Sodium Trifluoroacetate mediated Copper-Catalyzed aza-Michael addition of α,ß-unsaturated olefins with aromatic amines.

We present a sodium trifluoroacetate (CF3CO2Na) mediated copper-catalyzed aza-Michael addition of aromatic amines with activated olefins under mild, aqueous reaction conditions. This simplistic protocol employs a copper catalyst (10 mol%) and water as solvent. This transformation occurs precisely with aromatic substituted amines containing both electron-donating (EDG) and electron-withdrawing (EWG) groups. A broad range of substrates were tested under the optimized conditions, which are producing good to moderate yields.

Exposure to a phthalate mixture disrupts ovulatory progesterone receptor signaling in human granulosa cells in vitro†.

Exposure to phthalates disrupts ovarian function. However, limited studies have investigated the effects of phthalate mixtures on ovulation, especially in women. Human granulosa cells were used to test the hypothesis that exposure to a phthalate mixture (PHTmix) disrupts progesterone (P4)/progesterone receptor (PGR) signaling, which is a crucial pathway for ovulation. In addition, progestin and cyclic adenosine 3', 5'-monophosphate (cAMP) supplementation were tested as methods to circumvent phthalate toxicity. Granulosa cells from women undergoing in vitro fertilization were acclimated in culture to regain responsiveness to human chorionic gonadotropin (hCG; clinical luteinizing hormone analogue). Granulosa cells were treated with or without hCG, and with or without PHTmix (1-500 µg/ml; dimethylsulfoxide = vehicle control) for 0.5-36 h. In the supplementation experiments, cells were treated with or without R5020 (stable progestin), and with or without 8-Br-cAMP (stable cAMP analogue). Exposure to hCG + PHTmix decreased P4 levels and mRNA levels of steroidogenic factors when compared to hCG. This was accompanied by decreased mRNA levels of PGR and downstream P4/PGR ovulatory mediators (ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), C-X-C motif chemokine receptor 4 (CXCR4), pentraxin 3 (PTX3), and regulator of G protein signaling 2 (RGS2)) in the hCG + PHTmix groups compared to hCG. Exposure to hCG + PHTmix 500 µg/ml decreased cAMP levels and protein kinase A activity compared to hCG. Supplementation with progestin in the hCG + PHTmix 500 µg/ml group did not rescue toxicity, while supplementation with cAMP restored PGR levels and downstream P4/PGR mediator levels to hCG levels. These findings suggest that phthalate mixture exposure inhibits P4/PGR signaling in human granulosa cells via decreased steroidogenesis, cAMP levels, and protein kinase A activity. Restored P4/PGR signaling with cAMP supplementation provides a potential cellular target for intervention of phthalate-induced ovulatory dysfunction in women.

A single-cell gene expression atlas of human follicular aspirates: Identification of leukocyte subpopulations and their paracrine factors.

Leukocytes are in situ regulators critical for ovarian function. However, little is known about leukocyte subpopulations and their interaction with follicular cells in ovulatory follicles, especially in humans. Single-cell RNA sequencing (scRNA-seq) was performed using follicular aspirates obtained from four IVF patients and identified 13 cell groups: one granulosa cell group, one thecal cell group, 10 subsets of leukocytes, and one group of RBC/platelet. RNA velocity analyses on five granulosa cell populations predicted developmental dynamics denoting two projections of differentiation states. The cell type-specific transcriptomic profiling analyses revealed the presence of a diverse array of leukocyte-derived factors that can directly impact granulosa cell function by activating their receptors (e.g., cytokines and secretory ligands) and are involved in tissue remodeling (e.g., MMPs, ADAMs, ADAMTSs, and TIMPs) and angiogenesis (e.g., VEGFs, PGF, FGF, IGF, and THBS1) in ovulatory follicles. Consistent with the findings from the scRNA-seq data, the leukocyte-specific expression of CD68, IL1B, and MMP9 was verified in follicle tissues collected before and at defined hours after hCG administration from regularly cycling women. Collectively, this study demonstrates that this data can be used as an invaluable resource for identifying important leukocyte-derived factors that promote follicular cell function, thereby facilitating ovulation and luteinization in women.

Ovulatory upregulation of angiotensin-converting enzyme 2, a receptor for SARS-CoV-2, in dominant follicles of the human ovary.

