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.

Central Role for Glycolysis and Fatty Acids in LH-responsive Progesterone Synthesis.

Progesterone production by the corpus luteum is fundamental for establishing and maintaining pregnancy. The pituitary gonadotropin luteinizing hormone (LH) is recognized as the primary stimulus for luteal formation and progesterone synthesis, regardless of species. Previous studies demonstrated an elevation in abundance of genes related to glucose and lipid metabolism during the follicular to luteal transition. However, the metabolic phenotype of these highly steroidogenic cells has not been studied. Herein, we determined acute metabolic changes induced by LH in primary luteal cells and defined pathways required for progesterone synthesis. Untargeted metabolomics analysis revealed that LH induces rapid changes in vital metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway, de novo lipogenesis, and hydrolysis of phospholipids. LH stimulated glucose uptake, enhanced glycolysis, and flux of [U- 13 C 6 ]-labeled glucose-derived carbons into metabolic branches associated with adenosine 5'-triphosphate (ATP) and NADH/NADPH production, synthesis of nucleotides, proteins, and lipids, glycosylation of proteins or lipids, and redox homeostasis. Selective use of small molecule inhibitors targeting the most significantly changed pathways, such as glycolysis, TCA cycle, and lipogenesis, uncovered cellular metabolic routes required for LH-stimulated steroidogenesis. Furthermore, LH via the protein kinase A (PKA) pathway triggered post- translational modification of acetyl-CoA carboxylase alpha (ACACA) and ATP citrate lyase (ACLY), enzymes involved in de novo synthesis of fatty acids. Inhibition of ACLY and fatty acid transport into mitochondria reduced LH-stimulated ATP, cAMP production, PKA activation, and progesterone synthesis. Taken together, these findings reveal novel hormone-sensitive metabolic pathways essential for maintaining LHCGR/PKA signaling and steroidogenesis in ovarian luteal cells. Significance: The establishment and maintenance of pregnancy require a well-developed corpus luteum, an endocrine gland within the ovary that produces progesterone. Although there is increased awareness of intracellular signaling events initiating the massive production of progesterone during the reproductive cycle and pregnancy, there are critical gaps in our knowledge of the metabolic and lipidomic pathways required for initiating and maintaining luteal progesterone synthesis. Here, we describe rapid, hormonally triggered metabolic pathways, and define metabolic targets crucial for progesterone synthesis by ovarian steroidogenic cells. Understanding hormonal control of metabolic pathways may help elucidate approaches for improving ovarian function and successful reproduction or identifying metabolic targets for developing nonhormonal contraceptives.

Palmitate induces integrated stress response and lipoapoptosis in trophoblasts.

Maternal obesity increases the risk of childhood obesity and programs the offspring to develop metabolic syndrome later in their life. Palmitate is the predominant saturated free fatty acid (FFA) that is transported across the placenta to the fetus. We have recently shown that saturated FFA in the maternal circulation as a result of increased adipose tissue lipolysis in third trimester of pregnancy induces trophoblast lipoapoptosis. Here, we hypothesized that palmitate induces integrated stress response by activating mitogen-activated protein kinases (MAPKs), endoplasmic reticulum (ER) stress and granular stress and lipoapoptosis in trophoblasts. Choriocarcinoma-derived third-trimester placental trophoblast-like cells (JEG-3 and JAR) referred as trophoblasts were exposed to various concentrations of palmitate (PA). Apoptosis was assessed by nuclear morphological changes and caspase 3/7 activity. Immunoblot and immunofluorescence analysis was performed to measure the activation of MAPKs, ER stress and granular stress response pathways. Trophoblasts exposed to pathophysiological concentrations of PA showed a concentration-dependent increase in trophoblast lipoapoptosis. PA induces a caspase-dependent trophoblast lipoapoptosis. Further, PA induces MAPK activation (JNK and ERK) via phosphorylation, and activation of ER stress as evidenced by an increased phosphorylation eIF2α & IRE1α. PA also induces the activation of stress granules formation. Two pro-apoptotic transcriptional mediators of PA-induced trophoblast lipoapoptosis, CHOP and FoxO3 have increased nuclear translocation. Mechanistically, PA-induced JNK is critical for trophoblast lipoapoptosis. However, PA-induced activation of ERK and stress granule formation were shown to be cell survival signals to combat subcellular stress due to PA exposure. In conclusion, PA induces the activation of integrated stress responses, among which small molecule inhibition of JNK demonstrated that activation of JNK is critical for PA-induced trophoblast lipoapoptosis and small molecule activation of stress granule formation significantly prevents PA-induced trophoblast lipoapoptosis.

