Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

In vitro effect of visfatin on endocrine functions of the porcine corpus luteum.

Previously, we demonstrated the expression of visfatin in porcine reproductive tissues and its effect on pituitary endocrinology. The objective of this study was to examine the visfatin effect on the secretion of steroid (P4, E2) and prostaglandin (PGE2, PGF2α), the mRNA and protein abundance of steroidogenic markers (STAR, CYP11A1, HSD3B, CYP19A1), prostaglandin receptors (PTGER2, PTGFR), insulin receptor (INSR), and activity of kinases (MAPK/ERK1/2, AKT, AMPK) in the porcine corpus luteum. We noted that the visfatin effect strongly depends on the phase of the estrous cycle: on days 2-3 and 14-16 it reduced P4, while on days 10-12 it stimulated P4. Visfatin increased secretion of E2 on days 2-3, PGE2 on days 2-3 and 10-12, reduced PGF2α release on days 14-16, as well as stimulated the expression of steroidogenic markers on days 10-12 of the estrous cycle. Moreover, visfatin elevated PTGER mRNA expression and decreased its protein level, while we noted the opposite changes for PTGFR. Additionally, visfatin activated ERK1/2, AKT, and AMPK, while reduced INSR phosphorylation. Interestingly, after inhibition of INSR and signalling pathways visfatin action was abolished. These findings suggest a regulatory role of visfatin in the porcine corpus luteum.

Autophagy favors survival of corpora lutea during the long-lasting pregnancy of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

The corpus luteum (CL) is a transient endocrine gland that plays a crucial role in establishing and maintaining pregnancy. Although autophagy and apoptosis have been suggested as cooperative mechanisms, their interaction within the CL of pregnant mammals has not been thoroughly investigated. To understand the collaborative function of autophagy and apoptosis in the CL, we analyzed both mechanisms during pregnancy in the South American plains vizcacha, Lagostomus maximus. This rodent undergoes a decline in progesterone levels during mid-gestation, a reactivation of the hypothalamus-hypophysis-gonadal axis, and the incorporation of new functional secondary CL. Our analysis of autophagy markers BECLIN 1 (BECN1), SEQUESTOSOME1 (SQSTM1), Microtubule-associated protein light chain 3 (LC3B), and lysosomal-associated membrane protein 1 (LAMP1) and anti- and pro-apoptotic markers BCL2 and ACTIVE CASPASE 3 (A-C3) revealed interactive behaviors between both processes. Healthy primary and secondary CL exhibited positive expression of BECN1, SQSTM1, LC3B, and LAMP1, while regressed CL displayed enhanced expression of these autophagy markers along with nuclear A-C3. Transmission electron microscopy revealed a significant formation of autophagic vesicles in regressed CL during full-term pregnancy, whereas healthy CL exhibited a low number of autophagy vesicles. The co-localization between LC3B and SQSTM1 and LC3B with LAMP1 was observed in both healthy and regressed CL during pregnancy, while co-localization of BECN1 and BCL2 was only detected in healthy CL. LC3B and ACTIVE CASPASE 3 co-localization were detected in a subset of luteal cells within the regressing CL. We propose that autophagy could act as a survival mechanism in the CL, allowing the pregnancy to progress until full-term, while also serving as a mechanism to eliminate remnants of regressed CL, thereby providing the necessary space for subsequent follicular maturation.

Expression dynamics of adipokines during induced ovulation in the bovine follicles and early corpus luteum.

The study aimed to assess the local gene expression of adipokine members, namely vaspin, adiponectin, visfatin, resistin and their associated receptors - heat shock 70 protein 5 (HSPA5), adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) - in bovine follicles during the preovulatory period and early corpus luteum development. Follicles were collected before gonadotropin-releasing hormone (GnRH) treatment (0 h) and at 4, 10, 20, 25 and 60 h after GnRH application through transvaginal ovariectomy (n = 5 samples/group). Relative mRNA expression levels were quantified using real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin exhibited high mRNA levels immediately 4 h after GnRH application, followed by a significant decrease. Adiponectin mRNA levels were elevated at 25 h after GnRH treatment. AdipoR2 exhibited late-stage upregulation, displaying increased expression at 20, 25 and 60 h following GnRH application. Visfatin showed upregulation at 20 h post-GnRH application. In conclusion, the observed changes in adipokine family members within preovulatory follicles, following experimentally induced ovulation, may constitute crucial components of the local mechanisms regulating final follicle growth and development.

