Biology subjects

Douglas, N. C.

Publications and source records attributed to Douglas, N. C..

4 recordsLinked to original sources

bioRxiv2024

Ovarian disrupting effects and mechanisms of long- and short-chain per- and polyfluoroalkyl substances in mice

BackgroundThe extensive use of per- and polyfluoroalkyl substances (PFAS) has led to environmental contamination and bioaccumulation. Previous research linked PFAS exposure to female reproductive disorders, but the mechanism remains elusive. Further, most studies focused on legacy long-chain PFOA and PFOS, yet the reproductive impacts of other long-chain PFAS and short-chain alternatives are rarely explored. ObjectivesWe investigated the effects and mechanisms of long- and short-chain PFAS on the ovary and associated ovarian functions. MethodsA 3D in vitro ovarian follicle culture system and an in vivo mouse model, together with approaches of reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, RNA-sequencing, pharmacological treatment, in situ zymography, histology, in situ hybridization, analytical chemistry, and benchmark dose modeling (BMD), were used to test environmentally relevant exposure levels of six long- and short-chain PFAS on follicle maturation, hormone secretion, and ovulation. ResultsIn vitro exposure revealed that long-but not short-chain PFAS interfered with gonadotropin-dependent follicle maturation, ovulation, and hormone secretion. Mechanistically, long-chain perfluorononanoic acid (PFNA) acted as a peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonist in granulosa cells to disrupt follicle-stimulating hormone (FSH)-dependent follicle maturation, luteinizing hormone (LH)-stimulated ovulation, and associated gene regulatory pathways. In vivo mouse exposure confirmed the ovarian accumulation of PFNA and the mechanism of PPAR{gamma}-mediated ovarian toxicities of PFNA observed in vitro. The BMD analysis of in vitro and in vivo results suggested human relevant exposure levels of long-chain PFAS in our study pose an extra risk of ovarian defects, with follicular rupture as the most sensitive endpoint. DiscussionUsing in vitro follicle culture and in vivo mouse models, we discovered that long-chain PFAS interfere with gonadotropin-dependent follicle maturation, hormone secretion, and ovulation, posing a non-negligible risk to womens reproductive health including anovulation, irregular menstrual cycles, and sub- or infertility.

pharmacology and toxicology↗

bioRxiv2024

Dose-response functional and transcriptomic effects of follicle-stimulating hormone on ex vivo mouse folliculogenesis

The gonadotropin-dependent phase of ovarian folliculogenesis primarily requires follicle-stimulating hormone (FSH) to support one or multiple antral follicles, dependent on the species, to mature fully, enabling ovarian steroidogenesis, oogenesis, and ovulation to sustain female reproductive cycles and fertility. FSH binds to its membrane receptor in granulosa cells to activate various signal transduction pathways and gene regulatory networks. Poor female reproductive outcomes can result from both FSH insufficiency owing to genetic or non-genetic factors and FSH excess as encountered with ovarian stimulation in assisted reproductive technology (ART), but the underlying molecular mechanisms remain elusive. Herein, we conducted single-follicle and single-oocyte RNA sequencing analysis along with other approaches in an ex vivo mouse folliculogenesis and oogenesis system to investigate the effects of different concentrations of FSH on key follicular events. Our study revealed that a minimum FSH threshold is required for follicle maturation into the high estradiol-secreting preovulatory stage, and the threshold is moderately variable among individual follicles. FSH at subthreshold, threshold, and suprathreshold levels induced distinct expression patterns of follicle maturation-related genes and the follicular transcriptomics. The RNA-seq analysis identified novel genes and signaling pathways that may critically regulate follicle maturation. Suprathreshold FSH resulted in multiple ovarian disorders including premature luteinization, high production of androgen and proinflammatory factors, and reduced expression of energy metabolism-related genes in oocytes. Together, this study improves our understanding of gonadotropin-dependent folliculogenesis and provides crucial insights into how high doses of FSH used in ART may impact follicular health, oocyte quality, pregnancy outcome, and systemic health.

molecular biology↗

bioRxiv2022

Endometrial adhesion G protein-coupled receptors are dynamically expressed across the menstrual cycle and expression is altered by ovarian stimulation

