Atypical Psuedo-Demons-Meigs Syndrome Presenting As Acute Dyspnoea With Pseudomembranous Colitis.

Demons-Meigs syndrome is a rare clinical presentation of benign ovarian mass with hydrothorax and ascites. As ascites can be present in any ovarian mass, hydrothorax is a salient feature of the syndrome. The syndrome is subtyped as atypical in the absence of ascites from the triad. Nevertheless, it is labeled as pseudo-Demons-Meigs syndrome if the ovarian tumor is neoplastic rather than benign. The management of Demons-Meigs syndrome is complex and could be misleading due to pleural effusion and ascites, so an understanding of the syndrome is important. This case report is unique as it has two rare findings of neoplastic tumor and absence of ascites. Furthermore, this case is distinct as both ovaries are involved in malignant granulosa theca cell tumor with right-sided pleural effusion without ascites.

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.

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.

The Roles of Autophagy in the Genesis and Development of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common and frequent disease and always leads endocrine and metabolic disorder among women in reproductive age. Ovary is the main organ involved in polycystic ovary syndrome, and its function impairment will lead to reproductive dysfunction. Some recent studies have demonstrated that autophagy plays an important role in the pathogenesis of PCOS, and there are many different mechanisms that affect autophagy and the occurrence of PCOS, and they provide a new direction for us to predict the mechanism of PCOS. In this review, we discuss the role of autophagy in different ovarian cells: granulosa cells, oocytes, and theca cells, and introduce the important role that they play in the progress of PCOS. The main purpose of this review is to provide the research background and some relevant suggestions for our future work in autophagy and help us better explore the pathogenesis and autophagy mechanisms of PCOS. Furthermore, it will help us gain a new insight of the pathophysiology and treatment of PCOS.

Characterization of ovarian follicles, serum steroid hormone concentration, and steroidogenic gene expression profiles in the developing ovarian follicles in White King pigeons.

Paired pigeons only lay 2 eggs in a laying period, which is closely related to ovarian follicle development, but this process is not well understood. In this study, 60 pairs of 12-mo-old White King pigeons were selected and serum and follicles were collected at 4 stages of laying interval (LI), including the first (LI1), the third (LI3), the fifth (LI5), and the seventh day (LI7). Morphological results showed that paired pigeons normally had 2 preovulatory follicles and the second-largest follicle (F2) developed from LI3 and had been selected in LI5. Prehierarchical follicles were coupled and hierarchical, which was in accordance with its clutch size. The P4 concentration increased gradually from LI1 to LI5, reaching a maximum of 30.67 ng/mL in LI5 and decreasing to 27.83 ng/mL in LI7 (P < 0.05). The levels of T in LI1 and LI5 were higher than LI3 and LI7 (P < 0.05), although there was no significant difference in E2 in LI (P > 0.05), but it stayed at high levels. In the TCs of the largest follicle (F1), HSD3B1 mRNA and HSD17B1 mRNA levels peaked in LI7. The expression pattern of CYP17A1 and CYP19A1 was similar, increasing from LI3 to LI5 and then decreasing. In the TCs of F2, the expressions of HSD3B1 and CYP17A1 had no significant difference between LI5 and LI7 (P > 0.05), while the expression pattern of HSD17B1 and CYP19A1 was the opposite. In TCs of SF1, HSD3B1 mRNA level peaked in LI3 while CYP19A1 mRNA levels peaked in LI7. The expression of CYP17A1 had a minor change (P > 0.05) and the expression pattern of HSD17B1 was similar to F1. It was concluded that the morphological characteristics of follicles during the LI for the first time, including the number and diameter of small follicles (SFs) and hierarchical follicles in pigeon and the concentrations of steroid hormones and expressions of steroidogenic genes in TCs of different follicles could explain the growth and selection of 2 preovulatory follicles. This study facilitates further research into the regulation of ovulation and egg production in pigeons.

Single-Cell Transcriptomics Analysis Reveals a Cell Atlas and Cell Communication in Yak Ovary.

