Steroidogenic Cells Research Articles

Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 μM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors

BackgroundThe absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored.ResultsBy differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells.ConclusionsOur findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

A cholesterol-centric outlook on steroidogenesis.

Cholesterol, an essential and versatile lipid, is the precursor substrate for the biosynthesis of steroid hormones, and a key structural and functional component of organelle membranes in eukaryotic cells. Consequently, the framework of steroidogenesis across main steroidogenic cell types is built around cholesterol, including its cellular uptake, mobilization from intracellular storage, and finally, its transport to the mitochondria where steroidogenesis begins. This setup, which is controlled by different trophic hormones in their respective target tissues, allows steroidogenic cells to meet their steroidogenic need of cholesterol effectively without impinging on the basic need for organelle membranes and their functions. However, our understanding of the basal steroidogenesis (i.e., independent of trophic hormone stimulation), which is a cell-intrinsic trait, remains poor. Particularly, the role that cholesterol itself plays in the regulation of steroidogenic factors and events in steroid hormone-producing cells remains largely unexplored. This is likely because of challenges in selectively targeting the steroidogenic intracellular cholesterol pool in studies. New evidence suggests that cholesterol plays a role in steroidogenesis. These new findings have created new opportunities to advance our understanding in this field. In this book chapter, we will provide a cholesterol-centric view on steroidogenesis and emphasize the importance of the interplay between cholesterol and the mitochondria in steroidogenic cells. Moreover, we will discuss a novel mitochondrial player, prohibitin-1, in this context. The overall goal is to provide a stimulating perspective on cholesterol as an important regulator of steroidogenesis (i.e., more than just a substrate for steroid hormones) and present the mitochondria as a potential cell-intrinsic factor in regulating steroidogenic cholesterol homeostasis.

Downregulation of the Expression of Steroidogenic Acute Regulatory Protein and Aromatase in Steroidogenic KGN Human Granulosa Cells after Exposure to Bisphenol A.

Although increasing evidence of cause-and-effect relationship between BPA exposure and female reproductive disorders have been suggested through many studies, the precise biochemical and molecular mechanism(s) by which BPA interferes with steroidogenesis in the ovarian cells still remain unclear. Therefore, the purpose of this study was to discover the steroidogenic biomarker(s) associated with BPA treatment in human granulosa cell line, KGN. In this study, our results obtained via the analysis of steroidogenesis-related protein expression in KGN cells using quantitative polymerase chain reaction (qPCR) and western blot analyses revealed that the expression levels of steroidogenic acute regulatory (StAR) and aromatase decreased considerably and gradually after BPA treatment in a dose-dependent manner under BPA treatment. Further, remarkable decreases in their expression levels at the cellular levels were also confirmed via immunocytochemistry, and subsequent StAR and aromatase mRNA expression levels showed profiles similar to those observed for their proteins, i.e., both StAR and aromatase mRNA expression levels were significantly decreased under BPA treatment at concentrations ≥0.1 μM. We observed that follicle stimulating hormone upregulated StAR and aromatase protein expression levels; however, this effect was suppressed in the presence of BPA. Regarding the steroidogenic effects of BPA on KGN cells, controversies remain regarding the ultimate outcomes. Nevertheless, we believe that the results here presented imply that KGN cells have a good cellular and steroidogenic machinery for evaluating endocrine disruption. Therefore, StAR and aromatase could be stable and sensitive biomarkers in KGN cells for the cellular screening of the potential risk posed by exogenous and environmental chemicals to female reproductive (endocrine) function.

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

In vitro evaluation of exocytosis-associated SNARE molecules in human granulosa cells in polycystic ovary syndrome.

