Xiaoying Hou

Evidence That High Variation in Ovarian Reserves of Healthy Young Adults Has a Negative Impact on the Corpus Luteum and Endometrium During Estrous Cycles in Cattle

Abstract Low progesterone concentrations and diminished ovarian reserves (total number of healthy oocytes) during reproductive cycles are linked to infertility in single-ovulating species like cattle. However, the extent and mechanisms whereby the inherently high variation in ovarian reserves may negatively affect progesterone production are unknown. Cattle were chosen to address these questions because the size of their ovarian reserves can be predicted based on an antral follicle count (AFC) during follicular waves. The present study determined if progesterone concentrations, differentiation and function of the corpus luteum (CL), and endometrial thickness differed during reproductive cycles of age-matched healthy young adult cattle with low versus high AFC during follicular waves. The results showed that, despite enhanced LH secretion, progesterone concentrations were lower during estrous cycles for animals with low versus high AFC. Animals with low versus high AFC also had a decreased basal, LH-, and 25-hydroxycholesterol-induced capacity of luteal and granulosal cells to produce progesterone, reduced amounts of STAR and mRNAs for STAR and LH receptor in the CL, and no change in endometrial thickness during estrous cycles. Taken together, these results 1) supported the conclusion that high variation in ovarian reserves of young adults is associated with alterations in differentiation and function of the CL and 2) provided insight into the potential factors that may cause suboptimal luteal function (e.g., heightened LH secretion and desensitization of the LH receptor, diminished LH responsiveness, diminished STAR, inherent deficiency in capacity of granulosal cells to undergo luteinization) and infertility (e.g., low progesterone, poor endometrial growth) in individuals with diminished ovarian reserves.

AKT-independent Phosphorylation of TSC2 and Activation of mTOR and Ribosomal Protein S6 Kinase Signaling by Prostaglandin F2α

Prostaglandin F2α (PGF2α) is an important mediator of corpus luteum (CL) regression, although the cellular signaling events that mediate this process have not been clearly identified. It is established that PGF2α binds to a G-proteincoupled receptor (GPCR) to stimulate protein kinase C (PKC) and Raf-MEK-Erk signaling in luteal cells. The present experiments were performed to determine whether PGF2α stimulates the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathway in steroidogenic luteal cells. We demonstrate that PGF2α treatment results in a timeand concentration-dependent stimulation of the phosphorylation and activation of S6K1. The stimulation of S6K1 in response to PGF2α treatment was abolished by the mTOR inhibitor rapamycin. Treatment with PGF2α did not increase AKT phosphorylation but increased the phosphorylation of Erk and the tumor suppressor protein tuberous sclerosis complex 2 (TSC2), an upstream regulator of mTOR. The effects of PGF2α were mimicked by the PKC activator PMA and inhibited by U0126, a MEK1 inhibitor. The activation of mTOR/S6K1 and putative down stream processes involving the translational apparatus (i.e. 4EBP1 phosphorylation, release of 4EBP1 binding in m7G cap binding assays, and the phosphorylation and synthesis of S6) were completely sensitive to treatment with rapamycin, implicating mTOR in the actions of PGF2α. Taken together, our data suggest that GPCR activation in response to PGF2α stimulates the mTOR pathway which increases the translational machinery in luteal cells. The translation of proteins under the control of mTOR may have implications for luteal development and regression and offer new strategies for therapeutic intervention in PGF2α-target tissues.

Convergence of 3′,5′-Cyclic Adenosine 5′-Monophosphate/Protein Kinase A and Glycogen Synthase Kinase-3β/β-Catenin Signaling in Corpus Luteum Progesterone Synthesis

Progesterone secretion by the steroidogenic cells of the corpus luteum (CL) is essential for reproduction. Progesterone synthesis is under the control of LH, but the exact mechanism of this regulation is unknown. It is established that LH stimulates the LH receptor/choriogonadotropin receptor, a G-protein coupled receptor, to increase cAMP and activate cAMP-dependent protein kinase A (PKA). In the present study, we tested the hypothesis that cAMP/PKA-dependent regulation of the Wnt pathway components glycogen synthase kinase (GSK)-3beta and beta-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3beta at N-terminal Ser9 causing its inactivation and resulted in the accumulation of beta-catenin. Overexpression of N-terminal truncated beta-catenin (Delta90 beta-catenin), which lacks the phosphorylation sites responsible for its destruction, significantly augmented LH-stimulated progesterone secretion. In contrast, overexpression of a constitutively active mutant of GSK3beta (GSK-S9A) reduced beta-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of beta-catenin with the proximal promoter of the StAR gene, a gene that expresses the steroidogenic acute regulatory protein, which is a cholesterol transport protein that controls a rate-limiting step in steroidogenesis. Collectively these data suggest that cAMP/PKA regulation of GSK3beta/beta-catenin signaling may contribute to the acute increase in progesterone production in response to LH.

