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.

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.

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.

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.

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.

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 case-based or hands-on laboratory exercises with physiology lectures improves knowledge retention, but veterinary medicine students prefer case-based activities.

Didactic lectures are prevalent in physiology courses within veterinary medicine programs, but more active learning methods have also been utilized. Our goal was to identify the most appropriate learning method to augment the lecture component of our physiology course. We hypothesized that case-based learning would be well received by students and would be more effective at helping them learn physiological concepts compared with more traditional laboratory exercises. In this study, approximately one-half of the laboratory sessions for the two-semester course were dedicated to traditional hands-on laboratory exercises, whereas the remaining one-half of the sessions were dedicated to case-based exercises. The lecture portion of the course was not altered. Student attitudes were evaluated after each session and at the end of each semester via quantitative and qualitative survey questions. Student performance was evaluated using section exams and end-of-semester posttests. The vast majority of survey responses received were positive for both cased-based activities and traditional hands-on laboratories. In addition, participation in both types of active learning activities, but not lecture, was associated with retention of conceptual knowledge based on student performance between the section exams and posttests ( P < 0.002). These results indicate that both case-based learning and laboratory exercises are beneficial learning activities to incorporate into a lecture-based physiology course. However, positive survey responses were significantly greater following case-based activities vs. traditional hands-on laboratories, and only participation in case-based activities resulted in greater student performance on the posttest ( P < 0.04). Therefore, case-based activities may be the preferred supplemental learning activity for veterinary medical physiology.

VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance.

Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo.

Ept7 influences estrogen action in the pituitary gland and body weight of rats.

Estrogens control many aspects of pituitary gland biology, including regulation of lactotroph homeostasis and synthesis and secretion of prolactin. In rat models, these actions are strain specific and heritable, and multiple quantitative trait loci (QTL) have been mapped that impact the responsiveness of the lactotroph to estrogens. One such QTL, Ept7, was mapped to RNO7 in female progeny generated in an intercross between BN rats, in which the lactotroph population is insensitive to estrogens, and ACI rats, which develop lactotroph hyperplasia/adenoma and associated hyperprolactinemia in response to estrogen treatment. The primary objective of this study was to confirm the existence of Ept7 and to quantify the impact of this QTL on responsiveness of the pituitary gland of female and male rats to 17ß-estradiol (E2) and diethylstilbestrol (DES), respectively. Secondary objectives were to determine if Ept7 influences the responsiveness of the male reproductive tract to DES and to identify other discernible phenotypes influenced by Ept7. To achieve these objectives, a congenic rat strain that harbors BN alleles across the Ept7 interval on the genetic background of the ACI strain was generated and characterized to define the effect of administered estrogens on the anterior pituitary gland and male reproductive tissues. Data presented herein indicate Ept7 exerts a marked effect on development of lactotroph hyperplasia in response to estrogen treatment, but does not affect atrophy of the male reproductive tissues in response to hormone treatment. Ept7 was also observed to exert gender specific effects on body weight in young adult rats.

Effects of IL8 and immune cells on the regulation of luteal progesterone secretion.

Recent studies have suggested that chemokines may mediate the luteolytic action of prostaglandin F2α (PGF). Our objective was to identify chemokines induced by PGF in vivo and to determine the effects of interleukin 8 (IL8) on specific luteal cell types in vitro. Mid-cycle cows were injected with saline or PGF, ovaries were removed after 0.5-4 h, and expression of chemokine was analyzed by qPCR. In vitro expression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial, and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were cocultured with steroidogenic cells to determine their effect on progesterone production. IL8, CXCL2, CCL2, and CCL8 transcripts were rapidly increased following PGF treatment in vivo. The stimulatory action of PGF on IL8 mRNA expression in vitro was prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but stimulated ERK phosphorylation in neutrophils. In coculture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell progesterone synthesis. In contrast, activated PBMCs inhibited LH-stimulated progesterone synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis, involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum.

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.

Puberty classifications in beef heifers are moderately to highly heritable and associated with candidate genes related to cyclicity and timing of puberty.