Yaks (Bos grunniens) are the only bovine species that adapt well to the harsh high-altitude environment in the Qinghai-Tibetan plateau. However, the reproductive adaptation to the climate of the high elevation remains to be elucidated. Cell composition and molecular characteristics are the foundation of normal ovary function which determines reproductive performance. So, delineating ovarian characteristics at a cellular molecular level is conducive to elucidating the mechanism underlying the reproductive adaption of yaks. Here, the single-cell RNA-sequencing (scRNA-seq) was employed to depict an atlas containing different cell types with specific molecular signatures in the yak ovary. The cell types were identified on the basis of their specifically expressed genes and biological functions. As a result, a cellular atlas of yak ovary was established successfully containing theca cells, stromal cells, endothelial cells, smooth muscle cells, natural killer cells, macrophages, and proliferating cells. A cell-to-cell communication network between the distinct cell types was constructed. The theca cells were clustered into five subtypes based on their biological functions. Further, CYP11A1 was confirmed as a marker gene for the theca cells by immunofluorescence staining. Our work reveals an ovarian atlas at the cellular molecular level and contributes to providing insights into reproductive adaption in yaks.

AMH inhibits androgen production in human theca cells.

Both excessive ovarian production of AMH and androgen are important features of polycystic ovary syndrome (PCOS). Present study aimed to explore the direct effect of AMH on androgen production in human theca cells. Primary cultured human theca cells were treated with AMH, an ALK2 (the BMP type 1 receptor) inhibitor and an ALK5 (the TGFß type 1 receptor) inhibitor. AMH significantly suppresses the expression of the androgen synthesis-related enzyme CYP17A1 and reduces the production of androstenedione and testosterone in normal human theca cells and PCOS theca cells. Inhibitors of ALK2/3 and ALK5 antagonize the effect of AMH on the expression of CYP17A1. Although both ALK5 and ALK2 interact with AMHR2 in the presence of AMH, AMH activated neither TGFßR-Smads (Smad 2/3) nor BMPR-Smads (Smad 1/5/8). Our data suggested that AMH suppresses androgen synthesis-related enzyme CYP17A1 expression and inhibits androgen production in human theca cells, which process may be mediated by ALK2 and ALK5.

Squeezing the eggs to grow: The mechanobiology of mammalian folliculogenesis.

The formation of functional eggs (oocyte) in ovarian follicles is arguably one of the most important events in early mammalian development since the oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. While past studies have identified many genes that are critical to normal ovarian development and function, recent studies have highlighted the role of mechanical force in shaping folliculogenesis. In this review, we discuss the underlying mechanobiological principles and the force-generating cellular structures and extracellular matrix that control the various stages of follicle development. We also highlight emerging techniques that allow for the quantification of mechanical interactions and follicular dynamics during development, and propose new directions for future studies in the field. We hope this review will provide a timely and useful framework for future understanding of mechano-signalling pathways in reproductive biology and diseases.

Aberrant activation of KRAS in mouse theca-interstitial cells results in female infertility.

KRAS plays critical roles in regulating a range of normal cellular events as well as pathological processes in many tissues mediated through a variety of signaling pathways, including ERK1/2 and AKT signaling, in a cell-, context- and development-dependent manner. The in vivo function of KRAS and its downstream targets in gonadal steroidogenic cells for the development and homeostasis of reproductive functions remain to be determined. To understand the functions of KRAS signaling in gonadal theca and interstitial cells, we generated a Kras mutant (tKrasMT) mouse line that selectively expressed a constitutively active Kras G12D in these cells. Kras G12D expression in ovarian theca cells did not block follicle development to the preovulatory stage. However, tKrasMT females failed to ovulate and thus were infertile. The phosphorylated ERK1/2 and forkhead box O1 (FOXO1) and total FOXO1 protein levels were markedly reduced in tKrasMT theca cells. Kras G12D expression in theca cells also curtailed the phosphorylation of ERK1/2 and altered the expression of several ovulation-related genes in gonadotropin-primed granulosa cells. To uncover downstream targets of KRAS/FOXO1 signaling in theca cells, we found that the expression of bone morphogenic protein 7 (Bmp7), a theca-specific factor involved in ovulation, was significantly elevated in tKrasMT theca cells. Chromosome immunoprecipitation assays demonstrated that FOXO1 interacted with the Bmp7 promoter containing forkhead response elements and that the binding activity was attenuated in tKrasMT theca cells. Moreover, Foxo1 knockdown caused an elevation, whereas Foxo1 overexpression resulted in an inhibition of Bmp7 expression, suggesting that KRAS signaling regulates FOXO1 protein levels to control Bmp7 expression in theca cells. Thus, the anovulation phenotype observed in tKrasMT mice may be attributed to aberrant KRAS/FOXO1/BMP7 signaling in theca cells. Our work provides the first in vivo evidence that maintaining normal KRAS activity in ovarian theca cells is crucial for ovulation and female fertility.