Patients with polycystic ovarian morphology (PCOM) make up 20% cases forassisted reproductive technology (ART). Folliculogenesis is impaired in PCOS. Signaling molecules are involved in follicle development. Dysregulations of intrafollicular environment and signaling molecules are observed in PCOS. Granulosa cells (GCs) and oocytes secrete molecules into follicular fluid by exocytosis of SNAREs. The aim of this study is to evaluate vesicle transport and vesicle fusion proteins (SNAREs) in GCs from PCOS patients who have undergone IVF treatment. Follicular fluids were collected from patients who undergo IVF/ICSI with the diagnosis of male factor (n = 10) and PCOS (n = 10) patients. GCs were separated and cultured. Each group of GCs was stimulated with FSH-hCG. The cells were examined under electron microscope. Immunofluorescent labeling was performed on cells for Stx6, SNAP25, StxBP1, FSHr, and KITL. Integrated density was analyzed from images of Stx6, SNAP25, StxBP1, FSHr, and KITL. Intercellular communication occurs by signal molecules; Stx6, SNAP25, and StxBP1 fusion proteins involved in exocytosis were decreased in the GCs of PCOS. There was no increase in in vitro stimulation with FSH-hCG either. In the electron microscope, it was observed that exocytosis of the vesicles was disrupted. Exocytosis and vesicular dynamics are among the basic physiological functions of human steroidogenic granulosa cells. Follicle development is necessary for production of competentoocytes and ovulation. Understanding the pathophysiology of PCOS at follicular level is important for diseasemanagement. According to our findings, deficits in vesicular dynamics of human granulosa cells in may be central to the treatment strategy forPCOS patients.

Cholesterol Hydroperoxide Co-trafficking in Testosterone-generating Leydig Cells: GPx4 Inhibition of Cytotoxic and Anti-steroidogenic Effects

Trafficking of intracellular cholesterol (Ch) to and into mitochondria of steroidogenic cells is required for steroid hormone biosynthesis. This trafficking is typically mediated by one or more proteins of the steroidogenic acute regulatory (StAR) family. Our previous studies revealed that 7-OOH, a redox-active cholesterol hydroperoxide, could be co-trafficked with Ch to/into mitochondria of MA-10 Leydig cells, thereby inducing membrane lipid peroxidation (LPO) which impaired progesterone biosynthesis. These negative effects of 7-OOH were inhibited by endogenous selenoperoxidase GPx4, indicating that this enzyme could protect against 7-OOH-induced oxidative damage/dysfunction. In the present study, we advanced our Leydig focus to cultured murine TM3 cells and then to primary cells from rat testis, both of which produce testosterone. Using a fluorescent probe, we found that extensive free radical-mediated LPO occurred in mitochondria of stimulated primary Leydig cells during treatment with liposomal Ch+7-OOH, resulting in a significant decline in testosterone output relative to that with Ch alone. Strong enhancement of LPO and testosterone shortfall by RSL3 (a GPx4 inhibitor) and reversal thereof by Ebselen (a GPx4 mimetic), suggested that endogenous GPx4 was playing a key antioxidant role. 7-OOH in increasing doses was also cytotoxic to these cells, RSL3 exacerbating this in Ebselen-reversable fashion. Moreover, GPx4 knockdown increased cell sensitivity to LPO with reduced testosterone output. These findings, particularly with primary Leydigs (which best represent cells in intact testis) suggest that GPx4 plays a key protective role against peroxidative damage/dysfunction induced by 7-OOH co-trafficking with Ch.

Autophagic and apoptotic proteins in goat corpus luteum and the effect of Adiponectin/AdipoRon on luteal cell autophagy and apoptosis

The most abundant adipokine Adiponectin (APN) is present in ovaries. AdipoRon is a small molecule oral APN receptor agonist that binds and activates APN receptors. However, the function of APN/AdipoRon in regulation of luteal cell processes has not been elucidated. To investigate autophagic and apoptotic proteins in goat CLs and effects of APN/AdipoRon on goat luteal autophagy and apoptosis, goat CLs were collected during the early, mid and late luteal stages of the estrous cycle to evaluate autophagic and apoptotic protein patterns. LC3B, Beclin 1, Caspase-3 and Bax/Bcl-2 as well as p-AMPK were differentially abundant at different stages of CL development. All these proteins were primarily localized in large and small luteal steroidogenic cells. Then, isolated luteal steroidogenic cells were evaluated to ascertain the functions and mechanism of APN/AdipoRon in luteal autophagy and apoptosis. Treatment with AdipoRon (25 and 50 μM) and APN (1 μg/mL) for 48 h resulted in a decrease in cell viability and P4 level, increased autophagic and apoptotic proteins. Treatment with AdipoRon (25 μM) led to rapid and transient p-AMPK activation, with p-AMPK elevated at 30 min to 1 h with there being a return to a basal concentration at 2 h post-treatment. Moreover, treatment with AdipoRon led to an increase in autophagy by activating AMPK, which was markedly reduced with treatment with an AMPK inhibitor Compound C and siAMPK, however, abundances of apoptotic proteins were not affected by these treatments. In conclusion, autophagy and apoptosis are involved in the structural regression of goat CL. APN/AdipoRon led to a lesser cell viability and P4 concentration, and activated autophagy through induction of the AMPK while there was induction of apoptosis through an AMPK - independent pathway in goat luteal cells.