OBJECTIVE: To determine the temporal expression of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2, in dominant follicles throughout the periovulatory period in women and the regulatory mechanisms underlying ACE2 expression in human granulosa/lutein cells (hGLC). DESIGN: Experimental prospective clinical study and laboratory-based investigation. SETTING: University Medical Center and private in vitro fertilization center. PATIENT(S): Thirty premenopausal women undergoing surgery for tubal ligation and 16 premenopausal women undergoing in vitro fertilization. INTERVENTION(S): Administration of human chorionic gonadotropin (hCG) and harvesting of preovulatory/ovulatory follicles by timed laparoscopy, and collection of granulosa/lutein cells and cumulus cells at the time of oocyte retrieval. MAIN OUTCOME MEASURE(S): Expression and localization of ACE2 in granulosa cells and dominant follicles collected throughout the periovulatory period of the menstrual cycle and in hGLC using quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULT(S): ACE2 expression (mRNA and protein) is up-regulated in human ovulatory follicles after administration of hCG. ACE2 expression was higher in cumulus cells than in granulosa cells. hCG increased the expression of ACE2 in primary hGLC cultures; the increase was inhibited by RU486 (an antagonist for progesterone receptor and glucocorticoid receptor) and CORT125281 (a selective glucocorticoid receptor antagonist), but not by AG1478 (an EGF receptor tyrosine kinase inhibitor) or by dexamethasone. CONCLUSION(S): The hormone-regulated expression of ACE2 in granulosa cells suggests a potential role of ACE2 in the ovulatory process. These data also imply the possible impact of COVID-19 on a vital cyclic event of ovarian function and thus on women's overall reproductive health. However, SAR-CoV-2 infection in ovarian cells in vivo or in vitro has yet to be determined.

The FOS/AP-1 Regulates Metabolic Changes and Cholesterol Synthesis in Human Periovulatory Granulosa Cells.

FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.

FOS, a Critical Downstream Mediator of PGR and EGF Signaling Necessary for Ovulatory Prostaglandins in the Human Ovary.

Context: Fos null mice failed to ovulate and form a corpus luteum (CL) even when given exogenous gonadotropins, suggesting that ovarian Fos expression is critical for successful ovulation and CL formation. However, little is known about FOS in the human ovary. Objectives: To determine the expression, regulation, and function of FOS in human periovulatory follicles. Design/Participants: Timed periovulatory follicles were obtained from normally cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures: The in vivo expression after human chorionic gonadotropin (hCG) administration and in vitro regulation of FOS, JUN, JUNB, and JUND was evaluated at the mRNA and protein level. Binding of progesterone receptor (PGR) and FOS to their target genes was assessed by chromatin immunoprecipitation analyses. Prostaglandin E2 (PGE2) and progesterone were measured. Results: The expression of FOS, JUNB, and JUND drastically increased in ovulatory follicles after hCG administration. In human granulosa/lutein cell cultures, hCG increased the expression of FOS and JUN proteins. Inhibitors of PGR and epidermal growth factor (EGF) receptors reduced hCG-induced increases in the expression and phosphorylation of FOS. PGR bound to the FOS gene. A selective FOS inhibitor blocked hCG-induced increases in PGE2 and the expression of prostaglandin (PG) synthases and transporters (PTGES, SLCO2A1, and ABCC1). FOS bound to the promoter regions of these genes. Conclusions: The increase of FOS/activator protein 1 in human periovulatory follicles after hCG administration is mediated by collaborative actions of PGR and EGF signaling and critical for the upregulated expression of key ovulatory genes required for the rise in ovulatory PG in human granulosa cells.

Ovulatory Induction of SCG2 in Human, Nonhuman Primate, and Rodent Granulosa Cells Stimulates Ovarian Angiogenesis.

The luteinizing hormone (LH) surge is essential for ovulation, but the intrafollicular factors induced by LH that mediate ovulatory processes (e.g., angiogenesis) are poorly understood, especially in women. The role of secretogranin II (SCG2) and its cleaved bioactive peptide, secretoneurin (SN), were investigated as potential mediators of ovulation by testing the hypothesis that SCG2/SN is induced in granulosa cells by human chorionic gonadotropin (hCG), via a downstream LH receptor signaling mechanism, and stimulates ovarian angiogenesis. Humans, nonhuman primates, and rodents were treated with hCG in vivo resulting in a significant increase in the messenger RNA and protein levels of SCG2 in granulosa cells collected early during the periovulatory period and just prior to ovulation (humans: 12 to 34 hours; monkeys: 12 to 36 hours; rodents: 4 to 12 hours post-hCG). This induction by hCG was recapitulated in an in vitro culture system utilizing granulosa-lutein cells from in vitro fertilization patients. Using this system, inhibition of downstream LH receptor signaling pathways revealed that the initial induction of SCG2 is regulated, in part, by epidermal growth factor receptor signaling. Further, human ovarian microvascular endothelial cells were treated with SN (1 to 100 ng/mL) and subjected to angiogenesis assays. SN significantly increased endothelial cell migration and new sprout formation, suggesting induction of ovarian angiogenesis. These results establish that SCG2 is increased in granulosa cells across species during the periovulatory period and that SN may mediate ovulatory angiogenesis in the human ovary. These findings provide insight into the regulation of human ovulation and fertility.

