocked the ability of vaspin cell proliferation and enhanced caspase-3/7 activities. These results suggest that vaspin via mitogenic effect on porcine Gc acts as a new regulator of ovarian growth, development, or folliculogenesis. [ABSTRACT FROM AUTHOR]

Adiponectin is the most abundant plasma adipokine. It mainly derives from white adipose tissue and plays a key role in the control of energy metabolism thanks to its insulin-sensitising, anti-inflammatory, and antiatherogenic properties. In vitro and in vivo evidence shows that adiponectin could also be one of the hormones controlling the interaction between energy balance and fertility in several species, including humans. Indeed, its two receptors—AdipoR1 and AdipoR2—are expressed in hypothalamic–pituitary–gonadal axis and their activation regulates Kiss, GnRH and gonadotropin expression and/or secretion. In male gonads, adiponectin modulates several functions of both somatic and germ cells, such as steroidogenesis, proliferation, apoptosis, and oxidative stress. In females, it controls steroidogenesis of ovarian granulosa and theca cells, oocyte maturation, and embryo development. Adiponectin receptors were also found in placental and endometrial cells, suggesting that this adipokine might play a crucial role in embryo implantation, trophoblast invasion and foetal growth. The aim of this review is to characterise adiponectin expression and its mechanism of action in male and female reproductive tract. Further, since features of metabolic syndrome are associated with some reproductive diseases, such as polycystic ovary syndrome, gestational diabetes mellitus, preeclampsia, endometriosis, foetal growth restriction and ovarian and endometrial cancers, evidence regarding the emerging role of adiponectin in these disorders is also discussed. [ABSTRACT FROM AUTHOR]

The apelinergic system comprises two peptide ligands, apelin and ELABELA, and their cognate G-protein-coupled receptor, the apelin receptor APJ. Apelin is a peptide that was isolated from bovine stomach extracts; the distribution of the four main active forms, apelin-36, -17, -13, and pyr-apelin-13 differs between tissues. The mature form of ELABELA-32 can be transformed into forms called ELABELA-11 or -21. The biological function of the apelinergic system is multifaceted, and includes the regulation of angiogenesis, body fluid homeostasis, energy metabolism, and functioning of the cardiovascular, nervous, respiratory, digestive, and reproductive systems. This review summarises the mechanism of the apelinergic system in cell apoptosis. Depending on the cell/tissue, the apelinergic system modulates cell apoptosis by activating various signalling pathways, including phosphoinositide 3-kinase (PI3K), extracellular signal-regulated protein kinase (ERK1/2), protein kinase B (AKT), 5'AMP-activated protein kinase(AMPK), and protein kinase A (PKA). Apoptosis is critically important during various developmental processes, and any dysfunction leads to pathological conditions such as cancer, autoimmune diseases, and developmental defects. The purpose of this review is to present data that suggest a significant role of the apelinergic system as a potential agent in various therapies.

The apelinergic system, which includes the apelin receptor (APJ) as well as its two specific ligands, namely apelin and ELABELA (ELA/APELA/Toddler), have been the subject of many recent studies due to their pleiotropic effects in humans and other animals. Expression of these factors has been investigated in numerous tissues and organs-for example, the lungs, heart, uterus, and ovary. Moreover, a number of studies have been devoted to understanding the role of apelin and the entire apelinergic system in the most important processes in the body, starting from early stages of human life with regulation of placental function and the proper course of pregnancy. Disturbances in the balance of placental processes such as proliferation, apoptosis, angiogenesis, or hormone secretion may lead to specific pregnancy pathologies; therefore, there is a great need to search for substances that would help in their early diagnosis or treatment. A number of studies have indicated that compounds of the apelinergic system could serve this purpose. Hence, in this review, we summarized the most important reports about the role of apelin and the entire apelinergic system in the regulation of placental physiology and pregnancy.