Evaluation of Lipids and Lipid-Related Transcripts in Human and Ovine Theca Cells and an in Vitro Mouse Model Exposed to the Obesogen Chemical Tributyltin.

BACKGROUND: Exposure to obesogenic chemicals has been reported to result in enhanced adipogenesis, higher adipose tissue accumulation, and reduced ovarian hormonal synthesis and follicular function. We have reported that organotins [tributyltin (TBT) and triphenyltin (TPT)] dysregulate cholesterol trafficking in ovarian theca cells, but, whether organotins also exert lipogenic effects on ovarian cells remains unexplored. OBJECTIVE: We investigated if environmentally relevant exposures to organotins [TBT, TPT, or dibutyltin (DBT)] induce lipid dysregulation in ovarian theca cells and the role of the liver X receptor (LXR) in this effect. We also tested the effect of TBT on oocyte maturation and neutral lipid accumulation, and lipid-related transcript expression in cumulus cells and preimplantation embryos. METHODS: Primary theca cell cultures derived from human and ovine ovaries were exposed to TBT, TPT, or DBT (1, 10, or 50 ng/ml). The effect of these chemical exposures on neutral lipid accumulation, lipid abundance and composition, lipid homeostasis-related gene expression, and cytokine secretion was evaluated using liquid chromatography-mass spectrometry (LC-MS), inhibitor-based methods, cytokine secretion, and lipid ontology analyses. We also exposed murine cumulus-oocyte complexes to TBT and evaluated oocyte maturation, embryo development, and lipid homeostasis-related mRNA expression in cumulus cells and blastocysts. RESULTS: Exposure to TBT resulted in higher intracellular neutral lipids in human and ovine primary theca cells. In ovine theca cells, this effect was dose-dependent, independent of cell stage, and partially mediated by LXR. DBT and TPT resulted in higher intracellular neutral lipids but to a lesser extent in comparison with TBT. More than 140 lipids and 9 cytokines were dysregulated in TBT-exposed human theca cells. Expression of genes involved in lipogenesis and fatty acid synthesis were higher in theca cells, as well as in cumulus cells and blastocysts exposed to TBT. However, TBT did not impact the rates of oocyte maturation or blastocyst development. DISCUSSION: TBT induced dyslipidemia in primary human and ovine theca cells, which may be responsible for some of the TBT-induced fertility dysregulations reported in rodent models of TBT exposure. https://doi.org/10.1289/EHP13955.

Altered expression of angiogenic factors in dominant preovulatory follicles of dairy cattle treated with ACTH.

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.

Effects of exogenous energy on synthesis of steroid hormones and expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen.

Energy and the cAMP-response element binding protein (CREB)/steroidogenic acute regulatory protein (StAR) signaling pathway play important roles in steroid hormone production and follicular development in hens. This present study aimed to investigate the effects of exogenous energy on the synthesis of steroid hormones and the expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen. The primary theca cells of small yellow follicles were randomly divided into 6 treatments and cultured in medium with glucose concentrations of 1, 1.5, 3, 4.5, 6, and 7.5 mg/mL for 48 h. It was found that growth was robust and cell outlines were clear when cells were cultured with 1, 1.5, 3, and 4.5 mg/mL glucose, but cell viability was diminished and cell density decreased after exposure to glucose at 6 and 7.5 mg/mL for 48 h. Cell viability showed an increasing and then decreasing quadratic response to increasing glucose concentration in culture (r2 = 0.688, P < 0.001). The cell viability of theca cells cultured with 4.5 mg/mL glucose was greater than those cultured with 1, 1.5, 6, and 7.5 mg/mL glucose (P < 0.05). The concentration of estradiol in the medium containing 3 mg/mL glucose was higher than in medium containing 1, 1.5, and 6 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between progesterone concentrations and glucose concentrations (r2 = 0.522, P = 0.002). The concentration of progesterone in medium with 4.5 mg/mL glucose was higher than in medium with 1 and 7.5 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between the relative expression of CREB1 (r2 = 0.752, P < 0.001), StAR (r2 = 0.456, P = 0.002), CYP1B1 (r2 = 0.568, P < 0.001), and 3ß-HSD (r2 = 0.319, P = 0.018) in theca cells of laying hens and glucose concentrations after treatment with different glucose concentrations for 48 h. After treatment with 4.5 mg/mL glucose, the expression of StAR, CYP1B1, and 3ß-HSD genes were increased compared to treatment with 1, 1.5, 3, 6, and 7.5 mg/mL glucose (P < 0.001). There was an increasing and then decreasing quadratic correlation between glucose concentrations and protein expression of CREB1 (r2 = 0.819, P < 0.001), StAR (r2 = 0.844, P < 0.001), 3ß-HSD (r2 = 0.801, P < 0.001), and CYP11A1 (r2 = 0.800, P < 0.001) in theca cells of laying hens. The protein expression of CREB1, StAR, and 3ß-HSD in theca cells cultured with 4.5 mg/mL glucose was higher than in other groups (P < 0.001). The results indicate that the appropriate glucose concentration (4.5 mg/mL) can improve the synthesis of steroid hormones in theca cells of laying hens through the upregulation of key genes and proteins in the CREB/StAR signaling pathway.