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, invitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency.

Ovary of Zebrafish during Spawning Season: Ultrastructure and Immunohistochemical Profiles of Sox9 and Myostatin.

This study sought to examine the ovarian cellular and stromal components of the zebrafish (Danio rerio) throughout the spawning season using light and electron microscopic tools. The ovaries of zebrafish showed oocytes in all stages of follicular development and degeneration (atresia). Six stages of oogenesis were demonstrated: oogonia, early oocytes, late oocytes, vacuolated follicles, the yolk globule stage (vitellogenesis), and mature follicles. The SOX9 protein was expressed in the ooplasm of the primary and previtellogenic oocytes and the theca cell layer of the mature follicles. Myostatin was expressed in the granulosa and theca cells. Many stem cells in the ovarian stroma expressed myostatin and SOX9. During the spawning season, the EM results indicated that the zona radiata increased in thickness and was crossed perpendicularly by pore canals that contained processes from both oocytes and zona granulosa. The granulosa cells contained many mitochondria, rER, sER, and vesicles. Meanwhile, the thecal layer consisted of fibroblast-like cells. Atretic follicles could be demonstrated that involved both oocytes and their follicular walls. Several types of cells were distinguished in the ovarian stroma, including mast cells, telocytes, lymphocytes, fibroblasts, endocrine cells, macrophages, adipocytes, dendritic cells, and steroidogenic (stromal) cells. The ovary of the zebrafish serves as a model to investigate follicular development.

Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17β-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17β-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

OR08-01 Expression of Gonadal Differentiation Markers in Bilateral Macronodular Adrenocortical Disease

Abstract Disclosure: K. De Sousa: None. V. Pautasso: None. C. Duparc: None. S. Renouf: None. B. Ragazzon: None. F. Violon: None. L. Bouys: None. F. Bonnet-Serrano: None. M. Sibony: None. N. Driessens: None. S. Espiard: None. G. Raverot: None. J.Y. Bertherat: None. E. Louiset: None. H. Lefebvre: None. Context: Bilateral macronodular adrenocortical disease (BMAD) is a rare cause of primary Cushing syndrome, occurring mostly in women. ARMC5 and KDM1A mutations are responsible for distinct forms of BMAD. In particular, KDM1A mutations are associated with food-dependent Cushing syndrome. In addition, BMAD tissues can express illegitimate receptors, including LH receptor, that lead to aberrant control of cortisol secretion by various factors. Moreover, steroidogenic cells are able to abnormally produce bioactive factors, such as ACTH and the Leydig cell hormone INSL3. Since LH receptor, ACTH and INSL3 are known to be produced in gonads, we speculated on a gonadal-like differentiation of adrenocortical cells in BMAD tissues. Aim: The aim of our study was to investigate the presence of different gonadal markers in a series of BMAD tissues, and to search for secretion of adrenal and gonadal hormones by BMAD cells. Methodology: Gene expression of gonadal markers were evaluated by RNAseq. Immunohistochemical studies were performed on 41 BMAD and 9 normal adrenal samples to examine the presence of hormones (ACTH, INSL3, anti-Mullerian hormone [AMH]), the estrogen synthetizing enzyme aromatase, and transcription factors (GATA4, SOX9, FOXL2, MAGEA4) known to be expressed in testis or ovary. Perifusion experiments, coupled to immunoassays or liquid chromatography/mass spectrometry, were conducted to quantify hormone secretion by 2 adrenal explants. Results: BMAD tissues were collected from patients (60% women) without or with ARMC5 (32%) or KDM1A (13%) mutations. ARMC5-mutated tissues overexpressed FOXL2 and CYP19A1 (encoding aromatase) in comparison with other BMAD samples. In normal adrenals, cortisol-producing cells, located in the zona fasciculata, were immunonegative for INSL3, AMH, aromatase, GATA4 and SOX9, whereas FOXL2 and MAGEA4 immunostainings were detected in the cortex. In BMAH tissues, the presence of positive cells for ACTH (100% of BMAD), INSL3 (90%), AMH (50%), aromatase (77%), SOX9 (92%), GATA4 (97%), FOXL2 (100%) and MAGEA4 (100%) were observed in most of the samples regardless of the gender or mutational status. FOXL2 and MAGEA4 immunostainings were detected throughout BMAD tissues. Distribution of other gonadal markers was heterogenous in tissue sections with clusters of cells positive for ACTH, aromatase and GATA4 or ACTH and SOX9. Functional studies revealed pulsatile secretion of different glucocorticoids and INSL3 by BMAD explants. ACTH and hCG stimulated steroid production but were without effect on INSL3 release. Conclusion: These data show that steroidogenic cells in BMAD tissues exhibit a gonadal-type phenotype and abnormally secrete INSL3. They suggest that BMAD may result from a genetically driven defect occurring during early embryogenesis and affecting differentiation and development of adrenal tissues. Presentation: Friday, June 16, 2023