Ovarian stimulation (OS), utilized for the development of multiple ovarian follicles for IVF, induces supraphysiologic levels of E2 and an early rise in P4 that disrupt endometrial differentiation and decreases implantation rates or result in placental insufficiency and pregnancy complications. To improve pregnancy rates and reduce the risk of pregnancy complications associated with IVF, it is crucial to advance our molecular understanding of the molecular regulation of endometrial differentiation. Previous studies from our laboratory suggest G protein-coupled receptors (GPCRs) are important regulators of endometrial differentiation. To investigate this further, using a retrospective dataset, we identified all GPCRs expressed across the proliferative and secretory phase of the menstrual cycle and found that many members of the adhesion G protein-coupled receptor (ADGR) family are dynamically expressed. For each ADGR subfamily exhibiting differentially-expressed genes across the cycle, their expression was investigated by RT-PCR in the non-pregnant mouse uterus and decidua on E7.5 of pregnancy. For those genes expressed in the E7.5 decidua, their expression was further quantified by qPCR across early mouse pregnancy. The RT-PCR screen revealed expression of 13 ADGRs (4 of the 9 subfamilies) in E7.5 decidua and among these genes, many were differentially expressed between E0.5 and E5.5 or 6.5 and between E5.5 and E6.5. The dynamic expression of the ADGRs across the menstrual cycle and in early mouse pregnancy, suggests these ADGRs are E2- and/or P4-regulated genes. We therefore hypothesized that for these ADGR genes, mRNA expression would be disrupted in an OS cycle. This hypothesis was tested on endometrial biopsies collected in the secretory phase from prospective cohorts of women in natural and OS cycles. Consistent with the retrospective dataset, our data revealed that members of the ADGR gene family are expressed in the secretory phase of the natural menstrual cycle and for the first time, we show that their expression is altered by ovarian stimulation.

cell biology↗

bioRxiv2022

Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cell Secretome Alters Gene Expression and Upregulates Motility of Human Endometrial Stromal Cells

IntroductionCyclic regeneration of the endometrium, and its repair after parturition or injury, are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, the mechanisms remain unclear. This study tested the hypothesis that the secretome of MSCs from human BM and UC upregulates human endometrial stromal cell (HESC) proliferation, migration and invasion, and activates pathways to increase HESC motility. MethodsMSCs were purchased from ATCC (BM-MSC-1) and cultured from the BM aspirate of three healthy female donors (BM-MSC-2-4), and from umbilical cords of two healthy male term infants (UC-MSC-1-2). Indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system studied the effect of the BM-MSC and UC-MSC secretome on HESC proliferation, migration, and invasion. To study the effect of the MSC secretome on HESC gene expression, HESCs were exposed to the BM-MSC secretome via indirect co-culture for 24 h. Total RNA was extracted from HESCs for RNA sequencing (RNA-Seq). Differentially expressed genes (DEG) and significantly altered pathways were identified. MSigDB was used to identify the top 15 enriched biological pathways (padj < 0.05). RT-qPCR was performed to validate changes in mRNA expression of DEG common to both BM-MSC exposures. Given robust upregulation of CCL2 mRNA expression in HESCs exposed to the BM- and UC-MSC secretomes, transwell migration and invasion assays were performed to determine the effect of recombinant CCL2 on HESC motility. Statistical significance was defined as p<0.05. ResultsIndirect co-culture of HESCs with BM- or UC-MSCs resulted in significant increase in HESC migration and invasion regardless of the source of MSCs. However, effects on cellular proliferation varied among the MSC donors. Exposure of HESCs to the secretome of BM-MSCs changed the expression of 10,139 genes with FDR < 0.05. There was overlap among 4350 genes between HESCs exposed to BM-MSC-1 and BM-MSC-2. Within four biological pathways enriched in HESCs, 4 genes (CCL2, HGF, PLAU, and BDKRB2) were differentially expressed in HESCs that had been cocultured with BM-MSC-1 and BM-MSC-2. qRT-PCR showed significantly increased mRNA expression of CCL2 in HESCs exposed to BM-MSC-1 (5-fold) and BM-MSC-2 (7.7-fold). In contrast, the increase in HGF expression was significant after exposure to BM-MSC-2 (1.8-fold) but not BM-MSC-1. Exposure to the UC-MSC secretome had similar effects on HESC-derived CCL2 and HGF levels. CCL2 expression was significantly increased (6.5-fold) by UC-MSC-2 but not by UC-MSC-1; HGF expression was significantly increased (1.6-fold) by UC-MSC-2 but not by UC-MSC-1. Validation studies indicated that exposure to recombinant CCL2 for 48 hours significantly increased HESC migration (1.2-fold) and invasion (1.4-fold). These data suggest that CCL2 is a key factor in mediating MSC-induced HESC motility. ConclusionIncreased HESC motility by the secretome of BM- and UC-MSC appears to be mediated by paracrine and autocrine mechanisms, in part by upregulated CCL2 expression in HESC. Together, our data support the potential for leveraging the MSC secretome as a novel cell-free therapy in the treatment of disorders of endometrial regeneration.

cell biology↗