Findings indicate that adropin may regulate porcine follicular development through its actions on Gc and oocytes through its actions on gonadotropin and steroid treatment.
Abstract
Adropin, a peptide involved in energy homeostasis, acts via G protein-coupled receptor 19 (GPR19). This study examined the expression of adropin/GPR19 in porcine ovarian follicles and assessed the effects of adropin on granulosa cells (Gc) function and oocyte maturation in vitro. Levels of adropin/GPR19 were analyzed in porcine ovarian follicles and follicular fluid during the estrous cycle. Then, Gc isolated from medium-sized follicles were cultured with gonadotropins, steroids, or adropin (0.1-100 nM) to evaluate adropin/GPR19 expression, Gc function, and signaling pathways using pharmacological inhibitors. Additionally, the effect of adropin on oocyte maturation was assessed after in vitro maturation of cumulus-oocyte complexes from prepubertal pigs. We noted that adropin expression increased in the whole porcine ovarian follicle, while GPR19 decreased during the estrous cycle. Adropin protein expression and secretion to culture medium and GPR19 protein increased with gonadotropin and steroid treatment. Both proteins are localized to Gc and theca cells. Functionally, adropin reduced Gc viability via AKT signaling, inhibited proliferation and cell cycle progression via ERK1/2, suppressed estradiol secretion and CYP19A1 expression via PKA, increased progesterone secretion, cleaved caspase-3 expression and its activity, and inhibited autophagy-related markers. Adropinsimultaneously enhancing oocyte maturation, progesterone secretion, and lipid utilization in cumulus-oocyte complexes. These findings indicate that adropin may regulate porcine follicular development through its actions on Gc and oocytes.
Adropin is a new protein that regulates energy homeostasis. The serum and follicular fluid (FF) levels of adropin are decreased in women with polycystic ovarian syndrome (PCOS); however, its role in ovarian function is unknown. The aims were to determine the expression of adropin and its receptor G protein-coupled receptor 19 (GPR19) in human granulosa cells (GC), its immunolocalization and its in vitro effects on GC function. Blood plasma, FF and GC samples were obtained from normal weight, obese and diagnosed with or without PCOS women (n=8). The in vitro effects of adropin on GC proliferation, apoptosis, cell cycle progression, steroidogenesis were analyzed. The results revealed that adropin plasma concentration was decreased in obese patients, with a similar reduction observed in obese patients with PCOS, while GPR19 expression was decreased in the GC of obese and PCOS women, as well as in obese PCOS patients. We noted that in all investigated patients groups adropin reduced GC proliferation and cell cycle progression, negatively influenced steroidogenesis enzyme levels and promoted apoptosis. Such disruptions in GC function are likely to impair ovarian follicular maturation and contribute to the subfertility commonly observed in PCOS. Such alterations may ultimately affect oocyte competence and ovarian responsiveness, parameters that are clinically relevant for in vitro fertilization outcomes. Our findings suggest that adropin may act as a novel regulator of ovarian function and could contribute to the pathophysiology of PCOS, highlighting its potential clinical value as a marker of altered ovarian follicular function in affected women.
Patrycja Kurowska, M. Dawid, N. Respekta-Długosz et al.· Journal of Endocrinology· 0 citations
Neuromedin B (NMB), a neuropeptide, is increasingly recognized as a potential regulator of female reproductive processes. However, its role in porcine ovarian function and follicular development remains unclear. Given the indispensable roles of ovarian granulosa cells (GCs) in follicular growth, endocrine homeostasis, and oocyte maturation, this study investigated the expression pattern of NMB in porcine ovaries and explored the effects of NMB knockdown on GC function and the underlying mechanisms. The results indicated that NMB was expressed in porcine follicles at all development stages (<3 mm, 3-5 mm, >5 mm), with its expression level increasing with follicle diameter; furthermore, NMB was primarily expressed in GCs. Functional assays further revealed that NMB knockdown reduced cell viability and suppressed the expression of Cyclin B1 and PCNA; induced cell apoptosis by upregulating Bax and Caspase-3 expression while downregulating Bcl-2 expression; impaired E2 and P4 synthesis by reducing STAR, CYP11A1, HSD3B1, and CYP19A1 expression. Mechanistically, NMB knockdown compromised mitochondrial Ca2+ uptake and disrupted PKA/CREB signaling, as reflected by decreased expression levels of total and phosphorylated PKA and CREB proteins. Importantly, the functional impairments induced by NMB knockdown were alleviated by exogenous NMB supplementation. Collectively, these findings suggest that NMB may serve as an important regulator of cell viability, apoptosis and steroidogenesis through mitochondrial Ca2+ uptake and the PKA/CREB axis in GCs, thereby providing new mechanistic insights into follicular development and potential targets for improving porcine reproductive efficiency.