SAT014 Involvement Of Cytochrome P450 1B1 In Synthesis Of Neurosteroid Pregnenolone In Human Brain Cells

Abstract Disclosure: Y. Lin: None. G. Cheung: None. Z. Zhang: None. V. Papadopoulos: None. Neurosteroids, steroids synthesized in the nervous system, have important functions in modulating brain function, neuroprotection, and reducing neuroinflammation. The neurosteroid pregnenolone, the precursor to all other neurosteroids, is one of the most abundant neurosteroids in the brain. Still, the enzyme known to make it, CYP11A1, has been difficult to detect in the human brain. We recently reported that unlike in peripheral steroidogenic organs, pregnenolone is not synthesized by CYP11A1 in human brain cells but rather by another mitochondrial cytochrome P450 enzyme (J Biol Chem. 2022 Jul;298(7):102110). Therefore, we aimed to identify the enzyme responsible for producing pregnenolone in the current study. Upon bioinformatic analyses of publicly available datasets for mitochondrial CYP450s expressed in the human brain, we identified three potential candidates: CYP27A1, CYP1A1, and CYP1B1. Given that CYP27A1 is highly expressed in the brain and is involved in cholesterol metabolism, we first tested the effects of CYP27A1 inhibition via drug treatment and siRNA knockdown on pregnenolone synthesis in steroidogenic MGM-1 human glioma cells. Neither treatment with CYP27A1 inhibitors anastrozole or dexmedetomidine nor CYP27A1 siRNA knockdown resulted in a significant reduction in pregnenolone formation by MGM-1 cells, suggesting that CYP27A1 likely is not involved in pregnenolone synthesis in human brain cells. On the other hand, MGM-1 cells treated with the CYP1A1/CYP1B1 inhibitors α-napthoflavone and 2,4,3’,5’-tetramethoxystilbene showed a significant reduction in pregnenolone production in the presence of the pregnenolone precursor 22R-hydroxycholesterol. To investigate whether this effect was due to the inhibition of CYP1A1 or CYP1B1, we knocked down CYP1A1 or CYP1B1 in MGM-1 cells using siRNA. While CYP1A1 knockdown did not result in any significant changes to pregnenolone synthesis, CYP1B1 knockdown significantly decreased secreted pregnenolone formation by MGM-1 cells. Together, these results suggest that CYP1B1 is likely involved in the alternative pathway for pregnenolone synthesis in human brain cells. Presentation: Saturday, June 17, 2023

OR19-03 SIK2 Regulates Granulosa Cell Response to Exogenous Gonadotropins

Abstract Disclosure: E. Hayes: None. O. Lakomy: None. M. Hassan: None. R. Filzen: None. M. Rodriguez Esquivel: None. M. Foretz: None. C.O. Stocco: Grant Recipient; Self; NIH R01HD097202. Forty percent of cases of infertility are attributed to ovulatory defects. Successful ovulation requires the coordinated development of the ovarian follicle, the functional unit of the ovary that is composed of the oocyte surrounded by steroidogenic granulosa cells. However, the mechanisms that govern follicle development remain incompletely understood. Previously, our group showed that loss of salt-inducible kinase 2 (SIK2) in a murine knockout model resulted in a significantly increased number of ovulated oocytes in response to an ovarian hyperstimulation protocol. Here, we aimed to further characterize the reproductive phenotype of SIK2 knockout mice, as well as to understand the role of SIK2 in granulosa cells by creating and characterizing a granulosa cell-specific knockdown of SIK2 using the Cre-Lox system. Loss of SIK2 resulted in no changes in the number of litters or litter size as well as a normal estrus cycle. Interestingly, granulosa cell-specific knockdown of SIK2 resulted in a significantly increased number of ovulated oocytes in response to ovarian hyperstimulation. However, SIK2 knockout mice do not ovulate more oocytes under physiologic conditions. Furthermore, knockdown of SIK2 in granulosa cells resulted in significantly increased Cyp19a1 gene expression and serum estradiol concentration. The data reveal that SIK2 plays an important role in the regulation of the response of granulosa cells to gonadotropins. These data support the potential of SIK2 as a pharmacologic target for improving oocyte yield for women receiving gonadotropins during in vitro fertilization as a treatment to trigger follicular maturation and ovulation. SIK2-regulated mechanisms could reveal new targets for the development of innovative methods of contraception. Presentation Date: Saturday, June 17, 2023

Mechanisms of angioregression of the corpus luteum.