Adropin may regulate ovarian functions by improving antioxidant potential in adult mouse.

The corpus luteum (CL) is a temporary endocrine gland that synthesizes progesterone. The luteal progesterone plays a central role in the regulation of the estrous cycle as well as the implantation and maintenance of pregnancy. Our previous study showed the expression of adropin and its receptor, GPR19, in the luteal cells and its significant role in luteinization. The aim of the present study was to investigate the in vitro effect of adropin on hCG-induced ovarian functions in adult mice. We also evaluated the effect of exogenous treatment with adropin on ovarian steroidogenesis and anti-oxidant parameters, with special emphasis on CL function. Our results demonstrated that adropin acts synergistically with hCG to promote ovarian steroidogenesis and survival by increasing the expression of StAR, 3ß-HSD, and aromatase proteins and decreasing the BAX/BCL2 ratio. Exogenous adropin treatment increased progesterone production by increasing the expression of GPR19, StAR and 3ß-HSD enzymes in the mouse ovary. Also, adropin inhibited the luteal oxidative stress by increasing nuclear translocation of NRF-2 in CL, which resulted in increased HO-1 expression and SOD, catalase activity. Decreased oxidative stress might inhibit the translocation of NF-κB into the nucleus of luteal cells, resulting into increased survival and decreased apoptosis, as evident by decreased lipid peroxidation, BAX/BCL2 ratio, caspase 3, active caspase 3 expression, and TUNEL-positive cells in adropin treated mice. Our findings suggest that adropin can be a promising candidate that can enhance the survivability of the CL.

Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep.

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Corpus luteum presence in the bovine ovary increase intrafollicular progesterone concentration: consequences in follicular cells gene expression and follicular fluid small extracellular vesicles miRNA contents.

BACKGROUND: It is well described that circulating progesterone (P4) plays a key role in several reproductive events such as oocyte maturation. However, during diestrus, when circulating P4 is at the highest concentrations, little is known about its local impact on the follicular cells such as intrafollicular P4 concentration due to corpus luteum (CL) presence within the same ovary. Based on that, our hypothesis is that the CL presence in the ovary during diestrus alters intrafollicular P4 concentrations, oocyte competence acquisition, follicular cells gene expression, and small extracellular vesicles (sEVs) miRNAs contents. RESULTS: P4 hormonal analysis revealed that ipsilateral to the CL follicular fluid (iFF) presented higher P4 concentration compared to contralateral follicular fluid (cFF). Furthermore, oocyte maturation and miRNA biogenesis pathways transcripts (ADAMTS-1 and AGO2, respectively) were increased in cumulus and granulosa cells of iFF, respectively. Nevertheless, a RT-PCR screening of 382 miRNAs showed that three miRNAs were upregulated and two exclusively expressed in sEVs from iFF and are predicted to regulate cell communication pathways. Similarly, seven miRNAs were higher and two exclusively expressed from cFF sEVs and are predicted to modulate proliferation signaling pathways. CONCLUSION: In conclusion, intrafollicular P4 concentration is influenced by the presence of the CL and modulates biological processes related to follicular cell development and oocyte competence, which may influence the oocyte quality. Altogether, these results are crucial to improve our knowledge about the follicular microenvironment involved in oocyte competence acquisition.

Role of chemokines in regulating luteal and uterine functions in pregnant cows.

Pregnancy is intricately regulated by the interactions between various bioactive substances secreted by the conceptus, uterus, and corpus luteum (CL). Interferon-τ, synthesized and secreted by the conceptus, plays a central role in the interaction mechanism of maternal recognition in cows. Chemokines, chemotaxis mediators that are primarily secreted by immune cells, regulate various reproductive responses in various species. Although there are scattered reports on the potential roles of chemokines in the bovine CL and the uterus during the estrous cycle, there is little information on chemokines in these organs during pregnancy. Therefore, in this review, we discuss the possible physiological roles of chemokines in the CL and uterus of pregnant cows, focusing on our recent findings on chemokines and changes in their receptor expression in the CL and endometrium of cows at some stages of pregnancy.

Role of thyroid stimulating hormone in the maintenance and functioning of the human corpus luteum.