Luteinizing Hormone Stimulates Mammalian Target of Rapamycin Signaling in Bovine Luteal Cells via Pathways Independent of AKT and Mitogen-Activated Protein Kinase: Modulation of Glycogen Synthase Kinase 3 and AMP-Activated Protein Kinase

LH stimulates the production of cAMP in luteal cells, which leads to the production of progesterone, a hormone critical for the maintenance of pregnancy. The mammalian target of rapamycin (MTOR) signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. This study examined the actions of LH on the regulation and possible role of the MTOR signaling pathway in primary cultures of bovine corpus luteum cells. Herein, we demonstrate that activation of the LH receptor stimulates the phosphorylation of the MTOR substrates ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1. The actions of LH were mimicked by forskolin and 8-bromo-cAMP. LH did not increase AKT or MAPK1/3 phosphorylation. Studies with pathway-specific inhibitors demonstrated that the MAPK kinase 1 (MAP2K1)/MAPK or phosphatidylinositol 3-kinase/AKT signaling pathways were not required for LH-stimulated MTOR/S6K1 activity. However, LH decreased the activity of glycogen synthase kinase 3Beta (GSK3B) and AMP-activated protein kinase (AMPK). The actions of LH on MTOR/S6K1 were mimicked by agents that modulated GSK3B and AMPK activity. The ability of LH to stimulate progesterone secretion was not prevented by rapamycin, a MTOR inhibitor. In contrast, activation of AMPK inhibited LH-stimulated MTOR/S6K1 signaling and progesterone secretion. In summary, the LH receptor stimulates a unique series of intracellular signals to activate MTOR/S6K1 signaling. Furthermore, LH-directed changes in AMPK and GSK3B phosphorylation appear to exert a greater impact on progesterone synthesis in the corpus luteum than rapamycin-sensitive MTOR-mediated events.

Prostaglandin F2α Represses IGF-I-Stimulated IRS1/Phosphatidylinositol-3-Kinase/AKT Signaling in the Corpus Luteum: Role of ERK and P70 Ribosomal S6 Kinase

Little is known about the early intracellular events that contribute to corpus luteum regression. Experiments were designed to determine the effects of prostaglandin F2alpha (PGF2alpha) on phosphatidylinositol-3-kinase (PI3K)/Akt signaling in the corpus luteum in vivo and in vitro. Treatment of midluteal-phase cows with a luteolytic dose of PGF2alpha resulted in a rapid increase in ERK and mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K1) signaling and a rapid suppression of Akt phosphorylation in luteal tissue. In vitro treatment of primary cultures of luteal cells with PGF2alpha also resulted in an increase in ERK and mTOR/p70S6K1 signaling and a diminished capacity of IGF-I to stimulate PI3K, Akt, and protein kinase C zeta activation. Accounting for the reductions in PI3K and Akt activation observed in response to PGF2alpha treatment, we found that PGF2alpha promoted the phosphorylation of serine residues (307, 612, 636) in the insulin receptor substrate 1 (IRS1) peptide sequence in vivo and in vitro. Serine phosphorylation of IRS1 was associated with reduced formation of IGF-I-stimulated IRS1/PI3Kp85 complexes. Furthermore, treatment with inhibitors of the MAPK kinase 1/ERK or mTOR/p70S6K1 signaling pathways prevented PGF2alpha-induced serine phosphorylation of IRS1 and abrogated the inhibitory actions of PGF2alpha on Akt activation. Taken together, these experiments provide compelling evidence that PGF2alpha treatment stimulates IRS1 serine phosphorylation, which may contribute to a diminished capacity to respond to IGF-I. It seems likely that the rapid changes in phosphorylation events are among the early events that mediate PGF2alpha-induced corpus luteum regression.

Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells

CONTEXT Previous studies suggest that aging in women is associated with a reduction in hypoglycosylated forms of FSH. OBJECTIVE Experiments were performed to determine whether glycosylation of the FSHβ subunit modulates the biological activity of FSH in human granulosa cells. DESIGN AND SETTING Recombinant human FSH (hFSH) derived from GH3 pituitary cells was purified into fractions containing hypoglycosylated hFSH(21/18) and fully glycosylated hFSH(24). The response to FSH glycoforms was evaluated using the well-characterized, FSH-responsive human granulosa cell line, KGN at an academic medical center. INTERVENTIONS Granulosa cells were treated with increasing concentrations of fully- or hypoglycosylated FSH glycoforms for periods up to 48 hours. MAIN OUTCOME MEASURE(S) The main outcomes were indices of cAMP-dependent cell signaling and estrogen and progesterone synthesis. RESULTS We observed that hypoglycosylated FSH(21/18) was significantly more effective than fully glycosylated FSH(24) at stimulating cAMP accumulation, protein kinase A (PKA) activity, and cAMP response element binding protein (CREB) (S133) phosphorylation. FSH(21/18) was also much more effective than hFSH(24) on the stimulation CREB-response element-mediated transcription, expression of aromatase and STAR proteins, and synthesis of estrogen and progesterone. Adenoviral-mediated expression of the endogenous inhibitor of PKA, inhibited FSH(21/18)- and FSH(24)-stimulated CREB phosphorylation, and steroidogenesis. CONCLUSIONS Hypoglycosylated FSH(21/18) has greater bioactivity than fully glycosylated hFSH(24), suggesting that age-dependent decreases in hypoglycosylated hFSH contribute to reduced ovarian responsiveness. Hypoglycosylated FSH may be useful in follicle stimulation protocols for older patients using assisted reproduction technologies.

ATF3 Expression in the Corpus Luteum: Possible Role in Luteal Regression†

The present study investigated the induction and possible role of activating transcription factor 3 (ATF3) in the corpus luteum. Postpubertal cattle were treated at midcycle with prostaglandin F2α(PGF) for 0-4 hours. Luteal tissue was processed for immunohistochemistry, in situ hybridization, and isolation of protein and RNA. Ovaries were also collected from midluteal phase and first-trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to obtain purified small (SLCs) and large luteal cells (LLCs). Real-time PCR and in situ hybridization showed that ATF3 mRNA increased within 1 hour of PGF treatment in vivo. Western blot and immunohistochemistry demonstrated that ATF3 protein was expressed in the nuclei of LLC within 1 hour and was maintained for at least 4 hours. PGF treatment in vitro increased ATF3 expression only in LLC, whereas TNF induced ATF3 in both SLCs and LLCs. PGF stimulated concentration- and time-dependent increases in ATF3 and phosphorylation of MAPKs in LLCs. Combinations of MAPK inhibitors suppressed ATF3 expression in LLCs. Adenoviral-mediated expression of ATF3 inhibited LH-stimulated cAMP response element reporter luciferase activity and progesterone production in LLCs and SLCs but did not alter cell viability or change the expression or activity of key regulators of progesterone synthesis. In conclusion, the action of PGF in LLCs is associated with the rapid activation of stress-activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during luteal regression. ATF3 appears to affect gonadotropin-stimulated progesterone secretion at a step or steps downstream of PKA signaling and before cholesterol conversion to progesterone.

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.

Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions.

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these four cell types. Analysis of the RNA present in each bovine cell type using Affymetrix microarrays yielded new cell-specific genetic markers, functional insight into the behavior of each cell type via Gene Ontology Annotations and Ingenuity Pathway Analysis, and evidence of small and large luteal cell lineages using Principle Component Analysis. Enriched expression of select genes for each cell type was validated by qPCR. This expression analysis offers insight into cell-specific behaviors and the differentiation process that transforms somatic follicular cells into luteal cells.

Transcriptomes of bovine ovarian follicular and luteal cells

Affymetrix Bovine GeneChip® Gene 1.0 ST Array RNA expression analysis was performed on four somatic ovarian cell types: the granulosa cells (GCs) and theca cells (TCs) of the dominant follicle and the large luteal cells (LLCs) and small luteal cells (SLCs) of the corpus luteum. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE83524). Subsequent ANOVA determined genes that were enriched (≥2 fold more) or decreased (≤−2 fold less) in one cell type compared to all three other cell types, and these analyzed and filtered datasets are presented as tables. Genes that were shared in enriched expression in both follicular cell types (GCs and TCs) or in both luteal cells types (LLCs and SLCs) are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values is shown as a figure. These data have been further analyzed and interpreted in the companion article “Gene expression profiling of ovarian follicular and luteal cells provides insight into cellular identities and functions” (Romereim et al., 2017) [1].