The corpus luteum is a transient ovarian endocrine gland that produces the progesterone necessary for the establishment and maintenance of pregnancy. The formation and function of this gland involves angiogenesis, establishing the tissue with a robust blood flow and vast microvasculature required to support production of progesterone. Every steroidogenic cell within the corpus luteum is in direct contact with a capillary, and disruption of angiogenesis impairs luteal development and function. At the end of a reproductive cycle, the corpus luteum ceases progesterone production and undergoes rapid structural regression into a nonfunctional corpus albicans in a process initiated and exacerbated by the luteolysin prostaglandin F2α (PGF2α). Structural regression is accompanied by complete regression of the luteal microvasculature in which endothelial cells die and are sloughed off into capillaries and lymphatic vessels. During luteal regression, changes in nitric oxide transiently increase blood flow, followed by a reduction in blood flow and progesterone secretion. Early luteal regression is marked by an increased production of cytokines and chemokines and influx of immune cells. Microvascular endothelial cells are sensitive to released factors during luteolysis, including thrombospondin, endothelin, and cytokines like tumor necrosis factor alpha (TNF) and transforming growth factor β 1 (TGFB1). Although PGF2α is known to be a vasoconstrictor, endothelial cells do not express receptors for PGF2α, therefore it is believed that the angioregression occurring during luteolysis is mediated by factors downstream of PGF2α signaling. Yet, the exact mechanisms responsible for angioregression in the corpus luteum remain unknown. This review describes the current knowledge on angioregression of the corpus luteum and the roles of vasoactive factors released during luteolysis on luteal vasculature and endothelial cells of the microvasculature.

CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate

The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms’ tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

Prohibitin-1 is an ACTH-Regulated Protein in Human and Mouse Adrenocortical Cells and Plays a Role in Corticosteroid Production

Cell-intrinsic early events involved in different trophic hormone-induced steroidogenesis in their respective steroidogenic cell type are very similar. For example, the activation of the cAMP-PKA signaling pathway in response to trophic hormone stimulation and, subsequently, cholesterol transport to the mitochondria to initiate steroidogenesis is common to them. Recently, we have found that an evolutionarily conserved protein, prohibitin-1 (PHB1), is regulated by Luteinizing Hormone (LH) in murine Leydig cells and plays a role in interconnected cell signaling and mitochondrial steps pertaining to testosterone production. Among the primary steroidogenic tissues, PHB1 expression levels are highest in the adrenal cortex (The Human Protein Atlas); however, its regulation and role in adrenocortical cells are virtually unknown. We investigated the regulation and the role of PHB1 in adrenocortical cells in vitro using human HAC15 and mouse Y-1 cell culture models. It was found that Adrenocorticotrophic Hormone (ACTH) stimulation upregulates PHB1 levels in adrenocortical cells in a time-dependent manner. A similar effect on PHB1 levels was also observed in response to dibutyryl-cAMP stimulation, a cell-permeable analogue of cAMP (the second messenger for ACTH action). Moreover, manipulating PHB1 levels in adrenocortical cells affected mitochondria, lysosomes, and lipid droplet characteristics, modulated phospho-PKA and phospho-ERK1/2 levels, and altered corticosteroid production. This finding suggests that ACTH regulates PHB1 in adrenocortical cells and plays a role in corticosteroid production, which was previously unknown.

