E-cadherin-catenin complex in the rat ovary: cell-specific expression during folliculogenesis and luteal formation.

The cadherins and their cytoplasmic counterparts, the catenins, form the adherens junctions, which are of importance for tissue integrity and barrier functions. The development and maturation of the ovarian follicle is characterized by structural changes, which require altered expression or function of the components involved in cell-cell contacts. The present study examined the cell-specific localization and temporal expression of epithelial cadherin (E-cadherin) and alpha- and beta-catenin during follicular development, ovulation and corpus luteum formation in the immature gonadotrophin- and oestrogen-stimulated rat ovary. Immunohistochemistry and immunoblotting demonstrated the expression of E-cadherin in theca and interstitial cells of immature ovaries before and after injection of equine chorionic gonadotrophin (eCG). E-cadherin was not detected in granulosa cells, except in the preantral follicles located to the inner region of the ovary. The content of E-cadherin in theca and interstitial cells decreased after an ovulatory dose of hCG. Granulosa cells of apoptotic follicles did not express E-cadherin. Oestrogen treatment (diethylstilboestrol) of immature rats for up to 3 days did not result in a measurable expression of E-cadherin in granulosa cells. alpha- and beta-catenin were expressed in all ovarian compartments. The concentration of beta-catenin was constant during the follicular phase, whereas the content of alpha-catenin decreased in granulosa cells after treatment with diethylstilboestrol or hCG. The expression of alpha-catenin was also reduced in theca and interstitial cells after hCG. alpha- and beta-catenin were present in most ovarian cells at all stages of folliculogenesis. Therefore, the catenins have the potential to associate with different members of the cadherin family and to participate in the regulation of cytoskeletal structures and intracellular signalling. The restricted expression of E-cadherin in granulosa cells of preantral follicles indicates a role in the recruitment of these follicles to subsequent cycles. The specific decrease of alpha-catenin in granulosa cells and the reduction of both alpha-catenin and E-cadherin in theca cells of ovulatory follicles might reflect some of the molecular changes in cell-cell adhesion associated with ovulation and luteinization.

E-cadherin expression in human epithelial ovarian cancer and normal ovary.

The ovarian surface epithelium (OSE) is the origin of the majority of human ovarian cancers. These adenocarcinomas are characterized by initial local growth followed by spreading into the peritoneal cavity at later stages of tumor progression. The cell-adhesion molecule E-cadherin (E-cad) plays an important role in maintaining tissue integrity. Disappearance or impaired function of E-cad have often been associated with tumor formation and invasion in vivo and in vitro. The cell-specific expression of E-cad was investigated in normal human ovaries (n = 12), in benign (n = 5) and borderline (n = 4) ovarian epithelial tumors and in adenocarcinomas of different stages and histological grades (n = 18), by immunohistochemistry and immunoblotting. An ovarian cancer cell line (NIH-OVCAR3) was used as a reference. The epithelial origin of the cells was confirmed with cytokeratin (AE1/AE3) staining. In normal ovaries, the expression of E-cad was limited to inclusion cysts or deep clefts lined with OSE, whereas no staining of the OSE could be demonstrated at the surface of the ovary. In contrast, benign and borderline tumors uniformly expressed E-cad. This was observed in malignant tumors of all stages despite their degree of differentiation. E-cad was also present in metastasis from such tumors. The cell-specific expression of E-cad in inclusion cysts of normal ovaries and in epithelial layers of borderline tumors indicates a role for E-cad in the early events of the progression to a malignant phenotype. E-cad was not downregulated in later stages of ovarian cancer progression.

The expression of c-myc during follicular growth and luteal formation in the rat ovary in vivo.