Identification of Lipids and Cytokines in Plasma and Follicular Fluid before and after Follicle-Stimulating Hormone Stimulation as Potential Markers for Follicular Maturation in Cattle.

The process of follicle maturation leading to ovulation is a key milestone in female fertility. It is known that circulating lipids and cytokines play a role in the follicle's ability to go through follicular maturation and the ovulatory processes. However, the specific mechanisms are not well understood. We posit that dysregulation of granulosa cells influences the ovarian environment, which tries to adapt by changing released lipids and cytokines to achieve follicular maturation. Eleven non-lactating adult females underwent estrus synchronization with two injections of PGF2α 14 days apart. Daily blood samples were collected for 28 days to monitor steroid hormone production after the second injection. To understand the potential impacts of lipids and cytokines during ovulation, a low-dose FSH stimulation (FSHLow) was performed after resynchronization of cows, and daily blood samples were collected for 14 days to monitor steroid hormone production until ovariectomies. The lipidomic analysis demonstrated increased circulating diacylglycerides and triacylglycerides during the mid-luteal phase and after FSHLow treatment. Cholesteryl esters decreased in circulation but increased in follicular fluid (FF) after FSHLow. Increased circulating concentrations of TNFα and reduced CXCL9 were observed in response to FSHLow. Therefore, specific circulating lipids and cytokines may serve as markers of normal follicle maturation.

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.

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.

Ovarian inflammation mediated by Toll-like receptor 4 increased transcripts of maternal effect genes and decreased embryo development†.

Obese women are subfertile and have reduced assisted reproduction success, which may be due to reduced oocyte competence. We hypothesize that consumption of a high-fat/high-sugar diet induces ovarian inflammation, which is a primary contributor to decreased oocyte quality and pre-implantation embryo development. To test this hypothesis, C57BL/6 (B6) mice with a normal inflammatory response and C3H/HeJ (C3H) mice with a dampened inflammatory response due to dysfunctional Toll-like receptor 4 were fed either normal chow or high-fat/high-sugar diet. In both B6 and C3H females, high-fat/high-sugar diet induced excessive adiposity and hyperglycemia compared to normal chow-fed counterparts. Conversely, ovarian CD68 levels and oocyte expression of oxidative stress markers were increased when collected from B6 high-fat/high-sugar but not C3H high-fat/high-sugar mice. Following in vitro fertilization of in vivo matured oocytes, blastocyst development was decreased in B6-high-fat/high-sugar but not C3H high-fat/high-sugar mice. Expression of cumulus cell markers of oocyte quality were altered in both B6 high-fat/high-sugar and C3H high-fat/high-sugar. However, there were no diet-dependent differences in spindle abnormalities in either B6 or C3H mice, suggesting potential defects in cytoplasmic maturation. Indeed, there were significant increases in the abundance of maternal effect gene mRNAs in oocytes from only B6 high-fat/high-sugar mice. These differentially expressed genes encode proteins of the subcortical maternal complex and associated with mRNA metabolism and epigenetic modifications. These genes regulate maternal mRNA degradation at oocyte maturation, mRNA clearance at the zygotic genome activation, and methylation of imprinted genes suggesting a mechanism by which inflammation induced oxidative stress impairs embryo development.