Effects of grape phenolics, myricetin and piceatannol, on bovine granulosa and theca cell proliferation and steroid production in vitro.

Myricetin (a flavonol) and piceatannol (a stilbenoid) are naturally occurring phenolic compounds in red wine with cardio-protective and anti-carcinogenic effects, but their potential reproductive effects have not been investigated. Thus, the present study was designed to determine if myricetin and piceatannol can directly affect ovarian function using bovine granulosa cells (GC) and theca cells (TC) as in vitro model systems to evaluate effects on cell proliferation and steroid production. In Experiment 1 and 2, myricetin and piceatannol at 30 µM blocked insulin-like growth factor 1 (IGF1)-induced progesterone production by GC without affecting GC numbers. In contrast, myricetin stimulated IGF1-induced estradiol production, whereas piceatannol at 30 µM inhibited IGF1-induced estradiol production by 90% in GC. In Experiment 3 and 4, TC androstenedione and progesterone production and TC proliferation was inhibited by myricetin and piceatannol at 30 µM. In Experiment 5, piceatannol (30 µM) reduced the Fusarium mycotoxin, beauvericin (6 µM)-induced inhibition on progesterone production and cell proliferation. Myricetin (30 µM) reduced the inhibitory effect of beauvericin on estradiol but not progesterone production or cell proliferation. In conclusion, the red wine phenols, myricetin and piceatannol, directly affected GC and TC steroidogenesis, and were able to reduce some of the inhibitory effects of beauvericin on GC function.

Changes in fibroblast growth factor receptors-1c, -2c, -3c, and -4 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

The various fibroblast growth factors (FGF) regulate their function via binding to 4 main FGF receptor (FGFR) subtypes and their splice variants, FGFR1b, FGF1c, FGFR2b, FGFR2c and FGFR3c and FGFR4, but which of these FGFR are expressed in the granulosa (GC) and theca cells (TC), the 2 main cell layers of ovarian follicles, or change during follicular development is unknown. We hypothesized that FGFR1c, FGFR2c and FGFR3c (but not FGFR4) gene expression in GC (but not TC) would change with follicular development. Hence, the objective of this study was to determine if abundance of FGFR1c, FGFR2c, FGFR3c, and FGFR4 mRNA change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in Holstein cattle exhibiting regular estrous cycles. Estrous cycles of non-lactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or d 5 to 6 (n = 8) post-ovulation for GC and TC RNA extraction from small (1-5 mm), medium (5.1 to 8 mm) or large (8.1-18 mm) follicles for real-time PCR analysis. In GC, FGFR1c and FGFR2c mRNA relative abundance was greater in estrogen (E2)-inactive (ie, concentrations of E2 < progesterone, P4) follicles of all sizes than in GC from large E2-active follicles (ie, E2 > P4), whereas FGFR3c and FGFR4 mRNA abundance did not significantly differ among follicle types or days post-estrus. In TC, medium E2-inactive follicles had greater FGFR1c and FGFR4 mRNA abundance than large E2-active and E2-inactive follicles on d 5 to 6 post-ovulation whereas FGFR2c and FGFR3c mRNA abundance did not significantly differ among follicle types or day post-estrus. In vitro experiments revealed that androstenedione increased abundance of FGFR1c, FGFR2c and FGFR4 mRNA in GC whereas estradiol decreased FGFR2c mRNA abundance. Neither androstenedione nor estradiol affected abundance of the various FGFR mRNAs in cultured TC. Taken together, the findings that FGFR1c and FGFR2c mRNA abundance was less in GC of E2-active follicles and FGFR1c and FGFR4 mRNA was greater in TC of medium inactive follicles at late than at early growing phase of the first dominant follicle support an anti-differentiation role for FGF and their FGFR as well as support the idea that steroid-induced changes in FGF and their receptors may regulate selection of dominant follicles in cattle.

