To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and gamma-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (gamma-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes

This investigation was designed to study ovarian and hormonal changes in the rat after treatment with dehydroeplandrosterone (DHEA). We identified a heterogeneous experimental group of animals with respect to ovarian histology: group I, corpora lutea (Cls) + cysts; group II, CLs + no cysts; group III, no CLs + cysts; group IV, no CLs + no cysts. Histological sections of these ovaries showed healthy and atretic follicles in different stages of cytomorphosis and degeneration. The aforementioned histological groups were also heterogeneous according to their hormonal profiles. Serum androgens, estrogens, and prolactin concentrations are significantly increased in DHEA-treated animals as compared with controls. There was no significant difference in follicle stimulating hormone between rats with cysts and rats without cysts after DHEA treatment. After 20 days of DHEA treatment, rats with CLs have very high levels of luteinizing hormone. Luteinizing hormone and prolactin levels are significantly higher in rats with cysts than in rats without cysts after 10 days of DHEA treatment. As has been shown in this inquiry, androgens and estradiol levels in rats with cysts after DHEA treatment are higher than those in rats without cysts after DHEA treatment. Therefore, this study suggests that the ovarian cystic condition developed after DHEA treatment in rats, is associated with higher levels of circulating androgens, estradiol, and prolactin.

The abnormal regulation of the Ke 6 gene has been linked to the development of recessive polycystic kidney disease in the mouse. In this report, we have shown that Ke 6 is a 17␤-hydroxysteroid dehydrogenase and can regulate the concentration of biologically active estrogens and androgens. The Ke 6 enzyme is preferentially an oxidative enzyme and inactivates estradiol, testosterone, and dihydrotestosterone. However, the enzyme has some reductive activity and can synthesize estradiol from estrone. We find that the Ke 6 gene is expressed within the ovaries and testes. The presence of Ke 6 protein within the cumulus cells surrounding the oocyte places it in a strategic location to control the level of steroids to which the egg is exposed. Previously, it had been shown that glucocorticoids can induce renal cysts in the neonatal rodent, only when given at a narrow time window of postnatal kidney development. We propose that the reduction in the level of Ke 6 enzyme, which occurs in the cpk, jck, and pcy mice, may lead to abnormal elevations in local level of sex steroids, which either directly or indirectly via abnormal glucocorticoid metabolism result in recessive renal cystic disease, a developmental disorder of the kidney.The Ke 6 gene, encoded within the major histocompatibility complex, has been intimately linked to the development of cysts in the kidney and liver of mice (1-6). The expression of the Ke 6 gene is severely down-regulated in all murine models of PKD 1 that have been examined to date: cpk, jck (1, 3) and pcy mice (2). The inhibition of the Ke 6 gene expression using antisense deoxyoligonucleotides in embryonic kidneys in organ cultures gives rise to numerous cysts against a background of normal nephrogenesis (5, 6). Importantly, the human Ke 6 gene is located on chromosome 6p21 (7) where the human autosomal recessive PKD mutation has been mapped (8) and therefore there is a possibility that the Ke 6 gene is the primary mutation in autosomal recessive PKD. The structure of the mouse Ke 6 gene (2), cDNA (1), and protein (4) has been extensively characterized in our laboratory.Our earlier data base searches with the Ke 6 sequence showed that it belongs to the short-chain dehydrogenase/reductase family (1). At that time, Ke 6 was most similar to bacterial oxidoreductases with no close similarity to any mammalian oxidoreductase in the data base, leaving the exact function of Ke 6 unknown. Recently, we searched the data base again with Ke 6 and found it is similar to 17␤HSD4 (9). This prompted us to examine Ke 6 for 17␤HSD activity with various androgen and estrogen substrates.As reported here, we find that Ke 6 protein is an NAD-dependent 17␤HSD, making it the seventh member of this class of enzyme. It efficiently catalyzes the oxidation of estradiol, testosterone, and dihydrotestosterone and also the reduction of estrone to form biologically active estradiol. The identification of the substrate of Ke 6 allows us to postulate that regulated sex steroid metabolism plays a crucial role in the development o...

Immature 27-day-old female Sprague-Dawley rats were administered daily subcutaneous injections of dehydroepiandrosterone (DHEA, 5 mg/100 g BW) to induce the formation of ovarian follicular cysts. Groups of rats were killed on days 0, 10, 15, 20, 25, and 30. Ovaries from each group of rats were processed for light and electron microscopy and for follicular or cystic fluid hormone analysis. Normal antral follicle fluid, PMSG-treated preovulatory follicular fluid, and cystic fluids were analyzed for progesterone (P), estrone (E1), estradiol (E2), testosterone (T), delta 4-androstenedione (delta 4-A), 5 alpha-dihydrotestosterone (DHT), luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL). DHEA induced anovulation, acyclicity, and the formation of follicular cysts. In certain antral follicles, there was a dramatic increase in the quantities of smooth endoplasmic reticulum (SER) in the granulosa cells and many mitochondria had tubular cristae. Further depletion of granulosa cell number was associated with intense blebbing of the cytoplasm into the follicle antrum. Formation of the ovarian follicular cyst was completed when the entire cyst was lined by a single layer of transformed granulosa cells in contact via adhering, gap, and tight junctions. These cells had little cytoplasm, mitochondria with lamellar cristae, vast basal and apical bands of microfilaments, and an extensive array of smooth-surfaced endocytotic invaginations on the basal plasma membrane. These endocytotic pits may subsequently form smooth-surfaced vesicles and thereby serve as one mechanism for moving fluid from the ovarian interstitium into the cyst. Theca interna cells were rarely observed in the peripheral regions of the cyst. Abundant smooth muscle cells were located beneath the basement membrane of the epithelial cells comprising the cyst wall. These acquired morphological and physiological features may ensure persistence of the ovarian cyst and thus potentiate a chronic pathological condition. In this study it was also shown that progesterone, estrone, and estradiol as well as androgen concentration increased in the follicle after PMSG treatment. With DHEA treatment, the follicular cystic fluid concentrations of these steroids progressively increased to extremely high levels concurrent with the development of the follicular cysts.(ABSTRACT TRUNCATED AT 400 WORDS)