Adolescent exposure to a mixture of per- and polyfluoroalkyl substances (PFAS) depletes the ovarian reserve, increases ovarian fibrosis, and alters the hippo pathway in adult female mice.

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals known for their environmental persistence and resistance to biodegradation. This study investigated the impact of adolescent exposure to a PFAS mixture on adult ovarian function. Female CD-1 mice were orally exposed to vehicle control or a PFAS mixture (comprised of perfluorooctanoic acid [PFOA], perfluorooctanesulfonic acid [PFOS], undecafluoro-2-methyl-3-oxahexanoic acid [GenX/HFPO-DA], and perfluorobutanesulfonic acid [PFBS]) for 15 days. After a 42-day recovery period, reproductive hormones, ovarian fibrosis, and ovarian gene and protein expression were analyzed using ELISA, Picrosirius red (PSR) staining, qPCR, and immunoblotting, respectively. Results revealed that PFAS exposure did not affect adult body or organ weight, although ovarian weight slightly decreased. PFAS exposed mice exhibited a disturbed estrous cycle, with less time spent in proestrus than control mice. Follicle counting indicated a reduction in primordial and primary follicles. Serum analysis revealed no changes in steroid hormones, follicle-stimulating hormone, or anti-Müllerian hormone, but a significant increase in luteinizing hormone was observed in PFAS-treated mice. Ovaries collected from PFAS treated mice had increased mRNA transcripts for steroidogenic enzymes and fatty acid synthesis-related genes. PFAS exposure also increased collagen content in the ovary. Additionally, serum tumor necrosis factor-α levels were higher in PFAS treated mice. Finally, transcripts and protein abundance for Hippo pathway components were upregulated in the ovaries of the PFAS treated mice. Overall, these findings suggest that adolescent exposure to PFAS can disrupt ovarian function in adulthood.

Characterization of m6A Modifiers and RNA Modifications in Uterine Fibroids.

Uterine leiomyoma or fibroids are prevalent noncancerous tumors of the uterine muscle layer, yet their origin and development remain poorly understood. We analyzed RNA expression profiles of 15 epigenetic mediators in uterine fibroids compared to myometrium using publicly available RNA sequencing (RNA-seq) data. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key N6-methyladenosine (m6A) modifiers in fibroids and their matched myometrium, showing no significant differences in concordance with our RNA expression profiles. To determine RNA modification abundance, mRNA and small RNA from fibroids and matched myometrium were analyzed by ultra-high performance liquid chromatography-mass spectrometry identifying prevalent m6A and 11 other known modifiers. However, no aberrant expression in fibroids was detected. We then mined a previously published dataset and identified differential expression of m6A modifiers that were specific to fibroid genetic subtype. Our analysis also identified m6A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression, and protein profiles, we characterized and identified differentially expressed m6A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m6A modifications to fibroid pathology, there is a need for greater in-depth characterization of m6A marks and modifiers in a larger and diverse patient cohort.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

Central Role for Glycolysis and Fatty Acids in LH-responsive Progesterone Synthesis.

Progesterone production by the corpus luteum is fundamental for establishing and maintaining pregnancy. The pituitary gonadotropin luteinizing hormone (LH) is recognized as the primary stimulus for luteal formation and progesterone synthesis, regardless of species. Previous studies demonstrated an elevation in abundance of genes related to glucose and lipid metabolism during the follicular to luteal transition. However, the metabolic phenotype of these highly steroidogenic cells has not been studied. Herein, we determined acute metabolic changes induced by LH in primary luteal cells and defined pathways required for progesterone synthesis. Untargeted metabolomics analysis revealed that LH induces rapid changes in vital metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway, de novo lipogenesis, and hydrolysis of phospholipids. LH stimulated glucose uptake, enhanced glycolysis, and flux of [U- 13 C 6 ]-labeled glucose-derived carbons into metabolic branches associated with adenosine 5'-triphosphate (ATP) and NADH/NADPH production, synthesis of nucleotides, proteins, and lipids, glycosylation of proteins or lipids, and redox homeostasis. Selective use of small molecule inhibitors targeting the most significantly changed pathways, such as glycolysis, TCA cycle, and lipogenesis, uncovered cellular metabolic routes required for LH-stimulated steroidogenesis. Furthermore, LH via the protein kinase A (PKA) pathway triggered post- translational modification of acetyl-CoA carboxylase alpha (ACACA) and ATP citrate lyase (ACLY), enzymes involved in de novo synthesis of fatty acids. Inhibition of ACLY and fatty acid transport into mitochondria reduced LH-stimulated ATP, cAMP production, PKA activation, and progesterone synthesis. Taken together, these findings reveal novel hormone-sensitive metabolic pathways essential for maintaining LHCGR/PKA signaling and steroidogenesis in ovarian luteal cells. Significance: The establishment and maintenance of pregnancy require a well-developed corpus luteum, an endocrine gland within the ovary that produces progesterone. Although there is increased awareness of intracellular signaling events initiating the massive production of progesterone during the reproductive cycle and pregnancy, there are critical gaps in our knowledge of the metabolic and lipidomic pathways required for initiating and maintaining luteal progesterone synthesis. Here, we describe rapid, hormonally triggered metabolic pathways, and define metabolic targets crucial for progesterone synthesis by ovarian steroidogenic cells. Understanding hormonal control of metabolic pathways may help elucidate approaches for improving ovarian function and successful reproduction or identifying metabolic targets for developing nonhormonal contraceptives.