Dietary Coconut Oil Mitigates Hyperandrogenemia in Obese Female Pigs Due to Suppression of Androgen Steroidogenesis in the Adrenal Cortex and Theca Externa

Western-style diets (WSD) cause cardiometabolic disease and hyperandrogenemia in reproductive-age women. A promising alternative for improved fertility in obese women may derive from the nutraceutical, coconut oil. We hypothesized that dietary coconut oil included in a high fat diet would mitigate hyperandrogenemia due to obesity through depression of androgen steroidogenesis in theca externa and adrenal cortex. Seventeen sexually mature female Ossabaw pigs were divided into 3 diet groups: control (n=6; C; 2200 kcal/pig/day), WSD (n=5; WSD; 5000 kcal/pig/day), or high fat coconut oil diet (n=6; COC; 5000 kcal/pig/day). Ingredients in WSD and COC diets were the same except 9% of calories in WSD were from lard and 9% of calories in COC were from coconut oil. Pigs were fed for 9 cycles (~ 8 mos) and fasting blood was collected at cycle 0 (baseline), 7, and 9. After cycle 7, ovarian steroidogenesis was suppressed by oral progestogen for 18 days. On day 18 while ovarian steroidogenesis was still suppressed, an ACTH stimulation was performed. On day 19, progestogen was withdrawn and 7.5 µg/kg IM dexamethasone was given every 12 hours to suppress adrenal steroidogenesis. On day 20, 3000 IU/m2 human chorionic gonadotropin (hCG) was given IV. Pigs underwent a washout cycle post-stimulations and were euthanized when they had dominant ovarian follicles in cycle 9 for collection of follicular fluid (FF), theca externa, and adrenal cortex. FF was assessed for steroid hormones by LC-MS/MS. Adrenal cortex and theca cells were cultured as follows for 48 hours after which cells and media were collected: 1) theca: control, insulin (I; 100 ng/ml), LH (10 ng/ml), or LH+I (10 ng/ml + 100 ng/ml); 2) adrenal: control or 1 µM ACTH. WSD pigs had a protracted estrous cycle length and increased FF testosterone, dehydroepiandrosterone sulfate, androstenedione (A4), androstenediol, and allo-pregnanolone (Allo-Preg) compared to C pigs. Both serum and cell culture media A4 concentrations were higher in response to ACTH in WSD pigs. By 72 hours post-hCG, COC pigs had higher serum A4 than C or WSD pigs, but in vitro LH yielded decreased cell culture media A4 in both WSD and COC pigs. COC pigs had increased FF dihydrotestosterone and Allo-Preg compared to C pigs. These results suggest that dietary coconut oil administration in obese females may depress excessive androgen steroidogenesis by adrenal cortex but may only partially mitigate excessive androgen production by theca externa.

Histological description of aceh cattle ovary cryopreserved by various cryoprotectants

This research aimed to determine Aceh cattle ovarian follicle morphological integrity after vitrified by various cryoprotectants. Cryoprotectants used in this research were 30% ethylene glycol (EG), 30% dimethyl suphocide (DMSO), and combination of 15% EG + 15% DMSO. Prior to vitrification process, ovaries were cleansed by phosphate buffered saline (PBS) and then cut into ±1 mm³. Ovaries were consecutively submerged into the following liquid for 5 minutes each: PBS+ 0.25 M sucrose; PBS+ 0.5 M sucrose; PBS+ 0.5 M sucrose + 10% cryoprotectants; and PBS+ 0.5 M sucrose + 30% cryoprotectants. Then, ovaries were packed into straws with ±7 cm in length and ± 6 mm in diameter. Before kept in liquid nitrogen, ovaries were first exposed to nitrogen fume for 10 second. After being stored for 1 day, the ovaries were proceed for histological examination. The result showed that Aceh cattle ovarian follicle after vitrification using 30% EG yields the best morphological integrity. Cumulus oophorus, zona pellucida, granulose cell arrangement, theca interna, and theca externa cells were observed clearer in ovary which was vitrified with 30 % EG than those with 30% DMSO and combination of 15% EG + 15% DMSO. As conclusion, 30% EG was able to protect ovary morphological integrity better than 15 % EG + 15% DMSO and 30% DMSO. Furthermore, combination of 15% EG+ 15 % DMSO was relatively better in protecting ovary follicle morphological integrity compared to 30% DMSO.

Establishment of an in vitro culture model of theca cells from hierarchical follicles in ducks

Theca cells, including theca interna cells and theca externa cells, are vital components of ovarian follicles. The aim of the present study is to identify a reliable method for the in vitro culture of theca cells from duck ovarian hierarchical (F4-F2) follicles. We improved the method for cell separation by using trypsin to further remove granular cells, and we increased the concentration of fetal bovine serum used in in vitro culture to improve cytoactivity. Cell antibody immunofluorescence (IF) showed that all inoculated cells could be stained by the CYP17A1/19A1 antibody but not by the FSHR antibody, which could stain granulosa cells. Furthermore, morphological differences were observed between the outlines of theca interna and externa cells and in their nuclei. Growth curve and CYP17A1/19A1 mRNA relative expression analyses suggested that the growth profile of theca interna cells may have been significantly different from that of theca externa cells in vitro. Theca interna cells experienced the logarithmic phase on d1–d2, the plateau phase on d2–d3, and the senescence phase after d3, while theca externa cells experienced the logarithmic phase on d1–d3, the plateau phase on d3–d5, and the senescence phase after d5. Taken together, these results suggested that we have successfully established a reliable theca cell culture model and further defined theca cell characteristics in vitro.