The expression of CXCR4 is induced by the luteinizing hormone surge and mediated by progesterone receptors in human preovulatory granulosa cells.

The chemokine CXC motif ligand 12 (CXCL12) and its cognate receptor, CXCR4, have been implicated in the ovulatory process in various animal models. However, little is known about the expression and regulation of CXCL12 and CXCR4 and their functions during the ovulatory period in the human ovary. In this study, we characterized the expression patterns of CXCL12 and CXCR4 in preovulatory follicles collected before the luteinizing hormone (LH) surge and at defined hours after hCG administration in women with the regular menstrual cycle. The levels of mRNA and protein for CXCR4 were increased in granulosa cells of late ovulatory follicles, whereas CXCL12 expression was constant in follicles throughout the ovulatory period. Both CXCR4 and CXCL12 were localized to a subset of leukocytes around and inside the vasculature of human preovulatory follicles. Using a human granulosa cell culture model, the regulatory mechanisms and functions of CXCL12 and CXCR4 expression were investigated. Human chorionic gonadotropin (hCG) stimulated CXCR4 expression, whereas CXCL12 expression was not affected, mimicking in vivo expression patterns. Both RU486 (progesterone receptor antagonist) and CoCl2 (HIFs activator) blocked the hCG-induced increase in CXCR4 expression, whereas AG1478 (EGFR inhibitor) had no effect. The treatment with CXCL12 had no effect on granulosa cell viability but decreased hCG-stimulated CXCR4 expression.

Coordinated Regulation Among Progesterone, Prostaglandins, and EGF-Like Factors in Human Ovulatory Follicles.

Context: In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)-like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans. Objective: To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries. Design and Participants: Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures: The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured. Results: PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes. Conclusions: This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells.

Induction of proteinases in the human preovulatory follicle of the menstrual cycle by human chorionic gonadotropin.

OBJECTIVE: To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women to gain insight into their possible roles during ovulation and early luteinization. DESIGN: Experimental prospective clinical study and laboratory-based investigation. SETTING: University medical center and private IVF center. ANIMAL AND PATIENT(S): Thirty-eight premenopausal women undergoing surgery for tubal ligation and six premenopausal women undergoing assisted reproductive techniques. INTERVENTION(S): Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. MAIN OUTCOME MEASURE(S): Expression of mRNA for matrix metalloproteinase (MMPs) and the A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. RESULT(S): Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS9 mRNA increased in granulosa cells after hCG treatment, however, thecal cell expression for ADAMTS1 was unchanged, while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1, and ADAMTS9 to the granulosa and thecal cell layers. CONCLUSION(S): The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte.

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Initial experience with a donor egg bank.

OBJECTIVE: To report on the establishment of a commercial donor egg bank (CryoEggs International, LP) and to present our initial experience from the first four patients to receive eggs. DESIGN: Case report. SETTING: Private fertility clinic. PATIENT(S): The four recipient women were aged 43, 43, 40, and 33 years. All had cycle day FSH levels greater than 25 mIU/mL. All were given the option of fresh donor egg IVF but opted to use frozen donor oocytes. INTERVENTION(S): Purchased and quarantined frozen donor eggs were thawed and inseminated using intracytoplasmic sperm injection (ICSI). Subsequent embryos were transferred on day 3. MAIN OUTCOME MEASURE(S): Clinical pregnancy as defined by presence of cardiac activity. RESULT(S): There was a thawed egg survival rate of 76%, a fertilization rate of 74%, a pregnancy rate (PR) of 50%, with an average of 2.75 embryos per transfer and an implantation rate of 27%. CONCLUSION(S): Although very preliminary, these results indicate that more widespread use of frozen donor eggs obtained from a commercial egg bank may be feasible in the future, changing the landscape of donor egg IVF.