In vitro development of mechanically and enzymatically isolated cat ovarian follicles.

Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts.

Creating an Artificial 3-Dimensional Ovarian Follicle Culture System Using a Microfluidic System.

We hypothesized that the creation of a 3-dimensional ovarian follicle, with embedded granulosa and theca cells, would better mimic the environment necessary to support early oocytes, both structurally and hormonally. Using a microfluidic system with controlled flow rates, 3-dimensional two-layer (core and shell) capsules were created. The core consists of murine granulosa cells in 0.8 mg/mL collagen + 0.05% alginate, while the shell is composed of murine theca cells suspended in 2% alginate. Somatic cell viability tests and hormonal assessments (estradiol, progesterone, and androstenedione) were performed on days 1, 6, 13, 20, and 27. Confocal microscopy confirmed appropriate compartmentalization of fluorescently-labeled murine granulosa cells to the inner capsule and theca cells to the outer shell. Greater than 78% of cells present in capsules were alive up to 27 days after collection. Artificially constructed ovarian follicles exhibited intact endocrine function as evidenced by the production of estradiol, progesterone, and androstenedione. Oocytes from primary and early secondary follicles were successfully encapsulated, which maintained size and cellular compartmentalization. This novel microfluidic system successfully encapsulated oocytes from primary and secondary follicles, recapitulating the two-compartment system necessary for the development of the mammalian oocyte. Importantly, this microfluidic system can be easily adapted for sterile, high throughput applications.

New theca-cell marker insulin-like factor 3 is associated with premature ovarian insufficiency.

OBJECTIVE: To characterize circulating insulin-like factor 3 (INSL3) in different stages of ovarian insufficiency and its role in the evaluation of premature ovarian insufficiency (POI). DESIGN: Retrospective cohort study. SETTING: University-based center for reproductive medicine. PATIENT(S): A total of 145 women, including 48 patients with POI (25 IU/L < follicle-stimulating hormone [FSH] ≤40 IU/L), 49 with biochemical POI (bPOI) (10 IU/L < FSH ≤25 IU/L) and 48 age-matched control women with normal ovarian reserve (FSH <10 IU/L), retrospectively included from the reproductive hospital affiliated with Shandong University between 2017 and 2019. INTERVENTION(S): Levels of INSL3 in the serum and follicular fluid assayed with a commercial radioimmunoassay. MAIN OUTCOME MEASURE(S): Level of INSL3 in serum and follicular fluid among control women and patients with bPOI and POI, its association with different ovarian reserve markers, and its predictive value for bPOI and POI. RESULT(S): The serum INSL3 level continuously declined with the progress of ovarian insufficiency. It showed strong negative association with FSH (-0.655) and luteinizing hormone (-0.433), but positively correlated with antimüllerian hormone (0.617), inhibin B (0.400), antral follicle count (0.630), and testosterone (0.180). Additionally, the circulating INSL3 served as a good predictor for bPOI and POI. No statistically significant difference of INSL3 levels in follicular fluid was observed between bPOI patients and control women. CONCLUSION(S): For the first time our study has revealed an INSL3 deficiency in women with POI, indicating that circulating INSL3 could serve as a promising theca-cell specific marker for POI. Future research on the role of INSL3 in modulating follicular development, steroidogenesis, and POI pathogenesis is warranted.

Co-culture model reveals the characteristics of theca cells and the effect of granulosa cells on theca cells at different stages of follicular development.