Introduction: Pubertal attainment is critical to reproductive longevity in heifers. Previously, four heifer pubertal classifications were identified according to attainment of blood plasma progesterone concentrations > 1 ng/ml: 1) Early; 2) Typical; 3) Start-Stop; and 4) Non-Cycling. Early and Typical heifers initiated and maintained cyclicity, Start-Stop started and then stopped cyclicity and Non-Cycling never initiated cyclicity. Start-Stop heifers segregated into Start-Stop-Discontinuous (SSD) or Start-Stop-Start (SSS), with SSD having similar phenotypes to Non-Cycling and SSS to Typical heifers. We hypothesized that these pubertal classifications are heritable, and loci associated with pubertal classifications could be identified by genome wide association studies (GWAS). Methods: Heifers (n = 532; 2017 - 2022) genotyped on the Illumina Bovine SNP50 v2 or GGP Bovine 100K SNP panels were used for variant component estimation and GWAS. Heritability was estimated using a univariate Bayesian animal model. Results: When considering pubertal classifications: Early, Typical, SSS, SSD, and Non-Cycling, pubertal class was moderately heritable (0.38 ± 0.08). However, when heifers who initiated and maintained cyclicity were compared to those that did not cycle (Early+Typical vs. SSD+Non-Cycling) heritability was greater (0.59 ± 0.19). A GWAS did not identify single nucleotide polymorphisms (SNPs) significantly associated with pubertal classifications, indicating puberty is a polygenic trait. A candidate gene approach was used, which fitted SNPs within or nearby a set of 71 candidate genes previously associated with puberty, PCOS, cyclicity, regulation of hormone secretion, signal transduction, and methylation. Eight genes/regions were associated with pubertal classifications, and twenty-two genes/regions were associated with whether puberty was attained during the trial. Additionally, whole genome sequencing (WGS) data on 33 heifers were aligned to the reference genome (ARS-UCD1.2) to identify variants in FSHR, a gene critical to pubertal attainment. Fisher's exact test determined if FSHR SNPs segregated by pubertal classification. Two FSHR SNPs that were not on the bovine SNP panel were selected for additional genotyping and analysis, and one was associated with pubertal classifications and whether they cycled during the trial. Discussion: In summary, these pubertal classifications are moderately to highly heritable and polygenic. Consequently, genomic tools to inform selection/management of replacement heifers would be useful if informed by SNPs associated with cyclicity and early pubertal attainment.

Vascular contributions to early ovarian development: potential roles of VEGFA isoforms.

Vascularisation is an essential component of ovarian morphogenesis; however, little is known regarding factors regulating the establishment of vasculature in the ovary. Angiogenesis involving extensive endothelial cell migration is a critical component of vessel formation in the embryonic testis but vasculogenic mechanisms appear to play a prominent role in ovarian vascularisation. Vasculature has a strong influence on the formation of ovarian structures, and the early developmental processes of ovigerous cord formation, primordial follicle assembly and follicle activation are all initiated in regions of the ovary that are in close association with the highly vascular medulla. The principal angiogenic factor, vascular endothelial growth factor A (VEGFA), has an important role in both endothelial cell differentiation and vascular pattern development. Expression of VEGFA has been localised to ovigerous cords and follicles in developing ovaries and an increased expression of pro-angiogenic Vegfa isoform mRNA in relation to anti-angiogenic isoform mRNA occurs at the same time-point as the peak of primordial follicle assembly in perinatal rats. Elucidation of specific genes that affect vascular development within the ovary may be critical for determining not only the normal mechanisms of ovarian morphogenesis, but also for understanding certain ovarian reproductive disorders.

Fibroblast Growth Factor 21 Has a Diverse Role in Energetic and Reproductive Physiological Functions of Female Beef Cattle.

Fibroblast growth factor 21 (FGF21) has been identified in multiple mammalian species as a molecular marker of energy metabolism while also providing negative feedback to the gonads. However, the role of FGF21 in regulating the energetic and reproductive physiology of beef heifers and cows has yet to be characterized. Herein, we investigated the temporal concentrations of FGF21 in female beef cattle from the prepubertal period to early lactation. Circulating concentrations of FGF21, non-esterified fatty acids, plasma urea nitrogen, glucose, and progesterone were assessed. Ultrasonography was employed to determine the onset of puberty and resumption of postpartum ovarian cyclicity as well as to measure backfat thickness. Finally, cows and calves underwent the weigh-suckle-weigh technique to estimate rate of milk production. We have revealed that FGF21 has an expansive role in the physiology of female beef cattle, including pubertal onset, adaptation to nutritional transition, rate of body weight gain, circulating markers of metabolism, and rate of milk production. In conclusion, FGF21 plays a role in physiological functions in beef cattle that can be applied to advance the understanding of basic scientific processes governing the nutritional regulation of reproductive function but also provides a novel means for beef cattle producers to select parameters of financial interest.