VEGFA165 can rescue excess steroid secretion, inflammatory markers, and follicle arrest in the ovarian cortex of High A4 cows†.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFß secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Protein Kinase A and 5' AMP-Activated Protein Kinase Signaling Pathways Exert Opposite Effects on Induction of Autophagy in Luteal Cells.

In the absence of pregnancy the ovarian corpus luteum undergoes regression, a process characterized by decreased production of progesterone and structural luteolysis involving apoptosis. Autophagy has been observed in the corpus luteum during luteal regression. Autophagy is a self-degradative process important for balancing sources of cellular energy at critical times in development and in response to nutrient stress, but it can also lead to apoptosis. Mechanistic target of rapamycin (MTOR) and 5' AMP-activated protein kinase (AMPK), key players in autophagy, are known to inhibit or activate autophagy, respectively. Here, we analyzed the signaling pathways regulating the initiation of autophagy in bovine luteal cells. In vivo studies showed increased activating phosphorylation of AMPKα (Thr172) and elevated content of LC3B, a known marker of autophagy, in luteal tissue during PGF2α-induced luteolysis. In vitro, AMPK activators 1) stimulated phosphorylation of regulatory associated protein of MTOR (RPTOR) leading to decreased activity of MTOR, 2) increased phosphorylation of Unc-51-Like Kinase 1 (ULK1) and Beclin 1 (BECN1), at sites specific for AMPK and required for autophagy initiation, 3) increased levels of LC3B, and 4) enhanced colocalization of autophagosomes with lysosomes indicating elevated autophagy. In contrast, LH/PKA signaling in luteal cells 1) reduced activation of AMPKα and phosphorylation of RPTOR, 2) elevated MTOR activity, 3) stimulated phosphorylation of ULK1 at site required for ULK1 inactivation, and 4) inhibited autophagosome formation as reflected by reduced content of LC3B-II. Pretreatment with AICAR, a pharmacological activator of AMPK, inhibited LH-mediated effects on RPTOR, ULK1 and BECN1. Our results indicate that luteotrophic signaling via LH/PKA/MTOR inhibits, while luteolytic signaling via PGF2α/Ca2+/AMPK activates key signaling pathways involved in luteal cell autophagy.

Transcriptomic data of bovine ovarian granulosa cells of control and High A4 cows.

Microarray analysis using Affymetrix Bovine GeneChip 1.0 ST Array to determine RNA expression analysis was performed on somatic granulosa cells from two different groups of cows classified based on androstenedione concentration within the follicular fluid (Control vs High A4) of estrogen-active dominant follicles. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE97017 - RNA Expression Data from Bovine Ovarian Granulosa Cells from High or Low Androgen-Content Follicles). Subsequent ANOVA determined genes that were enriched (≥ 1.5 fold more) or decreased (≤ 1.5 fold less) in the High A4 granulosa cells compared to Control granulosa cells and analyzed filtered datasets of these differentially expressed genes are presented as tables. MicroRNAs that are differentially expressed in Control and High A4 granulosa cells are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values are shown as a figure. Ingenuity Pathway Analysis determined upstream regulators of differently expressed genes as presented in a table. These data have been further analyzed and interpreted in the companion article "A High-Androgen Microenvironment Inhibits Granulosa Cell Proliferation and Alters Cell Identity" (McFee et. al., 2021 [1].

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity.

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype.

Attainment and maintenance of pubertal cyclicity may predict reproductive longevity in beef heifers†.