Hongxia Li, Guangzhen Chen, Jia Li et al.· Theriogenology· 0 citations
Oocyte maturation is a critical stage in embryo development and female reproduction, and is regulated by adipokines- hormones produced by adipose tissue. Omentin-1 (ITLN1) is an adipokine known to regulate ovarian follicle function, however, its role in oocyte maturation remains unclear. This study investigated ITLN1 expression in porcine oocytes and cumulus cells before and after in vitro maturation (RT-qPCR, Western blotting, immunofluorescence) and the effect of ITLN1 on: oocyte maturation by assessing the percentage of oocytes in metaphase II (M2) (DAPI) and progesterone (P4) secretion (ELISA); glucose metabolism, including glucose transporter type 4 (GLUT4) expression, glucose and lactate secretion, and mitochondrial function (Seahorse Test); lipid metabolism, as assessed by lipid droplet area (Nile Red) and adipose differentiation-related protein and adipose triglyceride lipase (ATGL) expression; extracellular signal-regulated kinase 1/2 (ERK1/2) and AMP-activated protein kinase alpha 1 (PRKAA1) phosphorylation, which mediating the effects of ITLN1 on oocyte maturation. Moreover, the effect of ITLN1 on preimplantation embryo development was also examined. ITLN1 expression increased after maturation in both oocytes and cumulus cells, with strong immunofluorescence signals in both cells. ITLN1 increased oocyte maturation to the M2 stage and P4 secretion. It enhanced glucose metabolism by upregulating GLUT4 and reducing glucose and lactate levels, improved mitochondrial function, reduced lipid droplet area, upregulated ATGL expression, and stimulated ERK1/2 and PRKAA1 phosphorylation. However, ITLN1 had no significant effect on preimplantation embryo development. In conclusion, ITLN1 positively regulates porcine oocyte maturation in vitro through coordinated modulation of energy metabolism, mitochondrial activity, lipid utilization, and ERK1/2-PRKAA1 signalling.
K. Kubicka, N. Respekta-Długosz, Patrycja Kurowska et al.· Reproduction· 0 citations
This study investigates the regulatory role of p21-activated protein kinase 1 (PAK1) in estrogen secretion during ovulation induction in Bactrian camels, along with the associated molecular mechanisms. We evaluated the morphological differences of follicles between ovulatory and non-ovulatory camels through rectal examinations and B-ultrasound imaging. Using iTRAQ quantitative proteomics, we identified a significant upregulation of PAK1 protein in the ovarian tissues of the ovulatory group, followed by a bioinformatics analysis to explore its biological functions. Our results revealed distinct tissue-specific expression and distribution patterns of PAK1 within the hypothalamic-pituitary-gonadal (HPG) axis. In in vitro cultured ovarian granulosa cells, FTY720-induced activation of PAK1 significantly increased cell viability, reduced apoptosis rates, and decreased the expression of apoptosis-related molecules. Additionally, PAK1 activation enhanced the expression of key enzymes and receptors involved in estrogen synthesis, promoting estradiol secretion. Conversely, inhibiting PAK1 with IPA-3 resulted in opposing effects, exacerbating granulosa cell apoptosis and reducing estrogen synthesis. Overall, this study demonstrates that PAK1 is essential for regulating estrogen secretion by modulating granulosa cell apoptosis and the expression of key steroidogenic enzymes, ultimately influencing ovulation induction in Bactrian camels. These findings improve our understanding of the reproductive regulatory mechanisms in camels and provide a molecular framework for further investigations into ovulation induction and enhancements in reproductive performance.
Qi Wang, Tingting Ge, Caihui Si et al.· Journal of Steroid Biochemis...· 0 citations
The data demonstrate that MRO is poised to play a role in the coordination of periovulatory events through the EGF and progesterone signaling pathways through the EGF and progesterone pathways.
Ketan Shrestha, Patrick R. Hannon, M. Wynn et al.· Biology of Reproduction· 0 citations
Intrafollicular transfer of immature oocytes (IFIOT) is a promising embryo production strategy, but outcomes remain poor and inconsistent. We investigated the effects of the injection procedure and the number of injected cumulus-oocyte complexes (COCs) on the biochemical and molecular environment of follicles. First, dominant follicles from hormonally synchronized heifers were either not injected (NIFIOT), injected with medium only (IFIOTM), or injected with medium containing COCs (IFIOT-COCs). Follicular fluid (FF) was analyzed for cell-free DNA (cfDNA), oxidative stress markers (GPx activity and FRAP), steroid hormones, and metabolomic profiles using UPLC-HRMS. Injection procedure increased cfDNA levels and altered steroidogenesis, with higher progesterone levels, lower estradiol levels, and lower E2/P4 ratios in the injected follicles. Metabolomic analysis revealed changes in amino acid and anti-inflammatory pathways on the injected follicle compared to the non injected. In addition, follicles were injected with 25 or 50 COCs (IFIOT25 and IFIOT50) to assess the impact of oocyte number on the follicular environment. IFIOT25 follicles showed higher GPx activity and estradiol concentrations compared to IFIOT50, whereas IFIOT50 follicles exhibited the downregulation of redox- and stress-related genes (SOD1, GPX4, NFE2L2, HSP70, and CASP3) and lower embryo yield compared to IFIOT25 (42 vs 52%, respectively). Metabolomic analysis indicated alterations in lipid and energy metabolism, with key metabolites (creatine, l-acetylcarnitine, dodecanoic acid, and panthenol) reduced in the IFIOT50 group, suggesting impaired metabolic homeostasis at higher oocyte density. Overall, both the injection process and oocyte number influenced follicular physiology, potentially affecting oocyte quality, highlighting the need to refine IFIOT protocols.
N. R. Kussano, O. Faria, L. Pimenta et al.· Biology of Reproduction· 0 citations