Targeted deletion of NR2F2 and VCAM1 in theca cells impacts ovarian follicular development: insights into polycystic ovary syndrome?†.

Defining features of polycystic ovary syndrome (PCOS) include elevated expression of steroidogenic genes, theca cell androgen biosynthesis, and peripheral levels of androgens. In previous studies, we identified vascular cell adhesion molecule 1 (VCAM1) as a selective androgen target gene in specific NR2F2/SF1 (+/+) theca cells. By deleting NR2F2 and VCAM1 selectively in CYP17A1 theca cells in mice, we documented that NR2F2 and VCAM1 impact distinct and sometimes opposing theca cell functions that alter ovarian follicular development in vivo: including major changes in ovarian morphology, steroidogenesis, gene expression profiles, immunolocalization images (NR5A1, CYP11A1, NOTCH1, CYP17A1, INSL3, VCAM1, NR2F2) as well as granulosa cell functions. We propose that theca cells impact follicle integrity by regulating androgen production and action, as well as granulosa cell differentiation/luteinization in response to androgens and gonadotropins that may underlie PCOS.

Granulosa Cells Alone, Without Theca Cells, Can Mediate LH-induced Oocyte Meiotic Resumption.

Signaling in the granulosa cells of mammalian ovarian follicles is necessary for maintaining prophase arrest in the oocyte and for mediating the resumption of meiosis in response to luteinizing hormone (LH). However, the follicle also includes an outer layer of theca cells, some of which express receptors for LH. To investigate whether theca cells are required for maintaining meiotic arrest and reinitiating meiosis in response to LH, we mechanically separated the granulosa cells and oocyte from the theca and basal lamina. This was accomplished by cutting a slit in the outer surface of isolated follicles such that the mural granulosa cells and cumulus-oocyte complex were extruded from the theca shell, forming a lawn of cells on an organotypic membrane. The remnant of theca cells and basal lamina was then removed. The separation of the granulosa cells from the theca cells and basal lamina was demonstrated by immunofluorescence localization of endomucin (blood vessels of the theca) and laminin gamma (basal lamina). Cells comprising these granulosa cell-oocyte complexes expressed LH receptors and were connected by gap junctions. Oocytes within these granulosa cell complexes maintained meiotic arrest and resumed meiosis in response to LH, showing that the granulosa cells alone, without theca cells, transduce these signals. This semi-intact and mostly 2-dimensional preparation could facilitate imaging studies of follicle physiology.

In-vitro generation of follicle-like structures from human germ cell-like cells derived from theca stem cell combined with ovarian somatic cells.