Theca cells (TCs) play an important role in follicular development, which cannot be separated from granulosa cells (GCs). However, compared with mammals, the TCs and the effects of GCs on TCs at different follicular development stages (FDSs) have specific characteristics in avian species, but none of them have been clearly defined. In this study, we established an in vitro co-culture (with GC at the corresponding stage) model of goose TCs at different FDSs (pre-hierarchical, hierarchical and F1) by using a transwell system. The properties of TCs in co-culture at the three FDSs, including cell morphology, activity and intracellular lipid content, as well as the expression of key genes involved in de novo lipogenesis, steroidogenesis, proliferation and apoptosis, were examined and defined. We further compared the mono-culture and co-culture groups. After co-culture, the activity of TCs showed significant (p < .01) increases in all stages; moreover, in pre-hierarchical TCs, the expression levels of FAS, SREBP, 3ß-HSD and CCND1 were promoted, and PPARγ, CYP19, BCL2 and CAS3 were inhibited (p < .05); in the hierarchical TCs, the expression levels of PPARγ, FAS, CYP19, CCND1 and BCL2 were promoted, and SREBP, STAR, 3ß-HSD and CAS3 were inhibited (p < .05), whereas in the F1 TCs, the expression levels of PPARγ, FAS, 3ß-HSD, CYP19 and CCND1 were promoted, and STAR and CAS3 were inhibited (p < .05). These results suggested that GCs at the three FDSs have dynamic and complex influences on the physiological characteristics of TCs, and the influences on TCs at the three FDSs were varied.

Wilms' tumor (WT1) (±KTS) variants decreases the progesterone secretion of bovine ovarian theca cells.

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(-KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(-KTS) upregulation also decreased the mRNA expression of 3ß-HSD. In both variants, WT1(-KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3ß-HSD.

Active immunisation against GnRH as treatment for unilateral granulosa theca cell tumour in mares.

BACKGROUND: Stallion-like or aggressive behaviour in mares affected by unilateral granulosa theca cell tumour (GTCT) is well-known, but use of a GnRH-vaccine as an alternative to surgical removal of the neoplastic ovary has not been investigated. OBJECTIVES: To determine the effect of immunisation against GnRH on ovarian size, testosterone concentration, Anti-Müllerian hormone (AMH) concentration, and owner-reported behaviour in four mares affected by unilateral GTCT. STUDY DESIGN: Retrospective case report. METHODS: A presumptive diagnosis of GTCT was made in four mares based on clinical signs, behavioural changes, transrectal palpation, and ultrasonography. All mares were vaccinated twice with the GnRH-vaccine Improvac® on day 0 and on day 13-33. Further booster vaccinations were administered if aggressive behaviour recurred between days 15 and 498. Before and parallel to the vaccinations, serum levels of oestradiol, progesterone (P4), testosterone, and AMH were evaluated and transrectal ultrasonography was performed. RESULTS: In all horses, analysis of serum levels of oestradiol, progesterone, testosterone, and AMH confirmed the clinical diagnosis of GTCT. Serum levels of testosterone dropped to baseline levels following the first two of three vaccination in all mares. In addition, AMH serum values decreased shortly after the second vaccination in three of four mares, and in one of the four mares returned to baseline levels. No further GTCT linked behaviour was reported by the owners and the affected ovaries diminished in size in all four cases. MAIN LIMITATIONS: This report is a case series with a limited number of animals, no controls and no standardised immunisation protocol. CONCLUSIONS: Repeated vaccinations with the GnRH-vaccine Improvac® mitigated owner-reported behavioural abnormalities and stopped tumour growth in four mares affected by unilateral GTCT over the entire observation period which extends to 7 years in one mare.

Comparison of Human Antral Follicles of Xenograft versus Ovarian Origin Reveals Disparate Molecular Signatures.

The activation, growth, and maturation of oocytes to an ovulatory phase, termed folliculogenesis, is governed by the orchestrated activity of multiple specialized cell types within the ovary; yet, the mechanisms governing diversification and behavior of discrete cellular sub-populations within follicles are poorly understood. We use bulk and single-cell RNA sequencing to distinguish the transcriptional signature of prospectively isolated granulosa and theca/stroma cell subsets within human antral follicles derived from xenografts or ovaries. The analysis deconstructs phenotypic diversification within small (<4 mm) antral follicles, identifying secreted factors that are differentially enriched between mural and oophorus granulosa cells, and segregating stromal/support and steroidal activity between theca externa and interna, respectively. Multiple factors are differentially expressed in follicles of xenograft versus ovarian origin. These data capture a high-resolution transcriptional signature of granulosa and theca subpopulations and provide a systems-level portrait of cellular diversification in early antral human follicles.

