Localisation of potassium pumps in Drosophila ovarian follicles

It has been shown previously that, in Drosophila oogenesis, potassium ions are important for bioelectric phenomena as well as for other physiological and developmental processes. In the present study we determined the spatial distribution and activity of the Na+,K+)-pump and of ouabain-insensitive K+ pumps in plasma membranes of vitellogenic ovarian follicles (stage 10). We used the light microscopic anthroylouabain method as well as the cytochemical lead and cerium precipitation methods in combination with electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). (Na+,K+)-ATPase activity was predominantly observed on the oolemma as well as on the membranes of the columnar follicle cells covering the oocyte, whereas on the membranes of the nurse cells and of the squamous follicle cells covering the nurse cells the activity was very low. The highest activity of the (Na+,K+)-pump was found at the anterior and posterior ends of the oocyte, and this on the oolemma as well as on the membranes of the follicle cells located here. Strong activity of the ouabain-insensitive K+-pumps was observed on most of the oolemma (except at the anterior of the oocyte) and on the membranes of some nurse cells located next to the oocyte, whereas less activity was found on the other nurse cell membranes and on the membranes of all follicle cells. The suitability of the different methods used for determining the localisation as well as the activity of K+-pumps is discussed. We further discuss the nature of the ouabain-insensitive K+ pumps and the relevance of the observed distribution of K+-pumps for K+ uptake, extrafollicular ionic current flow, intercellular signalling and other developmental processes in Drosophila oogenesis.     

Suppression of apoptosis by gonadotropin, 17beta-estradiol, and epidermal growth factor in rainbow trout preovulatory ovarian follicles

In this study we present the first evidence for the occurrence of apoptotic cell death in ovarian follicles from teleost fish. Preovulatory ovarian follicles from mature hatchery-raised rainbow trout (Oncorhynchus mykiss) were collected and either immediately frozen in liquid nitrogen or incubated in serum-free medium at 18 degrees for 24 hr. The extent of ovarian apoptotic DNA fragmentation was determined using 3'-end labeling of DNA with [32P]dideoxy-ATP, size fractionation by agarose gel electrophoresis, and quantification of low-molecular-weight (<15 kb) DNA using autoradiography and liquid scintillation counting. The extent of apoptotic DNA fragmentation was eightfold greater in immediately frozen preovulatory follicles than in previtellogenic ovarian follicles collected from immature rainbow trout (P < 0.05), suggesting differences in the degree of apoptosis at different stages of follicular development. In preovulatory trout follicles, the extent of apoptotic DNA fragmentation was fivefold greater in follicles incubated for 24 hr. Treatment of incubated preovulatory follicles with either partially purified salmon gonadotropin SG-G100 (1 microg/ml) or epidermal growth factor (EGF; 100 ng/ml) suppressed apoptotic DNA fragmentation by 31 and 41%, respectively, in comparison to untreated incubated follicles (P < 0.01). Treatment of incubated follicles with 17beta-estradiol (1-100 ng/ml) caused a concentration-dependent suppression of apoptotic DNA fragmentation (P < 0.05). These results suggest that apoptosis is involved in teleost ovarian development and that several of the hormonal factors acting as follicle survival factors in mammalian and avian ovaries may play a similar role in teleost ovarian follicles.     

Permeability of ovarian follicles to electron-dense macromolecules

Ferritin (m.w. 500 000) perfused into the ovarian arteries of sheep permeated the theca interna and was also found in the membrana granulosa of both non-atretic and atretic Graafian follicles. Colloidal gold (m. w. 1 000 000) similarly perfused, was found in the theca interna but not in the granulosa. The results are discussed in terms of the blood-follicle barrier.     