We hypothesized the manner that heifers achieve puberty may indicate their future reproductive longevity. Heifers with discontinued or delayed cyclicity during puberty attainment may have irregular reproductive cycles, anovulation, and infertility in their first breeding season contributing to a shorter reproductive lifespan. Therefore, plasma progesterone (P4) was measured from weaning to breeding on 611 heifers born 2012-2017 and four pubertal classifications were identified: (1) Early; P4 ≥ 1 ng/ml < March 12 with continued cyclicity, (2) Typical; P4 ≥ 1 ng/ml ≥ March 12 with continued cyclicity, (3) Start-Stop; P4 ≥ 1 ng/ml but discontinued cyclicity, and (4) Non-Cycling; no P4 ≥ 1 ng/ml. Historical herd records indicated that 25% of heifers achieved puberty prior to March 12th in the 10 years prior to the study. Start-Stop and Non-Cycling yearling heifers were lighter indicating reduced growth and reproductive maturity traits compared with Early/Typical heifers. In addition, Non-Cycling/Start-Stop heifers were less responsive to prostaglandin F2 alpha (PGF2α) to initiate estrous behavior and ovulation to be artificially inseminated. Non-Cycling heifers had fewer reproductive tract score-5 and reduced numbers of calves born in the first 21-days-of-calving during their first breeding season. Within the Start-Stop classification, 50% of heifers reinitiated cyclicity with growth traits and reproductive parameters that were similar to heifers in the Early/Typical classification while those that remained non-cyclic were more similar to heifers in the Non-Cycling group. Thus, heifers with discontinued cyclicity or no cyclicity during puberty attainment had delayed reproductive maturity resulting in subfertility and potentially a shorter reproductive lifespan.

Maternal obesity alters placental lysophosphatidylcholines, lipid storage, and the expression of genes associated with lipid metabolism‡.

Dyslipidemia is a characteristic of maternal obesity and previous studies have demonstrated abnormalities in fatty acid oxidation and storage in term placentas. However, there is little information about the effect of pre-pregnancy obesity on placental lipid metabolism during early pregnancy. The objective of this study was to determine the relationship between lipid profiles and markers of metabolism in placentas from obese and lean dams at midgestation. Mice were fed a western diet (WD) or normal diet (ND) and lysophosphatidylcholines (LPCs) and/or phosphatidylcholines (PCs) were measured in dam circulation and placenta sections using liquid chromatography-tandem mass spectrometry and mass spectrometry imaging, respectively. In WD dam, circulating LPCs containing 16:1, 18:1, 20:0, and 20:3 fatty acids were increased and 18:2 and 20:4 were decreased. In WD placenta from both sexes, LPC 18:1 and PC 36:1 and 38:3 were increased. Furthermore, there were moderate to strong correlations between LPC 18:1, PC 36:1, and PC 38:3. Treatment-, spatial-, and sex-dependent differences in LPC 20:1 and 20:3 were also detected. To identify genes that may regulate diet-dependent differences in placenta lipid profiles, the expression of genes associated with lipid metabolism and nutrient transport was measured in whole placenta and isolated labyrinth using droplet digital PCR and Nanostring nCounter assays. Several apolipoproteins were increased in WD placentas. However, no differences in nutrient transport or fatty acid metabolism were detected. Together, these data indicate that lipid storage is increased in midgestation WD placentas, which may lead to lipotoxicity, altered lipid metabolism and transport to the fetus later in gestation.

Formation and characterization of lipid droplets of the bovine corpus luteum.

Establishment and maintenance of pregnancy depends on progesterone synthesized by luteal tissue in the ovary. Our objective was to identify the characteristics of lipid droplets (LDs) in ovarian steroidogenic cells. We hypothesized that LDs are a major feature of steroidogenic luteal cells and store cholesteryl esters. Whole bovine tissues, isolated ovarian steroidogenic cells (granulosa, theca, small luteal, and large luteal), and isolated luteal LDs were assessed for LD content, LD-associated proteins and lipid analyses. Bovine luteal tissue contained abundant lipid droplets, LD-associated perilipins 2/3/5, hormone-sensitive lipase, and 1-acylglycerol-3-phosphate O-acyltransferase ABHD5. Luteal tissue was enriched in triglycerides (TGs) compared to other tissues, except for adipose tissue. Luteal cells were distinguishable from follicular cells by the presence of LDs, LD-associated proteins, and increased TGs. Furthermore, LDs from large luteal cells were numerous and small; whereas, LDs from small luteal cells were large and less numerous. Isolated LDs contained nearly all of the TGs and cholesteryl esters present in luteal tissue. Isolated luteal LDs were composed primarily of TG, with lesser amounts of cholesteryl esters, diglyceride and other phospholipids. Bovine luteal LDs are distinct from LDs in other bovine tissues, including follicular steroidogenic cells.