BACKGROUND: The objective of this study was to induce the differentiation of human theca stem cells (hTSCs) into germ cell-like cells (hGCLCs) and assess their developmental progression following in vitro 3D culture with ovarian somatic cells within the follicle-like structures. To achieve this, the hTSCs were isolated from small antral follicles of three patients of varying ages and were then seeded in a differentiation medium for 40 days. The differentiated hGCLCs were subsequently aggregated with somatic ovarian cells (cumulus cells and hTSCs) in a ratio of 1:10 and cultured in a growth medium in a suspension culture dish. In addition to examining the morphologies, sizes, and viabilities of the differentiated hGCLCs, this study also analyzed the expression of DAZL and GDF9 proteins within the follicle-like structures. RESULTS: After 12 days, the hTSCs began to differentiate into hGCLCs, with their shapes changing from spindle-shaped to spherical. The sizes of hGCLCs increased during the differentiation period (from 25 µm to 50 µm). The survival rate of the hGCLCs after differentiation and in vitro development in primordial follicle-like structures was 54%. Unlike hTSCs, which did not express the DAZL protein, the hGCLCs and follicle-like structures successfully expressed DAZL protein (P-value < 0.05). However, hGCLCs poorly expressed the GDF9 protein. Further, the culture of hGCLCs in primordial follicle-like structures significantly increased GDF9 expression (P-value < 0.05). CONCLUSION: In conclusion, our study demonstrated that 3D cultures with ovarian somatic cells in follicle-like structures caused the successful differentiation of reproducible hGCLCs from hTSCs derived from three patients of different ages. Moreover, this method not only enhanced the in vitro development of hGCLCs but also presented a novel approach for co-culturing and developing in vitro oocyte like cells, ultimately leading to the production of artificial follicles.

Classification of Atretic Small Antral Follicles in the Human Ovary.

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan.

Expression of genes and enzymes involved in ovarian steroidogenesis in relation to human follicular development.

Introduction: Granulosa cells (GCs) and theca cells (TCs) play a pivotal role in human ovarian steroidogenesis, facilitating the conversion of cholesterol into sex steroids that regulate normal reproductive function. This study aims to explore the expression patterns of key enzymes that govern human ovarian steroidogenesis throughout follicle development, employing both genomic and immunological methodologies. Methods: Follicles and GCs obtained from women undergoing ovarian tissue cryopreservation (OTC) and in vitro fertilisation treatment were utilized. Gene expression data were obtained from a Chinese study using RNA sequencing and from microarray data generated in our laboratory to comprehensively analyse gene expression profiles across distinct stages of follicular development. To corroborate the localisation of key enzymes within GCs and TCs, immunohistochemistry analyses utilizing colourimetric and fluorescent techniques were conducted. Results: Steroidogenesis-related enzymes displayed low gene expression levels during early follicle development. However, a notable upregulation of HSD3B2 was observed in GCs as follicles progressed to the antral/preovulatory stage, confirmed consistently using both microarray and RNA sequencing methodologies. Furthermore, immunohistochemical analyses effectively demonstrated that HSD3B2 were not only expressed in GCs, but co-localised with CYP17A1 within a specific subset of TCs surrounding human small antral follicles. Contributing to an enhanced progesterone production during the second half of the follicular phase was a significant upregulation of CYB5A in both microarray and RNA-seq datasets as follicles transition from the antral stage to the pre-ovulatory stage. Moreover, an augmented expression of DHCR24 and LDLR in both types of data, along with HMGCR expression expression in the microarray data, indicates increased substrate availability for ovarian steroidogenesis. Discussion: This study confirms and extends that GCs gradually augment expression of HSD3B2 thereby enhancing their capacity for progesterone synthesis as follicles reach the size of selection at around 10 mm in diameter. This is supported by the expression CYB5A and possibly augmented availability of steroid precursors. A subset of TCs exhibit concurrent expression of CYP17A1 and HSD3B2, collectively contributing to the synthesis of 17-hydroxyprogesterone. These data significantly enhance our understanding of the dynamic regulation of progesterone throughout the process of follicular development.

Biomolecular composition of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination.

The study aimed to analyze changes in biomolecular composition of granulosa and theca interna cells of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination. Medium antral follicles (n = 4/each group) were cultured alone (C; control) or in the presence of 1α,25(OH)2D3 (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination, VD and I (VD+I). Then paraplast-embedded ovarian follicles were used for Fourier Transform Infrared (FTIR) spectroscopy and respective histological stainings. FTIR analysis revealed changes in the content of fibrous proteins (mainly collagens) within theca interna following vitamin D3 and insulin co-administration that was verified by Masson's trichrome staining. Treatment-dependent differences were also observed in the secondary structure of proteins, indicating enhanced conversion to α-helices in response to vitamin D3 and insulin action/interaction in both follicular compartments. In the granulosa and theca interna layers, tendency to lower DNA content in the VD+I group was noted and confirmed by Fulgen's staining. Finally, altered monosaccharides production in both follicular layers was found. Based on FTIR results, it is possible to attribute the observed alterations to biological processes that could be regulated by vitamin D3 and insulin in the porcine ovarian follicles.