Detection and clinical correlations of ras gene mutations in human ovarian tumors

In epithelial ovarian neoplasms K-ras codon 12 gene mutations show a wide variation fluctuating between 4-39% in invasive carcinomas and 20-48% in borderline malignant tumors. In this study, we showed the pattern of point mutations in codon 12 of the K-ras, H-ras and N-ras genes, using polymerase chain reaction restriction fragment length polymorphism analysis in 74 tissue specimens of Greek patients with epithelial ovarian tumors. K-ras and H-ras gene mutations were detected in 11/48 (23%) and 3/48 (6%) cases with primary invasive ovarian carcinomas, respectively, while N-ras gene mutations were not found. No mutation of K-, H- and N-ras genes was detected in 23 ovarian cystadenomas. In 1 out of 3 borderline ovarian tumors (33%) we found an H-ras gene mutation. The prevalence of mutations in K-ras gene was 1/8 (13%) in mucinous, 7/29 (24%) in serous, 1/3 (33%) in endometrioid and 2/8 (25%) in clear-cell adenocarcinomas and in H-ras gene 1/8 (13%) in mucinous and 2/29 (7%) in serous adenocarcinomas. Analysis of the results revealed no significant correlation between ras gene mutations and clinicopathological parameters or clinical outcome of this primary invasive ovarian carcinoma population. Our present data suggest that ras gene mutations in invasive ovarian carcinomas occur in 29% of Greek patients and are not associated with the differentiation of the epithelial cells or the response of patients to adjuvant platinum-based chemotherapy.     

Structural changes occurring during atresia in sheep ovarian follicles

The structural changes that characterize primary, secondary and tertiary atresia in sheep Graafian follicles have been studied by means of histological, histochemical and ultrastructural techniques. In primary atresia vacuoles representing swollen endoplasmic reticulum are prominent along the antral border together with disorganized granulosa cells containing pyknotic nuclei. Phagocytic cells, which increase in number as atresia progresses, were seen within the membrana granulosa and are considered to be transformed granulosa cells. Even in follicles classified as nonatretic, a few antral vacuoles and occasional pyknotic nuclei are present. During secondary atresia there is a large increase in the number of cells with pyknotic nuclei; many of these nuclei had been extruded and had fused to form the characteristic Feulgen-positive atretic bodies found along the edge of the antral cavity. These bodies usually have a diameter of up to 15 mum but occasionally reached as much as 400 mum. A second area of degeneration is frequently present in the membrana granulosa, two or three cell layers from the basal lamina, and it is at this level that exfoliation of granulosa cells occurs in tertiary atresia. In contrast to the membrana granulosa, there are during secondary atresia, only slight indications of degeneration in the cumulus. In tertiary atresia the membrana granulosa is highly disorganized; the atretic bodies are often fewer in number than at earlier stages. The basal lamina remains essentially intact. It is at this stage that the first clear signs of degeneration occur in the theca interna. Despite some disintegration of the cumulus, the integrity of the oocyte is maintained and its nucleus remains vesicular. Changes in the thecal microcirculation may plan a key role in atresia: adjacent to the basal lamina of non-atretic follicles, there is a well-developed capillary network which is significantly reduced as atresia progresses.     

Engrafted human T and B lymphocytes form mixed follicles in lymphoid organs of human/mouse and human/rat radiation chimera

BACKGROUND: We recently described a new approach that enables the generation of human/mouse chimera by adoptive transfer of human peripheral blood mononuclear cells into lethally irradiated normal strains of mice or rats, radioprotected with bone marrow from donors with severe combined immune deficiency. In such human/mouse chimera, a marked humoral response to recall antigens, as well as a significant primary response to keyhole limpet hemocyanin, has been generated. METHODS: In the present study, the organ distribution of the engrafted human cells in the human/mouse and human/rat chimera was investigated by immunohistochemistry. RESULTS: Our results show that the T cells seem to be distributed throughout the reticular endothelial system, almost behaving like particles without any homing specificity. The B cells, however, can barely be found in internal organs, such as the liver or the pancreas, and are concentrated in the secondary lymphoid system (e.g., spleen, lymph node, and nonencapsulated lymphoid tissue). The B cells, together with the engrafted human T cells, form mixed lymphoid follicles. CONCLUSIONS: The different homing patterns exhibited by the T and B lymphocytes indicate that the homing receptors on human B cells might be cross-reactive with their mouse counterparts, in contrast to the human T cells, which seem to be unable to interact with the mouse homing receptors. The presence of human B and T lymphocytes in close proximity to each other in the lymphoid tissues is in accordance with the ability of human/BALB radiation chimera to mount significant primary human antibody responses.     