Trafficking of cholesterol from lipid droplets to mitochondria in bovine luteal cells: Acute control of progesterone synthesis.

The corpus luteum is a transient endocrine gland that synthesizes and secretes the steroid hormone, progesterone, which is vital for establishment and maintenance of pregnancy. Luteinizing hormone (LH) via activation of protein kinase A (PKA) acutely stimulates luteal progesterone synthesis via a complex process, converting cholesterol via a series of enzymatic reactions, into progesterone. Lipid droplets in steroidogenic luteal cells store cholesterol in the form of cholesterol esters, which are postulated to provide substrate for steroidogenesis. Early enzymatic studies showed that hormone sensitive lipase (HSL) hydrolyzes luteal cholesterol esters. In this study, we tested whether HSL is a critical mediator of the acute actions of LH on luteal progesterone production. Using LH-responsive bovine small luteal cells our results reveal that LH, forskolin, and 8-Br cAMP-induced PKA-dependent phosphorylation of HSL at Ser563 and Ser660, events known to promote HSL activity. Small molecule inhibition of HSL activity and siRNA-mediated knock down of HSL abrogated LH-induced progesterone production. Moreover, western blotting and confocal microscopy revealed that LH stimulates phosphorylation and translocation of HSL to lipid droplets. Furthermore, LH increased trafficking of cholesterol from the lipid droplets to the mitochondria, which was dependent on both PKA and HSL activation. Taken together, these findings identify a PKA/HSL signaling pathway in luteal cells in response to LH and demonstrate the dynamic relationship between PKA, HSL, and lipid droplets in luteal progesterone synthesis.

A primate perspective on oocytes and transgenerational PCOS.

'Androgenized' rodent models are widely used to explore the pathophysiology underlying human polycystic ovary syndrome (PCOS), including reproductive and metabolic dysfunction. Based on a recent study using a dihydrotestosterone (DHT)-treated murine model, it has been proposed that prenatal androgen excess alone can predispose to transgenerational transmission of PCOS. From RNA sequencing analysis of metaphase II (MII) oocytes of androgenized lineages, the authors speculated that oocyte factors, including up-regulation of cytotoxic granulosa-associated RNA binding protein-like 1 (TiaL1), are sufficient to promote disease transfer across generations. Although this is an intriguing concept, it was not considered in the context of earlier publications in which the transcriptomes of human MII oocytes from PCOS women undergoing IVF were compared with women without PCOS. In one of these papers, a number of differentially expressed genes in PCOS MII oocytes (TIAL1 was not differentially expressed) were found to have putative response elements in their promoters for androgen receptors and peroxisome proliferating receptor gamma, providing a mechanism for how excess androgens and/or metabolic defects associated with PCOS might affect female germ cells.

Luteinizing hormone regulates the phosphorylation and localization of the mitochondrial effector dynamin-related protein-1 (DRP1) and steroidogenesis in the bovine corpus luteum.

The corpus luteum is an endocrine gland that synthesizes and secretes progesterone. Luteinizing hormone (LH) activates protein kinase A (PKA) signaling in luteal cells, increasing delivery of substrate to mitochondria for progesterone production. Mitochondria maintain a highly regulated equilibrium between fusion and fission in order to sustain biological function. Dynamin-related protein 1 (DRP1), is a key mediator of mitochondrial fission. The mechanism by which DRP1 is regulated in the ovary is largely unknown. We hypothesize that LH via PKA differentially regulates the phosphorylation of DRP1 on Ser616 and Ser637 in bovine luteal cells. In primary cultures of steroidogenic small luteal cells (SLCs), LH, and forskolin stimulated phosphorylation of DRP1 (Ser 637), and inhibited phosphorylation of DRP1 (Ser 616). Overexpression of a PKA inhibitor blocked the effects of LH and forskolin on DRP1 phosphorylation. In addition, LH decreased the association of DRP1 with the mitochondria. Genetic knockdown of the DRP1 mitochondria receptor, and a small molecule inhibitor of DRP1 increased basal and LH-induced progesterone production. Studies with a general Dynamin inhibitor and siRNA knockdown of DRP1 showed that DRP1 is required for optimal LH-induced progesterone biosynthesis. Taken together, the findings place DRP1 as an important target downstream of PKA in steroidogenic luteal cells.

