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.

Serum Amyloid A is not obligatory for high-fat, high-sucrose, cholesterol-fed diet-induced obesity and its metabolic and inflammatory complications.

Several studies in the past have reported positive correlations between circulating Serum amyloid A (SAA) levels and obesity. However, based on limited number of studies involving appropriate mouse models, the role of SAA in the development of obesity and obesity-related metabolic consequences has not been established. Accordingly, herein, we have examined the role of SAA in the development of obesity and its associated metabolic complications in vivo using mice deficient for all three inducible forms of SAA: SAA1.1, SAA2.1 and SAA3 (TKO). Male and female mice were rendered obese by feeding a high fat, high sucrose diet with added cholesterol (HFHSC) and control mice were fed rodent chow diet. Here, we show that the deletion of SAA does not affect diet-induced obesity, hepatic lipid metabolism or adipose tissue inflammation. However, there was a modest effect on glucose metabolism. The results of this study confirm previous findings that SAA levels are elevated in adipose tissues as well as in the circulation in diet-induced obese mice. However, the three acute phase SAAs do not play a causative role in the development of obesity or obesity-associated adipose tissue inflammation and dyslipidemia.

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.

Core Binding Factors are essential for ovulation, luteinization, and female fertility in mice.

Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfbflox/flox;Esr2cre/+;Runx2flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.