Naturally occurring variability in larval period length is associated with intraspecific differences in late tadpoles of a South American true toad

AbstractThe larval period in anurans is highly plastic. Even though larval period length and larval size have been the most studied characteristics showing intraspecific variability, other features such as morphology, behavior, food preferences and physiology have also been described as plastic. Metamorphic changes are triggered by thyroid and interrenal hormones, and environmental signals modulate the onset of metamorphosis via the hypothalamic–pituitary–interrenal axis. The South American bufonid toad Rhinella arenarum from nearby Buenos Aires City has a larval period of 30–120 days under the same environmental conditions. The goal of this study was to evaluate life‐history traits in tadpoles from different localities and potential morphological differences among tadpoles with different larval period length. For this purpose, we reared under natural conditions tadpoles from two different localities nearby Buenos Aires City and in three different years measured their size at the end of metamorphosis and recorded their larval period length. The external appearance, histo‐morphology and morphometry of the skin and kidney/interrenal complex were studied in metamorphs with short and long larval periods (SLP and LLP, respectively). We observed a larval period lasting 30–120 days in tadpoles from different localities or years. SLP metamorphs were smaller, darker, had less protruding eyes without eyelids, a thinner epidermis and fewer skin glands than LLP metamorphs. The secretion of skin glands from SLP tadpoles had acidic glycoconjugates. Kidney size was similar between LLP and SLP metamorphs, but the former had an adult‐like histomorphology. Interrenal gland in LLP early and late climax tadpoles had cells with a high nucleus/cell diameter ratio, a sign of highly active steroidogenic cells. Overall, these results show that intraspecific variability in size, skin and interrenal gland development is associated with differences in larval period length. Distinct skin gland number and secretion, along with distinct interrenal histology at late climax, could have physiological consequences in the post‐metamorphic life.

The Adrenal Gland of Squamata (Reptilia): A Comparative Overview.

The adrenal gland is a complex endocrine organ composed of two components: a steroidogenic tissue, which produces steroid hormones, and a chromaffin tissue, which mainly produces norepinephrine and epinephrine. Through evolution, their relationships with each other changed. They begin as isolated chromaffin and steroidogenic cell aggregates, typical of fish, and end with the advanced compact gland, typical of mammals, which consists of an external steroidogenic cortical zone and an internal chromaffin medullary zone. The adrenal gland of reptiles is unique because, with few exceptions, it is near the gonads and genital ducts, and the chromaffin and steroidogenic tissues are closely associated. However, the degree of mixing is variable. For example, in Squamata, the mixing degree of chromaffin and steroidogenic tissues, their reciprocal position in the gland, and the relative quantities of norepinephrine and epinephrine secreted by the chromaffin cells are extremely variable. This variability could be related to the phylogenetic history of the species. After a brief discussion of the adrenal gland and its main functions in vertebrates, this overview will examine the general characteristics of the adrenal gland of squamates, the differences in morphology of the gland, and the possible relationships with the phylogeny of the different species.

Classic genetic and hormonal switches during fetal sex development and beyond.

Critical genetic and hormonal switches characterize fetal sex development in humans. They are decisive for gonadal sex determination and subsequent differentiation of the genital and somatic sex phenotype. Only at the first glace these switches seem to behave like the dual 0 and 1 system in computer sciences and lead invariably to either typically male or female phenotypes. More recent data indicate that this model is insufficient. In addition, in case of distinct mutations, many of these switches may act variably, causing a functional continuum of alterations of gene functions and -dosages, enzymatic activities, sex hormone levels, and sex hormone sensitivity, giving rise to a broad clinical spectrum of biological differences of sex development (DSD) and potentially diversity of genital and somatic sex phenotypes. The gonadal anlage is initially a bipotential organ that can develop either into a testis or an ovary. Sex-determining region Y (SRY) is the most important upstream switch of gonadal sex determination inducing SOX9 further downstream, leading to testicular Sertoli cell differentiation and the repression of ovarian pathways. If SRY is absent (virtually "switched off"), e. g., in 46,XX females, RSPO1, WNT4, FOXL2, and other factors repress the male pathway and promote ovarian development. Testosterone and its more potent derivative, dihydrotestosterone (DHT) as well as AMH, are the most important upstream hormonal switches in phenotypic sex differentiation. Masculinization of the genitalia, i. e., external genital midline fusion forming the scrotum, growth of the genital tubercle, and Wolffian duct development, occurs in response to testosterone synthesized by steroidogenic cells in the testis. Müllerian ducts will not develop into a uterus and fallopian tubes in males due to Anti-Müllerian-Hormone (AMH) produced by the Sertoli cells. The functionality of these two hormone-dependent switches is ensured by their corresponding receptors, the intracellular androgen receptor (AR) and the transmembrane AMH type II receptor. The absence of high testosterone and high AMH is crucial for anatomically female genital development during fetal life. Recent technological advances, including single-cell and spatial transcriptomics, will likely shed more light on the nature of these molecular switches.