PURPOSE: To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. METHODS: hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2 mU/L). Cells were cultured at 37 °C in DMEM, supplemented with 5% FBS. The cells were treated with 1 nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. RESULTS: The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. CONCLUSIONS: Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.

Prostaglandins as local regulators of ovarian physiology in ruminants.

Prostaglandins are synthesized from arachidonic acid through the catalytic activities of cyclooxygenase, while the production of different prostaglandin types, prostaglandin F2 alpha (PGF) and prostaglandin E2 (PGE), are regulated by specific prostaglandin synthases (PGFS and PGES). Prostaglandin ligands (PGF and PGE) bind to specific high-affinity receptors and initiate biologically distinct signalling pathways. In the ovaries, prostaglandins are known to be important endocrine regulators of female reproduction, in addition to maintaining local function through autocrine and/or paracrine effect. Many research groups in different animal species have already identified a variety of factors and molecular mechanisms that are responsible for the regulation of prostaglandin functions. In addition, prostaglandins stimulate their intrafollicular and intraluteal production via the pathway of prostaglandin self-regulation in the ovary. Therefore, the objective of the review article is to discuss recent findings about local regulation patterns of prostaglandin ligands PGF and PGE during different physiological stages of ovarian function in domestic ruminants, especially in bovine. In conclusion, the discussed local regulation mechanisms of prostaglandins in the ovary may stimulate further research activities in different methodological approaches, especially during final follicle maturation and ovulation, as well as corpus luteum formation and function.

Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000 nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function.

PGF2alpha Inhibits 20alpha-HSD Expression by Suppressing CK1alpha-induced ERK and SP1 Activation in the Corpus Luteum of Pregnant Mice.

Prostaglandin F2α (PGF2α) is a luteolytic hormone that promotes parturition in mammals at the end of pregnancy by reducing progesterone secretion from the corpus luteum (CL). In rodents and primates, PGF2α rapidly converts progesterone to 20α-hydroxyprogesterone (20α-OHP) by promoting 20α-hydroxysteroid dehydrogenase (20α-HSD) expression. However, the specific mechanism of 20α-HSD regulation by PGF2α remains unclear. Casein Kinase 1α (CK1α) is a CK1 family member that regulates a variety of physiological functions, including reproductive development. Here, we investigated the effects of CK1α on pregnancy in female mice. Our experiments showed that CK1α is expressed in mouse CL, and its inhibition enhanced progesterone metabolism, decreased progesterone levels, and affected mouse embryo implantation. Further, CK1α mediated the effect of PGF2α on 20α-HSD in mouse luteal cells in vitro. Our results are the first to show that CK1α affects the 20α-HSD mRNA level by affecting the ERK signalling pathway to regulate the expression of the transcription factor SP1. These findings improve our understanding of PGF2α regulation of 20α-HSD.

Immunoexpression of vascular endothelial growth factor and vWF-regulating angiogenesis in cyclic corpus luteum of Indian buffalo.

The present study was conducted to localize the immunoexpression of VEGF-A (Vascular Endothelial Growth Factor) and von Willebrand factor (vWF) in corpora lutea of healthy buffaloes (24) collected from local slaughterhouses. CL collected were categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal phase (stage III, 12 to 16 days, n = 6) and regressing phase (stage IV, 17 to 20 days, n = 6). The percent positive immunostaining for VEGF-A was significantly (p < 0.05) higher in mid-luteal phase than the other three stages of CL. However, it was higher in early luteal phase as well indicated intense angiogenesis in both early and mid-luteal phases. The number of capillary endothelium expressing vWF was significantly (p < 0.05) highest in mid-luteal phase among all the phases. However, in late luteal phase, the percent area positive for VEGF-A immunostaining was reduced but it was significantly (p < 0.05) higher than corpus albicans phase. Thus, in regressing phase or corpus albicans, it was lowest and reduced considerably. However, in late luteal phase, the number of capillaries with vWF immunoexpression reduced significantly (p < 0.05) but it was lowest in corpus albicans phase. Therefore, the immunotaining pattern for VEGF-A and vWF concluded that there was a spositive linear correlation between the two, that is, as the VEGF-A expression was increased, the number of vWF positive capillaries also increased and vice versa. The VEGF-A expressed by the luteal parenchyma in different stages of development and regression of corpus luteum was thus observed to be involved in promoting the angiogenesis and luteal cell proliferation as supported by vWF expressed by endothelium of proliferating capillaries in buffalo corpus luteum throughout the estrous cycle.