Comparing ovarian expression of sperm acrosome associated 3 protein in young and adult queens.

The purpose of this research was to quantify sperm acrosome associated 3 protein expression in the ovaries of young (3.0 ± 0.9 months, n = 11) and adult (10.4 ± 2.8 months, n = 11) queens. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded feline ovarian sections. Ovaries were obtained following routine ovariohysterectomy of queens. Cellular expression of sperm acrosome associated 3 protein was measured in primordial, primary, secondary, and tertiary follicles using an image-analysis software's red, green, and blue stack and manual thresholding functions. The oocyte nucleus, ooplasm, granulosa cells, and theca cells were outlined using the freehand selection tool and mean grey value was recorded. Results from each cellular location were compared between age groups using a Student's t-test and between follicle stages using an analysis of variance. Compared to adult queens, younger queens had significantly greater sperm acrosome associated 3 protein expression in granulosa cells of primary, secondary, and tertiary follicles. Also, theca cells of secondary and tertiary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. The oocyte nucleus of primordial, primary, and secondary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. However, sperm acrosome associated 3 protein expression within the ooplasm did not differ significantly between age groups of any follicle type. More research is needed to determine what role sperm acrosome associated 3 protein may play in female fertility in animals as well as what mechanisms regulate ovarian sperm acrosome associated 3 protein expression over time.

Real-life exposure to Fusarium toxins deoxynivalenol and zearalenone triggers apoptosis and activates NLRP3 inflammasome in bovine primary theca cells.

Cattle are deemed less susceptible to mycotoxins due to the limited internal exposure resulting from rumen microbiota activity. However, the significant amounts of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) frequently detected in bovine follicular fluid samples suggest that they could affect ovarian function. Both mycotoxins trigger several patterns of cell death and activate the NLRP3 inflammasome in the intestine. In vitro studies have reported a number of adverse effects on bovine oocytes. However, the biological relevance of such findings with regard to realistic concentrations of DON and ZEN in bovine follicular fluid is still not clear. Hence, it is important to better characterize the effects of dietary exposure to DON and ZEN on the bovine ovary. Using bovine primary theca cells, this study investigated the effects of real-life patterns for bovine ovary exposure to DON and ZEN, but also DON metabolite DOM-1, on cell death and NLRP3 inflammasome activation. Exposure to DON starting from 0.1 µM significantly decreased theca cell viability. The kinetics of phosphatidylserine translocation and loss of membrane integrity showed that ZEN and DON, but not DOM-1, induce an apoptotic phenotype. qPCR analysis of the expression of NLRP3, PYCARD, IL-1ß, IL-18, and GSDMD in primary theca cells at concentrations of mycotoxin previously reported in cow follicular fluid clearly indicated that DON and DOM-1 individually and in mixture, but not ZEN, activate NLRP3 inflammasome. Altogether, these results suggest that real-life dietary exposure of cattle to DON may induce inflammatory disorders in the ovary.

Developmental and hormonal regulation of FBN1 and OR4M1 mRNA in bovine granulosa cells.

Recent studies have reported hormonal regulation of expression of fibrillin 1 (FBN1), the gene that encodes asprosin, in bovine theca cells, however, hormonal regulation of gene expression of FBN1 and the asprosin receptor, olfactory receptor 4M1 (OR4M1), has not been evaluated in granulosa cells (GC). This study was designed to characterize FBN1 and OR4M1 gene expression in GC during development of bovine dominant ovarian follicles, and to determine the hormonal regulation of FBN1 and OR4M1 mRNA expression in GC. GC FBN1 mRNA abundance was greater (P < 0.05) in medium (5.1-8 mm) estrogen inactive (EI) follicles than in large (>8.1 mm) or small (1-5 mm) EI follicles. In comparison, GC OR4M1 mRNA abundance was greater (P < 0.05) in small EI follicles than in large or medium EI follicles. Abundance of OR4M1 mRNA in GC of follicles collected on days 3 to 4 (early growth phase) and on days 5 to 6 (late growth phase) was similar, whereas FBN1 mRNA abundance was greater (P < 0.05) on days 5 to 6 vs days 3 to 4. Hormonal regulators for FBN1 mRNA abundance in cultured small-follicle GC were identified: TGFß1 causing a 2.45-fold increase, WNT3A causing a 1.45-fold increase, and IGF1 causing a 65% decrease. Steroids, leptin, insulin, growth hormone, follicle stimulating hormone, fibroblast growth factor 9 and epidermal growth factor had no effect on FBN1 mRNA abundance. Abundance of OR4M1 mRNA in GC was regulated by progesterone with 3.55-fold increase, but other hormones did not affect GC OR4M1 mRNA abundance. Findings indicate that both FBN1 and OR4M1 gene expression are hormonally and developmentally regulated in bovine follicles, and thus may affect asprosin production and its subsequent role in ovarian follicular function in cattle.