Duration of treatment with progesterone and regression of persistent ovarian follicles in cattle

The objective of the present study was to determine the duration of elevated concentrations of progesterone necessary to induce atresia of persistent ovarian follicles. Heifers were administered 25 mg of PGF2alpha on d 6 and 7 (d 0 = d of synchronized estrus) and a norgestomet implant from d 6 to 14. Ovaries were monitored by ultrasonography, and blood samples were collected on d 3, 5, 7, 9, 11, and 12 and daily from d 14 until ovulation. On d 12, heifers received either two progesterone-releasing intravaginal devices (PRID) for 6 h (6-h; n = 5), two PRID for 24 h (24-h; n = 5), or no treatment (CON; n = 5). Blood samples were collected at 15-min intervals from h -6 to 30 (PRID insertion = h 0) and analyzed for concentrations of LH. Characteristics of LH secretion were determined for consecutive 6-h periods (Period 0 to 5). Hourly blood samples, collected from h 0 to 29, were analyzed for concentrations of 17beta-estradiol (estradiol) and progesterone. The dominant ovarian follicles present on d 7 increased in size to 15.4+/-.3 mm on d 12 ("persistent follicle"). Following removal of the PRID and norgestomet implants, atresia of persistent follicles and ovulation of new follicles were induced in one of five and in four of five heifers in the 6-h and 24-h treatments, respectively. Persistent follicles ovulated after withdrawal of norgestomet in all other heifers. Concentrations of progesterone were increased from h 1 to 7 in the 6-h and h 1 to 26 in the 24-h treatment. Frequency of LH pulses was reduced (P < .05) during Periods 1 to 2 in the 6-h and Periods 1 to 5 in the 24-h treatment relative to the CON treatment. By h 10, concentrations of estradiol in the 6-h and 24-h treatments were lower (P < . 05) than in the CON treatment. This suppression continued through h 29 in the 24-h treatment (P < .05), whereas concentrations in the 6-h treatment were intermediate to those of the CON and 24-h treatments after h 14. Suppression of pulsatile LH release and estradiol secretion was evident with 6 and 24 h of treatment with progesterone, but only the 24-h treatment induced atresia of persistent follicles in a majority of the heifers.     

Messenger RNA synthesis, transport, and storage in silkmoth ovarian follicles

The effect on liver microsomal enzyme activity of three steroid contraceptive drug (SCD) combinations was compared in rats, mice and guinea-pigs. Lynestrenol plus mestranol, norethisterone plus mestranol and norethynodrel plus mestranol were given orally for 4 consecutive days (acute treatment) or 30 days (chronic treatment) at various doses eliciting an experimentally controlled antifertility activity which varied in its extent. In rats and mice all the combined treatments (with the exception of norethynodrel plus mestranol in mice) were active as inducers of liver microsomal enzymes. This induction seems to be mediated mainly by the progestogenic compounds. Oestrogens showed a very poor effect bordering on significance only in a few cases. No effect on liver microsomal protein or cytochrome P 450 concentration was obtained after treatment with doses capable of increasing the microsomal enzyme activity. The activity of the liver microsomal enzymes did not appear to be reduced immediately (2 h) after the last administration of the SCD given during 4 or 30 days. Contraceptive treatments at doses capable of eliciting complete antifertility activity were inactive on liver microsomal enzyme activity in guinea-pigs.     

Radiation-induced apoptosis in human ovarian carcinoma cells growing as a monolayer and as multicell spheroids

Response to external gamma irradiation was studied in a human ovarian carcinoma cell line (OVCAR 3) growing as a monolayer and as multicell spheroids. Necrosis and apoptosis were documented using Trypan-blue uptake and acridine-orange staining, respectively, and apoptosis was quantified using a terminal deoxynucleotidyl transferase assay. Exposure of OVCAR 3 cells growing as a monolayer to 137Cs gamma radiation at a dose of 10 Gy produced 30-40% apoptosis 72 hr after irradiation. Cell-cycle analysis of irradiated cells showed an accumulation of cells in G2/M phase 24 hr after irradiation and then a decline at 48 hr in conjunction with apoptosis onset. The loss of G0/G1 cells in irradiated cultures suggested a preferential entry into apoptosis. No increase in apoptotic cell number was observed in OVCAR 3 spheroids after irradiation, and the cells probably died as a result of necrosis. When spheroids were disrupted immediately after irradiation to obtain a cell suspension, minor apoptosis was observed in association with a marked increase in TB-positive cell number after 96 hr of incubation following irradiation. Thus, a relationship was found between radiation-induced apoptosis and the cell cycle. Results with spheroids suggested the possible involvement of cell-to-cell interactions in apoptosis regulation.     