Differential expression of angiogenesis-related genes in goat uterus and corpus luteum during pregnancy.

Vascularization and the control of luteal and endometrial development are regulated by hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF) during pregnancy. In this study, the mRNA and protein expression levels of HIFs (HIF1A, HIF2A and HIF3A) and VEGF in goat uterine and ovarian tissues during various stages of pregnancy were evaluated. A total of 42 Hair goats were used and were allocated into six groups, namely embryo-positive (G1), early pregnancy (G2), mid-term pregnancy (G3), late pregnancy (G4), oocyte-positive group (G5) and diestrus group (G6). The mRNA expression of the examined genes was evaluated by RT-qPCR, and protein expression was evaluated by immunohistochemistry (IHC). In caruncles, HIF1A mRNA expression was greater in G1, G2 and G4 than in G3 (p < .05). HIF1A and HIF2A expression was greater in G1 than in G5 (p < .05). In cotyledons, HIF1A, HIF2A and HIF3A mRNA expression was greater in G2 and G3 compared to G4 (p < .05). In luteal tissue, HIF1A mRNA expression was greater in G1 and G2 than in G3 and G4 (p < .05). In the immunohistochemical examination, HIF1A, HIF2A, HIF3A and VEGF immunoreactions were detected in uterine and luteal tissues. Findings suggest that HIFs and VEGF are involved in the regulation of ovarian functions as well as the processes of implantation and placentation.

ERK1/2-SOX9/FOXL2 axis regulates ovarian steroidogenesis and favors the follicular-luteal transition.

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals.

Adropin may regulate corpus luteum formation and its function in adult mouse ovary.

BACKGROUND: Adropin, a unique peptide hormone, has been associated with the regulation of several physiological processes, including glucose homeostasis, fatty acid metabolism, and neovascularization. However, its possible role in ovarian function is not understood. Our objective was to examine the expression of adropin and its putative receptor, GPR19, in the ovaries of mice at various phases of the estrous cycle. METHODS: Immunohistochemistry and western blot analysis were performed to explore the localization and changes in expression of adropin and GPR19 in the ovaries during different phases of the estrous cycle in mice. Hormonal assays were performed with ELISA. An in vitro study was performed to examine the direct effect of adropin (10, 100 ng/ml) on ovarian function. RESULTS: A western blot study showed that adropin and GPR19 proteins were maximum during the estrus phase of the estrous cycle. Interestingly, adropin and GPR19 displayed intense immunoreactivity in granulosa cells of large antral follicles and corpus luteum. This suggested the possible involvement of adropin in corpus luteum formation. Adropin treatment stimulated progesterone synthesis by increasing GPR19, StAR, CYP11A1, and 3ß-HSD expressions, while it decreased estrogen synthesis by inhibiting 17ß-HSD and aromatase protein expressions. Moreover, adropin treatment upregulated the cell cycle arrest-CDK inhibitor 1B (p27kip1), pERK1/2, and angiogenic protein (EG VEGF) that are involved in the process of luteinization. CONCLUSIONS: Adropin GPR19 signaling promotes the synthesis of progesterone and upregulates the expression of p27kip1, EG VEGF, and erk1/2, resulting in cell cycle arrest and neovascularization, which ultimately leads to corpus luteum formation.

Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis.

Progesterone is an essential steroid hormone that is required to initiate and maintain pregnancy in mammals and serves as a metabolic intermediate in the synthesis of endogenously produced steroids, including sex hormones and corticosteroids. Steroidogenic luteal cells of the corpus luteum have the tremendous capacity to synthesize progesterone. These specialized cells are highly enriched with lipid droplets that store lipid substrate, which can be used for the synthesis of steroids. We recently reported that hormone-stimulated progesterone synthesis by luteal cells requires protein kinase A-dependent mobilization of cholesterol substrate from lipid droplets to mitochondria. We hypothesize that luteal lipid droplets are enriched with steroidogenic enzymes and facilitate the synthesis of steroids in the corpus luteum. In the present study, we analyzed the lipid droplet proteome, conducted the first proteomic analysis of lipid droplets under acute cyclic adenosine monophosphate (cAMP)-stimulated conditions, and determined how specific lipid droplet proteins affect steroidogenesis. Steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 and 3 beta-hydroxysteroid dehydrogenase (HSD3B), were highly abundant on lipid droplets of the bovine corpus luteum. High-resolution confocal microscopy confirmed the presence of active HSD3B on the surface of luteal lipid droplets. We report that luteal lipid droplets have the capacity to synthesize progesterone from pregnenolone. Lastly, we analyzed the lipid droplet proteome following acute stimulation with cAMP analog, 8-Br-cAMP, and report increased association of HSD3B with luteal lipid droplets following stimulation. These findings provide novel insights into the role of luteal lipid droplets in steroid synthesis.