Sphingosine-1-phosphate regulation of luteinising hormone-induced steroidogenesis and proliferation of bovine theca cells in vitro.

CONTEXT: Sphingosine-1-phosphate (S1P) is synthesised by follicle granulosa cells under the influence of follicle-stimulating hormone and seems to be necessary for the biological effects of this gonadotrophin. AIMS: To determine if luteinising hormone (LH) increases S1P production and if this sphingolipid, either induced by LH or added to culture media, regulates steroidogenesis and cell viability in bovine theca cells. METHODS: We used bovine theca cell cultures treated with: S1P (0, 0.1, 1 and 10µM; Experiment 1), LH (0, 0.02, 0.2 and 2ngmL-1 ; Experiment 2) and LH (0.02ngmL-1 ) plus a sphingosine kinase inhibitor (SKI-178; 0, 5 and 10µM; Experiment 3). KEY RESULTS: Treatment with S1P did not affect (P >0.05) theca cell viability or their ability to produce progesterone and testosterone. LH (0.02ngmL-1 ) increased (P <0.05) S1P production, and stimulated the expression of phosphorylated sphingosine kinase-1 (pSPHK1). However, the inhibition of SPHK1, by a specific SPHK1 inhibitor (SKI-178), reduced (P <0.05) cell viability and progesterone secretion. Additionally, the use of SKI-178 increased theca cell testosterone production (P<0.05). CONCLUSIONS: S1P added to culture media did not affect cell viability or steroid synthesis. However, LH stimulated the production of S1P, by increasing phosphorylation of SPHK1 in theca cells. This intracellular S1P was inhibitory on testosterone production but augmented progesterone and viable cell number. IMPLICATIONS: These results suggest a novel signalling pathway for LH in theca cells and underline the importance of S1P in the regulation of steroid synthesis.

Co-culture of human cryopreserved fragmented ovarian tissue with theca progenitor cells derived from theca stem cells.

PURPOSE: Despite the significant advances in the in vitro development of human primordial follicles, it is still a challenging approach with great potential for improvements. Therefore, the present study aimed to investigate the effect of a feeder layer of human theca progenitor cells (hTPCs) on the development of primordial follicles embedded in human ovarian tissue. METHODS: Fragments of frozen-thawed ovarian tissue were activated using the vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) and kit ligand for 24 h. Then, the specimens were divided into the co-culture and mono-culture groups and were cultured with and without a hTPC feeder layer for 6 days, respectively. Afterward, the follicles were counted and classified, and the hormone levels and expression levels of apoptosis- and folliculogenesis-related genes were assessed. RESULTS: Both culture groups showed significant follicle growth (P < 0.05). However, the co-culture group had a significantly higher number of growing follicles compared to the other group (P < 0.05). Moreover, the expression levels of ZP1, ZP2, ZP3, BMP-7, AMH, and GDF9 were significantly higher in the co-culture group compared to the other group (P < 0.05), while the expression levels of P53 and CASP3 were significantly lower (P < 0.05). Also, the concentrations of estradiol, progesterone, testosterone, and androstenedione were significantly higher in the co-culture group compared to the other group (P < 0.05). CONCLUSION: The present study results provided novel evidence on the direct role of hTPCs in the growth and development of human primordial follicles. However, there is a need for future studies to illustrate the underlying mechanisms. Schematic summary of the results. According to our results, the expression of ZP1, ZP2, ZP3, and GDF9 in the oocytes, AMH in the granulosa cells, and BMP4 in the theca cells of the co-culture group were significantly higher than those of the mono-culture and non-culture groups, while the expression of apoptotic genes (BAX, CASP3, and P53) was significantly lower. Moreover, the co-culture group showed significantly increased levels of estradiol, progesterone, testosterone, and androstenedione in its culture media compared to the mono-culture groups.