Sheep with ovarian androgen excess have fibrosis and follicular arrest with increased mRNA abundance for steroidogenic enzymes and gonadotropin receptors.

An androgen excess ovarian micro-environment may limit follicle progression in sheep. Two populations of ewes with divergent follicular fluid androstenedione (A4) were identified in a flock in Jordan: High A4; (A4) ≥ 30 ng/mL, (N = 12) or Control A4 (Control); A4 ≤ 15 ng/mL; (N = 12). We hypothesized High A4 ewes would have increased steroidogenic enzyme mRNA abundance, inflammation, and follicular arrest. Messenger RNA abundance for steroidogenic enzymes StAR, CYP17A1, CYP11A1, and HSD3B1 were increased in theca cells while CYP17A1, CYP19A1, and HSD3B1 were increased in granulosa cells in High A4 ewes compared to Control. Gonadotropin receptor mRNA expression for LHCGR was increased in theca and FSHR in granulosa in High A4 ewes. Messenger RNA expression of FOS when reduced, increases expression of CYP17A1 which was observed in High A4 granulosa cells compared to Control. Furthermore, High A4 ewes had greater numbers of primordial follicles (P < 0.001) and fewer developing follicles compared to Control before, and after 7 d of culture, indicating follicular arrest was not alleviated by cortex culture. Increased fibrosis in the ovarian cortex was detected in High A4 ewes relative to Control (P < 0.001) suggesting increased inflammation and altered extracellular matrix deposition. Thus, this High A4 ewes population has similar characteristics to High A4 cows and women with polycystic ovary syndrome suggesting that naturally occurring androgen excess occurs in multiple species and may be a causative factor in follicular arrest and subsequent female sub- or infertility.

Excess androgen (androstenedione; A4) in ewes can result in ovarian follicular arrest and fibrosis contributing to anovulation in sheep. We have identified a naturally occurring ovarian A4 excess in a sheep population with similar characteristics to High A4 cows, both of which are similar to that in women with polycystic ovary syndrome indicating that several mammalian species experience naturally occurring androgen excess resulting in infertility or follicle arrest. Somatic cells, theca and granulosa, surrounding the egg in High A4 ewes had increased expression of steroidogenic enzymes, similar to that seen in High A4 cows, permitting more ovarian cells to manufacture androgens, which may be the cause of androgen excess. Thus, naturally occurring androgen-excess in domestic livestock females can be utilized as models to research the causes of androgen excess and determine the mechanisms that result in follicular arrest and sub- or infertility.

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.

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.

The balance of proangiogenic and antiangiogenic VEGFA isoforms regulate follicle development.

Vascular endothelial growth factor A (VEGFA) has been extensively studied because of its role in follicular development and is a principal angiogenic factor essential for angiogenesis. Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, VEGFA might influence follicle growth via the regulation of angiogenesis. However, VEGFA might also influence follicular development through nonangiogenic mechanisms, since its expression has been localized in nonvascular follicles and cells. Alternative mRNA splicing of eight exons from the VEGFA gene results in the formation of various VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms (VEGFA_XXX) are encoded by exon 8a, whereas a sister set of isoforms (VEGFA_XXXB) with antiangiogenic properties is encoded by exon 8b. The antiangiogenic VEGFA_XXXB isoforms comprise the majority of VEGFA expressed in most tissues, whereas expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. Although proangiogenic and antiangiogenic isoforms can now be distinguished from one another, many studies evaluating VEGFA in ovarian and follicular development up to now have not differentiated proangiogenic VEGFA from antiangiogenic VEGFA. Experiments from our laboratory indicate that proangiogenic VEGFA promotes follicle recruitment and early follicular development and antiangiogenic VEGFA inhibits these processes. The balance of proangiogenic versus antiangiogenic VEGFA isoforms is thus of importance during follicle development. Further studies are warranted to elucidate the way that this balance regulates follicular formation and progression.