Vascular endothelial growth factor A isoforms modulate follicle development in peripubertal heifers independent of diet through diverse signal transduction pathways.

Follicular progression during peripuberty is affected by diet. Vascular endothelial growth factor A (VEGFA) induces follicle progression in many species; however, there are limited studies to determine if diet may alter the effects of angiogenic VEGFA165-stimulated follicle progression or antiangiogenic VEGFA165b follicle arrest. We hypothesized that diet affects the magnitude of angiogenic and antiangiogenic VEGFA isoform actions on follicular development through diverse signal transduction pathways. To test this hypothesis, beef heifers in our first trial received Stair-Step (restricted and refeeding) or control diets from 8 to 13 months of age. Ovaries were collected to determine follicle stages, measure vascular gene expression and conduct ovarian cortical cultures. Ovarian cortical cultures were treated with phosphate-buffered saline (control), 50 ng/ml VEGFA165, VEGFA165b, or VEGFA165 + VEGFA165b. The Stair-Step heifers had more primordial follicles (P < 0.0001), greater messenger RNA abundance of vascular markers VE-cadherin (P < 0.0001) and NRP-1 (P < 0.0051) than controls at 13 months of age prior to culture. After culture, VEGFA isoforms had similar effects, independent of diet, where VEGFA165 stimulated and VEGFA165b inhibited VEGFA165-stimulated follicle progression from early primary to antral follicle stages. In vitro cultures were treated with VEGFA isoforms and signal transduction array plates were evaluated. VEGFA165 stimulated expression of genes related to cell cycle, cell proliferation, and growth while VEGFA165b inhibited expression of those genes. Thus, VEGFA isoforms can act independently of diet to alter follicle progression or arrest. Furthermore, follicle progression can be stimulated by VEGFA165 and inhibited by VEGFA165b through diverse signal transduction pathways.

Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro†.

Yes-associated protein 1 (YAP1) is a major component of the Hippo signaling pathway. Although the exact extracellular signals that control the Hippo pathway are currently unknown, increasing evidence supports a critical role for the Hippo pathway in embryonic development, regulation of organ size, and carcinogenesis. Granulosa cells (GCs) within the ovarian follicle proliferate and produce steroids and growth factors, which facilitate the growth of follicle and maturation of the oocyte. We hypothesize that YAP1 plays a role in proliferation and estrogen secretion of GCs. In the current study, we examined the expression of the Hippo signaling pathway in bovine ovaries and determined whether it was important for GC proliferation and estrogen production. Mammalian STE20-like protein kinase 1 (MST1) and large tumor suppressor kinase 2 (LATS2) were identified as prominent upstream components of the Hippo pathway expressed in granulosa and theca cells of the follicle and large and small cells of the corpus luteum. Immunohistochemistry revealed that YAP1 was localized to the nucleus of growing follicles. In vitro, nuclear localization of the downstream Hippo signaling effector proteins YAP1 and transcriptional co-activator with PDZ-binding motif (TAZ) was inversely correlated with GC density, with greater nuclear localization under conditions of low cell density. Treatment with verteporfin and siRNA targeting YAP1 or TAZ revealed a critical role for these transcriptional co-activators in GC proliferation. Furthermore, knockdown of YAP1 in GCs inhibited follicle-stimulating hormone (FSH)-induced estradiol biosynthesis. The data indicate that Hippo pathway transcription co-activators YAP1/TAZ play an important role in GC proliferation and estradiol synthesis, two processes necessary for maintaining normal follicle development.

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.