The Mycotoxin De-Epoxy-Deoxynivalenol (DOM-1) Increases Endoplasmic Reticulum Stress in Ovarian Theca Cells.

Deoxynivalenol (DON) is a major mycotoxin present in animal feed and negatively affects growth and reproduction in farm species, including pigs and cattle. The mechanism of DON action involves the ribotoxic stress response (RSR), and it acts directly on ovarian granulosa cells to increase cell death. In ruminants, DON is metabolized to de-epoxy-DON (DOM-1), which cannot activate the RSR but has been shown to increase cell death in ovarian theca cells. In the present study, we determined if DOM-1 acts on bovine theca cells through endoplasmic stress using an established serum-free cell culture model and to assess whether also DON activates endoplasmic stress in granulosa cells. The results show that DOM-1 increased the cleavage of ATF6 protein, increased the phosphorylation of EIF2AK3, and increased the abundance of cleaved XBP1 mRNA. Activation of these pathways led to an increased abundance of mRNA of the ER stress target genes GRP78, GRP94, and CHOP. Although CHOP is widely associated with autophagy, inhibition of autophagy did not alter the response of theca cells to DOM-1. The addition of DON to granulosa cells partially increased ER stress pathways but failed to increase the abundance of mRNA of ER stress target genes. We conclude that the mechanism of action of DOM-1, at least in bovine theca cells, is through the activation of ER stress.

The characterization and therapeutic applications of ovarian theca cells: An update.

Theca cells perform a range of roles during folliculogenesis. So far, little is known about their recruitment process and function since early research has mainly focused on the interactions between granulosa cells and the oocyte, leaving theca cells unfairly forgotten in the understanding of ovarian physiology and pathogenesis. Given that research on theca cells has greatly emerged in recent years, this review of literature aims to discuss the established theoretical concepts with the most recent findings about theca cells' characterization and origins, in vitro culture applications as models for fertility preservation and pharmacological/toxicological studies, its importance in unraveling pathogenic pathways, and stem-cell-based bioengineering for hormonal replacement therapies. Isolation and in vitro culture techniques for theca cells have led to essential advancements in their characterization as a specific cell population. Unraveling the origins of theca cells during the in vivo differentiation process in the adult ovary will assist the development of hormonal replacement therapies, reestablishment of fertility, and treatments for diseases such as premature ovarian insufficiency and polycystic ovarian syndrome, which seem to be directly influenced by theca cells.

Ligusticum chuanxiong promotes the angiogenesis of preovulatory follicles (F1-F3) in late-phase laying hens.

Ligusticum chuanxiong (CX) is a traditional Chinese medicine that is widely planted throughout the world. CX is one of the most important and commonly used drugs to enhance blood circulation. The preovulatory follicles in laying hens have a large number of blood arteries and meridians that feed the follicles' growth and maturation with nutrients, hormones, and cytokines. With the extension of laying time, preovulatory follicles angiogenesis decreased gradually. In this study, we studied the mechanism of CX on preovulatory follicles angiogenesis in late-phase laying hens. The results show that CX extract can increase the angiogenesis of preovulatory follicles (F1-F3) of late-phase laying hens. CX extract can promote vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation in preovulatory follicles theca layers, promote the proliferation, invasion and migration through PI3K/AKT and RAS/ERK signaling pathways in primary follicle microvascular endothelial-like cells (FMECs). In addition, CX extract can up-regulate the expression of hypoxia inducible factor α (HIF1α) in granulosa cells (GCs) and granulosa layers through PI3K/AKT and RAS/ERK signaling pathways, thereby promoting the secretion of vascular endothelial growth factor A (VEGFA). In conclusion, the current study confirmed the promoting effect of CX extract on the preovulatory follicles angiogenesis, which sets the stage for the design of functional animal feed for late-phase laying hens.

Human theca arises from ovarian stroma and is comprised of three discrete subtypes.