Alteration in localization of steroid hormone receptors and coregulatory proteins in follicles from cows with induced ovarian follicular cysts

Cystic ovarian disease (COD) is an important cause of infertility in cattle. The altered follicular dynamics and cellular differentiation observed in COD may be mediated through a disruption of the expression of steroid receptors and their associated transcriptional cofactors. The aim of this study was to determine the protein expression profiles of ESR1, ESR2, PGR, AR, NCOA3, NCOR2, and PHB2 (REA) in ovarian follicles in an experimental model of COD induced by the administration of ACTH. Ovaries were collected and follicles were dissected from heifers during the follicular phase (control) or from heifers treated with ACTH to induce the formation of ovarian follicular cysts. Ovaries were fixed, sectioned, and stained immunohistochemically for steroid receptors and the associated transcription factors. The relative expression of ESR1 was similar in follicular cysts and in tertiary follicles from both control and cystic cows and was significantly higher than in secondary follicles. The expression of ESR2 in the granulosa was higher in cystic follicles. No differences were seen for PGR. The expression of androgen receptor was significantly increased in tertiary follicles with lower immunostaining in cysts. The expression of NCOA3 was observed in the granulosa and theca with a significantly increased expression in the theca interna of cystic follicles. The highest levels of NCOR2 expression in granulosa, theca interna, and theca externa were observed in cysts. In granulosa cells, NCOR2 levels increase progressively as follicles mature and the treatment had no effect. In summary, ovaries from animals with induced COD exhibited altered steroid receptor expression compared with normal animals, as well as changes in the expression of their regulators. It is reasonable to suggest that in conditions characterized by altered ovulation and follicular persistence, such as COD, changes in the intra-ovarian expression of these proteins could play a role in their pathogenesis.

332. GYCOMIC ANALYSES OF THE GRANULOSA AND THECA OF BOVINE OVARIAN FOLLICLES

Development of ovarian follicles involves changes in cell function and remodelling of the follicular wall. Remodelling necessitates changes in the extracellular matrix, including proteoglycans (PGs). PGs contain glycosaminoglycans (GAGs) covalently bound to a protein core. The length of GAG chains in PGs and the degree and pattern of sulphation differs between cell types and change as cells alter their phenotype. PGs that have been identified in follicles include the chondroitin sulphate (CS) PG, versican and inter-a trypsin inhibitor, and the heparan sulphate (HS) PGs, perlecan and type XVIII collagen. The latter two are found in focimatrix, the follicular and the thecal subendothelial basal laminas. To examine GAGs composition in follicles, bovine antral follicles of various sizes were collected. Follicles were dissected and a biopsy taken for histological classification of health. Theca layers and granulosa cells were collected separately and analysed by fluorophore-assisted carbohydrate (FACE) analysis of GAGs following digestion to disaccharides with chondroitinase ABC, hyaluronidase, heparinase, and heparitinases I and II. Four non GAG sugars and 12 different GAG derived disaccharides were identified and quantitated on a per DNA basis. Healthy versus atretic follicles for each cell type were compared and correlation analyses were also undertaken. Immunohistochemistry using CS specific antibodies was also conducted. There was no effect of size on the GAG content for either granulosa or theca cells. The 4- and 6- sulphated CS sugars were the most abundant following digestion in all tissues. Theca had higher levels than granulosa cells of HS derived disaccharides and also of un- or 4- or 6- N-sulphated CS derived disaccharides. Some sulphated CS moieties localised uniquely to the stroma surrounding blood vessels in the theca externa. Atretic follicles had lower amounts of disaccharides derived from HS in both granulosa and thecal cells, suggesting that heparanase may be activated upon atresia.

Ultrastructural features of the follicular wall in developing follicles of the sexually immature ostrich (Struthio camelus)

The ultrastructure of the follicular wall in primordial, previtellogenic and vitellogenic follicles of the sexually immature ostrich is described in the present study. The follicular wall consists of a zona radiata, granulosa cell layer, basal lamina and thecal layer. Cytoplasmic processes from the plasma membranes of the granulosa cell layer and the ovocyte form the zona radiata in previtellogenic and vitellogenic follicles. The granulosa cell layer transforms from simple cuboidal epithelium in primordial follicles to simple columnar or pseudostratified columnar epithelium in previtellogenic and vitellogenic follicles. Transosomes were observed along the apical and lateral plasma membranes of granulosa cells. The thecal layer in previtellogenic and vitellogenic follicles consists of interna and externa components. The fibroblasts in the theca externa contain microfilaments, which are thought to be actin filaments. The study revealed ultrastructural features, which are associated with the transportation of yolk precursors and nutrients into the ovoplasm. In addition, the study indicates that, although the cells in the theca externa contain microfilaments, they do not possess the ultrastructural characteristics of smooth muscle cells.

Ultrastructural observations of previtellogenic ovarian follicles of dove

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.

Cloning and Characterization of a Rat Ortholog of MMP-23 (Matrix Metalloproteinase-23), a Unique Type of Membrane-Anchored Matrix Metalloproteinase and Conditioned Switching of Its Expression during the Ovarian Follicular Development

In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.