The processes of folliculogenesis and formation of corpora lutea involve proliferation and differentiation of the follicular cells. The expression of several oncogenes is associated with the proliferative phase in many cell types. The present study examined the expression and hormonal regulation of the c-myc proto-oncogene during follicular development and the luteal phase of pseudopregnancy. Follicular development was initiated by pregnant mare's serum gonadotropin (PMSG) in immature rats followed two days later by the injection of human chorionic gonadotropin (hCG) to induce ovulation and luteal formation. Ovaries were collected at different time points and the content and distribution of c-myc mRNA/protein were examined. C-myc increased rapidly after the administration of both PMSG and hCG, but the effect of PMSG was less pronounced. The increase after PMSG was transient and localized primarily to the granulosa cells of developing follicles. The ovulatory dose of hCG resulted in a rapid and substantial increase of c-myc mRNA and protein with maximal levels at 1 h and 2-4 respectively. At this stage, the c-myc protein was localized to the follicular cells, the surface epithelium and, to some extent, to the interstitial tissue. There was a subsequent decrease prior to ovulation. The luteal phase was characterized by decreasing levels of c-myc with increasing luteal age. In order to examine the involvement of specific hormones in the regulation of c-myc, hypophysectomized, immature rats were injected sequentially with estradiol (E2) and follicle-stimulating hormone (FSH). Hypophysectomy resulted in a decrease of c-myc compared with intact animals. The administration of E2 resulted in an increase of c-myc mRNA and protein. The subsequent treatment with FSH did not result in a further increase and the levels remained at the same level as with E2 only. However, an ovulatory dose of hCG to E2 and FSH primed animals resulted in an additional increase of c-myc mRNA and protein. The levels after E2 and FSH were considerably lower compared with those of untreated ovaries of intact, immature animals, suggesting the involvement of other endocrine and paracrine factors. The presence of proliferating cell nuclear antigen in cell extracts indicated that the expression of c-myc was associated with phases of increased proliferation of follicular cells after hormonal stimulation. The results demonstrate that c-myc is regulated by hormones (E2, gonadotropins) in the rat ovary during follicular development to the preovulatory stage. The pronounced increase prior to ovulation also suggests a role for c-myc in the regulation of proliferative events involved in luteal formation.

Expression of CCAAT enhancer binding protein-alpha (C/EBP alpha) in the rat ovary: implications for follicular development and ovulation.

The development of follicles from the antral to the preovulatory stage in the mammalian ovary involves both proliferation and differentiation of cells. These processes are coordinated by endocrine, paracrine, and autocrine factors in a time- and cell-specific pattern. Each stage of development is characterized by expression of specific genes the products of which are involved in different cellular processes, e.g., signal transduction (cAMP-dependent protein kinases), steroidogenesis (cytochrome P450 enzymes). At the nuclear level, the signaling pathway from external stimuli converges to modify the mechanisms of transcription factors. One such factor, CCAAT enhancer binding protein-alpha (C/EBPalpha), participates in differentiation processes of several organs, e.g., liver, adipose tissue, and gut. We have previously demonstrated that C/EBPalpha is expressed in rat ovarian follicles in a cell-, time-, and hormone-specific manner. This increase in granulosa cells was concomitant with the more differentiated phenotype. The aim of the present study was to explore the function of C/EBPalpha in the rat ovary. To achieve this, the expression of C/EBPalpha in follicular cells was attenuated in vivo by the local administration of antisense oligonucleotides (AS) into the ovarian bursa, i.e., the sac-like structure surrounding the ovary of immature rats. This administration resulted in an impaired response to subsequent injections of exogenous gonadotropins (PMSG, hCG) with an attenuated expression of C/EBPalpha protein and finally a decreased ovulation rate. Furthermore, the morphology of the AS-treated ovary was altered with large, oocyte-containing follicles at a time when ovaries exposed to sense (S) oligonucleotides demonstrated newly formed corpora lutea. AS also affected the expression of the proto-oncogene c-myc, which was elevated by this treatment. The administration of S was without effects. Thus, C/EBPalpha seems to be a necessary factor for follicular development in the rat ovary.

Meiotic arrest in incompetent rat oocytes is not regulated by cAMP.

Fully grown, but not growing, mammalian oocytes spontaneously resume meiosis in vitro. Resumption of meiosis, also known as oocyte maturation, is associated with a drop in intraoocyte concentrations of cAMP followed by activation of the maturation promoting factor (MPF). Microtubule-associated-protein (MAP) kinase has been suggested as a substrate for the active p34cdc2 kinase, the catalytic subunit of MPF. Our study was designed to explore the mechanism of regulation of meiotic arrest in growing rat oocytes. Confirming previous observations we showed that in our rat colony oocytes do not acquire the competence to spontaneously resume meiosis earlier than 22 days postpartum. We further demonstrated that follicle-enclosed oocytes from 20-day-old female rats fail to resume meiosis in response to luteinizing hormone, follicle-stimulating hormone, a gonadotropin-releasing hormone analog, or forskolin, all of which are known to induce maturation in competent oocytes. Immunoblot analysis using highly specific anti p34cdc2 antibodies revealed that incompetent oocytes express the catalytic subunit of MPF at amounts that are not different from that found in competent oocytes. In addition, highly specific anti MAP kinase antibodies detected the presence of similar quantities of two isoforms (42 and 44 kDa) of MAP kinase in competent and incompetent oocytes. Measurements of cAMP revealed that as compared to competent oocytes, incompetent oocytes contain somewhat lower levels of this nucleotide (1.42 +/- 0.3 and 1.17 +/- 0.07 fmole/oocyte, respectively). However, considering the difference in protein content, the calculated concentrations seem to be similar. Furthermore, similar to competent oocytes, intracellular concentrations of cAMP in incompetent oocytes dropped significantly (from 1.17 +/- 0.07 to 0.77 +/- 0.12 fmole/oocyte) 2 hr after isolation from the follicle. We hereby suggest that (a) in mammals, similar to amphibians, the term meiotic incompetence can be extended to include inability to resume meiosis in response to hormonal stimulation; (b) it is not the lack of p34cdc2 or downstream regulatory elements, such as MAP kinase, that prevents growing oocytes from resuming meiosis; and (c) unlike fully grown oocytes, resumption of meiosis in growing oocytes is not subjected to negative regulation by cAMP.