Theca cells serve multiple essential functions during the growth and maturation of ovarian follicles, providing structural, metabolic, and steroidogenic support. While the function of theca during folliculogenesis is well established, their cellular origins and the differentiation hierarchy that generates distinct theca sub-types, remain unknown. Here, we performed single cell multi-omics analysis of primary cell populations purified from human antral stage follicles (1-3 mm) to define the differentiation trajectory of theca/stroma cells. We then corroborated the temporal emergence and growth kinetics of defined theca/stroma subpopulations using human ovarian tissue samples and xenografts of cryopreserved/thawed ovarian cortex, respectively. We identified three lineage specific derivatives termed structural, androgenic, and perifollicular theca cells, as well as their putative lineage-negative progenitor. These findings provide a framework for understanding the differentiation process that occurs in each primordial follicle and identifies specific cellular/molecular phenotypes that may be relevant to either diagnosis or treatment of ovarian pathologies.

Effects of melatonin on development and hormone secretion of sheep theca cells in vitro.

Theca cells (TCs) play a unique role in the structure and function of the ovary. They are not only the structural basis of the follicle but also the androgen-secreting cells in female mammals, which can affect the normal development and atresia of the follicle. The results showed that melatonin receptor (MTR) MT1 and MT2 were expressed on sheep TCs. In the present study, the effects of different concentrations of MT at 0, 10-10, 10-8, 10-6 and 10-4 M/L on sheep TCs with regards to the antioxidant levels, proliferation, apoptosis and steroid hormone secretion were investigated. The results showed that in sheep TCs, all concentrations of MT significantly decreased reactive oxygen species (ROS) concentration and BAX expression; increased Cat, Sod1, and BCL-2 expression. The proliferation viability of TCs was significantly inhibited in all groups except for 10-10 M/L MT, and the expression of cyclin D1 and CDK4 was significantly reduced. MT significantly increased StAR expression and progesterone secretion in TCs, but there was no significant effect on androgen secretion and CYP11A1, CYP17A1 and 3ß-HSD expression in all groups. MT-induced progesterone secretion was completely inhibited by Luzindole (a nonspecific MT1 and MT2 inhibitor) and partially inhibited by 4p-PDOT (specific MT2 inhibitor). MT-induced progesterone secretion can be inhibited by LY294002 (PI3K/AKT pathway inhibitor). This study indicated that MT inhibits apoptosis and proliferation of in vitro cultured sheep TCs, which has implications for slowing ovarian atresia and aging. MT activates the PI3K/Akt pathway to mediate the synthesis and secretion of progesterone by TCs. This study provides a basis for further exploration of the role of TCs on follicle development and ovarian steroid hormone secretion.

Low temperature plasma protects against inflammatory agents-mediated dysfunction of theca cells via enhancing MANF expression.

BACKGROUND: Low temperature plasma (LTP) exerts a protective effect in inflammation via enhancing MANF expression. Hyperactivation and dysfunction of theca cells induced by inflammatory agents is accompanied by polycystic ovary syndrome (PCOS), which is a common reproductive and endocrine disorder. However, the effect of LTP on theca cells is still unknown. METHODS AND RESULTS: Theca cells were stimulated with IL-1ß or TNF-α for 12 h, then treated with LTP for 100 s. After 8 h, medium supernatant and theca cells were collected. Production of androgen from theca cells were detected by ELISA. The PCNA and Annexin V levels in theca cells were detected by using immunofluorescent staining. The levels of PCNA, BCL-2 and BAX were evaluated by western blot and qPCR. MTT assay was used to detect the viability of theca cells. The proportions of apoptosis of theca cells were detected by Flow cytometry. The mRNA levels of androgenic genes were detected by qPCR. The MANF levels in medium supernatant and cell lysate were detected by using ELISA, western and qPCR. BIP and CHOP expressions were detected by using western blot and qPCR. We found that LTP irradiation decreased inflammatory agents-induced upregulation of androgen and androgenic genes in theca cells. And LTP irradiation relieves IL-1ß or TNF-α-induced pathological proliferation and apoptosis in theca cells. In terms of mechanism, LTP irradiation increased MANF level in theca cells to inhibit BIP and CHOP expression. CONCLUSION: These evidences suggest the protective effect of LTP on theca cells in inflammatory microenvironment, and LTP has the potential clinical application of PCOS.