Basic fibroblast growth factor induces proliferation and collagen production by fibroblasts derived from the bovine corpus luteum†.

Cyclic regression of the ovarian corpus luteum, the endocrine gland responsible for progesterone production, involves rapid matrix remodeling. Despite fibroblasts in other systems being known for producing and maintaining extracellular matrix, little is known about fibroblasts in the functional or regressing corpus luteum. Vast transcriptomic changes occur in the regressing corpus luteum, among which are reduced levels of vascular endothelial growth factor A (VEGFA) and increased expression of fibroblast growth factor 2 (FGF2) after 4 and 12 h of induced regression, when progesterone is declining and the microvasculature is destabilizing. We hypothesized that FGF2 activates luteal fibroblasts. Analysis of transcriptomic changes during induced luteal regression revealed elevations in markers of fibroblast activation and fibrosis, including fibroblast activation protein (FAP), serpin family E member 1 (SERPINE1), and secreted phosphoprotein 1 (SPP1). To test our hypothesis, we treated bovine luteal fibroblasts with FGF2 to measure downstream signaling, type 1 collagen production, and proliferation. We observed rapid and robust phosphorylation of various signaling pathways involved in proliferation, such as ERK, AKT, and STAT1. From our longer-term treatments, we determined that FGF2 has a concentration-dependent collagen-inducing effect, and that FGF2 acts as a mitogen for luteal fibroblasts. FGF2-induced proliferation was greatly blunted by inhibition of AKT or STAT1 signaling. Our results suggest that luteal fibroblasts are responsive to factors that are released by the regressing bovine corpus luteum, an insight into the contribution of fibroblasts to the microenvironment in the regressing corpus luteum.

Progesterone signaling in the regulation of luteal steroidogenesis.

The corpus luteum is the major source of progesterone, the essential hormone for female reproductive function. While progesterone activity has been the subject of extensive research for decades, characterization of non-canonical progesterone receptor/signaling pathways provided a new perspective for understanding the complex signal transduction mechanisms exploited by the progesterone hormone. Deciphering these mechanisms has significant implications in the management of luteal phase disorders and early pregnancy complications. The purpose of this review is to highlight the complex mechanisms through which progesterone-induced signaling mediates luteal granulosa cell activity in the corpus luteum. Here, we review the literature and discuss the up-to-date evidence on how paracrine and autocrine effects of progesterone regulate luteal steroidogenic activity. We also review the limitations of the published data and highlight future research priorities.

Prostaglandin F2α regulates mitochondrial dynamics and mitophagy in the bovine corpus luteum.

Prostaglandins are arachidonic acid-derived lipid mediators involved in numerous physiological and pathological processes. PGF2α analogues are therapeutically used for regulating mammalian reproductive cycles and blood pressure, inducing term labor, and treating ocular disorders. PGF2α exerts effects via activation of calcium and PKC signaling, however, little is known about the cellular events imposed by PGF2α signaling. Here, we explored the early effects of PGF2α on mitochondrial dynamics and mitophagy in the bovine corpus luteum employing relevant and well characterized in vivo and in vitro approaches. We identified PKC/ERK and AMPK as critical protein kinases essential for activation of mitochondrial fission proteins, DRP1 and MFF. Furthermore, we report that PGF2α elicits increased intracellular reactive oxygen species and promotes receptor-mediated activation of PINK-Parkin mitophagy. These findings place the mitochondrium as a novel target in response to luteolytic mediator, PGF2α. Understanding intracellular processes occurring during early luteolysis may serve as a target for improving fertility.