Expression and hormonal regulation of the CCAAT enhancer binding protein-alpha during differentiation of rat ovarian follicles.

Follicular development involves both proliferation and differentiation of thecal and granulosa cells. The process is regulated by gonadotropins and paracrine and autocrine factors, including steroid hormones, presumably by the induction of different genes at specific time points. In the present study, the expression and distribution of the CCAAT enhancer-binding protein-alpha (C/EBP alpha) were studied in immature ovaries and in ovaries in which follicular growth and development were initiated with PMSG, whereas ovulation and luteal formation were induced by the injection of hCG. Ovaries were collected before and at different time points after PMSG (0, 6, 24, and 48 h) and hCG (0.25, 1, 3, 10, and 24 h) treatment for analyses of the contents of C/EBP alpha mRNA and protein and the cell-specific immunohistochemical localization of the protein. C/EBP alpha mRNA increased to maximal levels 24 h after PMSG treatment. The effect was specific for the ovary, as C/EBP alpha mRNA in the uterus did not change. C/EBP alpha mRNA decreased 10 h after hCG treatment and increased again in newly formed corpora lutea. Immunohistochemistry and immunoblotting demonstrated a similar increase in C/EBP alpha during follicular development. To examine the involvement of specific hormones in the regulation of C/EBP alpha, hypophysectomized immature rats were injected sequentially with estradiol and FSH. This treatment resulted in a substantial increase in C/EBP alpha mRNA and protein. These results demonstrate that C/EBP alpha is hormonally regulated in the ovary and suggest a role for C/EBP alpha during differentiation of ovarian cells and follicular development.

Nest ventilation explains gas composition in the nest-chamber of the European bee-eater.

The European bee-eater Merops apiaster builds a nest in soil banks comprising a horizontal corridor (159 cm) leading to a 3.4 L nest chamber. An adult and six eggs and/or nestlings occupy this nest for 60 d. Incubation and development are asynchronous. In order to evaluate the gas exchange pattern of the occupied nest, we measured the O2 diffusive conductance of the nest with the ambient environment (GNO2), its internal O2 and CO2 pressures (PNO2 and PNCO2), temperature (TN) and observed the sequences of development and the frequency of parental visits. We also measured the rate of O2 uptake (MO2) of the eggs, nestlings and adults. The GNO2 was 10.9 ml[STPD]/(h.Torr). The PNO2 dropped and the PNCO2 increased during incubation and growth to a maximal change of 24 and 23 Torr, respectively. Maximal MO2 of an egg and a nestling were 5.4 and 146 ml[STPD]/h, respectively. The MO2 of an adult at rest and at TN (27 degrees C) was 90.5 ml[STPD]/h. From clutch size, sequence of nest events, and the above values we have estimated a maximal nest chamber ventilation (VN) of 0.8 L/min necessary to prevent lowering PNO2 to 74 Torr below ambient PO2. This convection is achieved chiefly by the parental feeding visits (up to 40/h). Thus, movement along the corridor convects 1.2 L per visit.

Response of male and female Siberian hamsters towards pups.

The influence of sex, reproductive maturity, and parental experience on the behavior of Siberian hamsters (Phodopus sungorus sungorus) toward strange pups was examined in this study. Immature, mature, or parentally experienced male and female Siberian hamsters were presented with 3- to 8-day-old pups. We recorded pup carrying during the initial 10 min, nesting with pups after 8 or 14 h, and attacking pups during the test period. Among inexperienced animals, more mature males attacked pups than did immature males or mature females. Parental experience significantly decreased pup attacks by males and increased nesting with pups by both males and females. Repeated testing did not modify the responses to pups. We conclude that in Siberian hamsters both caretaking and aggressive responses to pups may be shown by immature and mature animals of both sexes. Parental experience resulted in increased caretaking by males and females and decreased aggressive responses by males.