Exploration of the effects of goose TCs on GCs at different follicular stages using a co-culture model.

Granulosa cells (GCs) play a critical role in follicular development, which cannot be separated from the assistance of theca cells (TCs). In the present study, we used a transwell system to develop three stages of goose GCs in vitro mono-culture and co-culture models, and we analyzed the morphology, activity, intracellular lipid content and the expression of core genes involved in de novo lipogenesis (DNL), steroidogenesis, proliferation and apoptosis of the GCs. In the co-culture group, the activity of all three stages of GCs showed significant (P<0.01) changes, and they had a strong (P<0.01) correlation with culture time; further, the intracellular lipid deposition of hierarchical GCs was significantly different (P<0.01) between the two methods. Moreover, after co-culture, in pre-hierarchical GCs, the expression of SREBP, CYP11 and 3ßHSD was promoted (P<0.01). In hierarchical GCs, the expression of ACC, SREBP, STAR, CYP11, 3ßHSD and CCND1 was promoted at 48 h, but they were inhibited (P<0.05) at 96 h. In F1 GCs, the expression of ACC, FAS, SREBP, CYP11, BCL2 and CAS3 was inhibited (P<0.01). The results indicate that goose TCs had complex and time-dependent effects on the biological function of GCs at each corresponding stage, and the effects were distinct in the different stages. In addition, DNL, steroidogenesis, proliferation and apoptosis in hierarchical and F1 GCs might have some synergistic relationships in the effects of TCs on GCs. Furthermore, we speculated that TCs might play an important role in the differentiation and maturation of GCs during follicular development.

Developmental and hormonal regulation of ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 gene expression in ovarian granulosa and theca cells of cattle.

Ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) is a multi-domain nuclear protein that plays an important role in epigenetics and tumorigenesis, but its role in normal ovarian follicle development remains unknown. Thus, the present study evaluated if UHRF1 mRNA abundance in bovine follicular cells is developmentally and hormonally regulated, and if changes in UHRF1 are associated with changes in DNA methylation in follicular cells. Abundance of UHRF1 mRNA was greater in granulosa cells (GC) and theca cells (TC) from small (<6 mm) than large (≥8 mm) follicles and was greater in small-follicle GC than TC. In GC and TC, fibroblast growth factor 9 (FGF9) treatment increased (P < 0.05) UHRF1 expression by 2-fold. Also, luteinizing hormone (LH) and insulin-like growth factor 1 (IGF1) increased (P < 0.05) UHRF1 expression in TC by 2-fold, and forskolin (an adenylate cyclase inducer) alone or combined with IGF1 increased (P < 0.05) UHRF1 expression by 3-fold. An E2F transcription factor inhibitor (E2Fi) decreased (P < 0.05) UHRF1 expression by 44% in TC and by 99% in GC. Estradiol, progesterone, and dibutyryl-cAMP decreased (P < 0.05) UHRF1 mRNA abundance in GC. Treatment of GC with follicle-stimulating hormone (FSH) alone had no effect but when combined with IGF1 enhanced the UHRF1 mRNA abundance by 2.7-fold. Beauvericin (a mycotoxin) completely inhibited the FSH plus IGF1-induced UHRF1 expression in small-follicle GC. Treatments that increased UHRF1 mRNA (i.e., FGF9) in GC tended to decrease (by 63%; P < 0.10) global DNA methylation, and those that decreased UHRF1 mRNA (i.e., E2Fi) in GC tended to increase (by 2.4-fold; P < 0.10) global DNA methylation. Collectively, these results suggest that UHRF1 expression in both GC and TC is developmentally and hormonally regulated, and that UHRF1 may play a role in follicular growth and development as well as be involved in ovarian epigenetic processes.