Polysystic ovary syndrome--loss of the apoptotic mechanism in the ovarian follicles?

Polycystic ovary syndrome (PCOS) is the most prevalent female endocrinopathy and the largest single cause of anovulatory infertility. The PCOS is characterized by multiple small antral follicles arrested in their development but nonatretic and viable. The hyperexpression of some growth factors (e.g. EGF/TGF alpha) in PCOS, considered to be survival or antiapoptotic factors, led to the hypothesis of their involvement in the blocking of apoptosis and atresia leading to an accumulation of multiple small antral follicles. Diminished FSH stimulation and accumulation of androgens could explain the arrest of progress to the preovulatory stage. Further investigation of the pathogenesis of PCOS is needed on the modulation of tumour suppressor and apoptosis genes such as p53, BAX or the APO/FAS system and the over expression of survival genes such as BCL2.     

Detection of pro-opiomelanocortin mRNA in human and rat caudal medulla by RT-PCR

Pro-opiomelanocortin (POMC) mRNA has been localized in the NTS of the rat, but not in the human or other species. Here, we report that RT-PCR amplification of human caudal medulla RNA generated a distinct band on agarose gels corresponding in size and sequence to the predicted 742-bp POMC PCR product. The 742-bp signal was undetectable following amplification of cortex, amygdala or caudate nucleus RNA. An homologous, 678-bp band was amplified from rat caudal medulla and, unexpectedly, from other brain regions. Competitive RT-PCR demonstrated that POMC cDNA from rat cortex, striatum and cerebellum was 17%, 22% and 45% of caudal medulla levels. These data indicate that the POMC gene is expressed in human caudal medulla and suggest that small amounts of POMC mRNA are present in regions other than the hypothalamus and NTS of rat brain.     

Paracrine mechanisms of ovarian follicle apoptosis

Early diagnosis of dementia can be difficult. Quantitative EEG (qEEG) in combination with cognitive tasks shows promise for improving diagnostic accuracy. The study of task induced qEEG changes in normal ageing is a prerequisite for differentiating these changes from those which are specific to dementia. Sixteen young adults (mean age 28.8+/-5.6 years) and 16 healthy cognitively normal older subjects (mean age 73.4+/-7.9 years) participated in the study. EEG recordings were made while subjects were in a relaxed (or resting) state, and also while they performed arithmetic and language tasks. From the resting to the arithmetic conditions, there was decreased alpha activity and increased delta and beta-3 activity for both subject groups. Changes in alpha and delta activity were found in almost all sites and could be associated with arousal. Increase in beta-3 activity was focal, appearing only in the posterior region of the brain and it could be assumed that this area is highly involved in arithmetic processes. In the young adult group, theta activity increased from the resting to the arithmetic conditions, while in the older group theta activity changes were in the opposite direction. From the resting to the language condition, there was increased delta activity and decreased alpha and beta-1 activity for both subject groups. Changes in alpha and delta activity were again found in almost all sites. The decrease in beta-1 activity was found in only four sites, but these were not specific to a region of the brain known to be involved in language processing. This consistency in qEEG changes during cognitive tasks suggests that the method can be applied to the investigation of cognitive deficits associated with a number of neurological syndromes.     

Effect of human and murine interferon-alpha on steroid production by rat ovarian cells

The effect of interferon on the rat ovarian cell function was investigated. Cells from the ovary of juvenile rats were used as a model to investigate the effect of IFN-alpha on the secretion of estradiol and testosterone. In addition the effect of human IFN-alpha (hIFN-alpha) on the secretion of testosterone by the rat adult testis was studied. Present results show that leukocyte hIFN-alpha decreased the human chorionic gonadotropin (hCG) stimulated secretion of estradiol and testosterone by ovarian cells, and the production of testosterone by testis cells. Basal secretion of steroids was affected later and in less proportion than the hCG-dependent production. The IFN-alpha obtained from murine leukocytes, also inhibited the response of ovarian cells to the hCG stimulus.The nature of this effect in the secretion of the steroids is dose and time-dependent. The incubation of hIFN-alpha with an specific antibody completely blocked the effect of the cytokine on ovarian cells.     

Rate of decline of cortisol concentrations in ovarian follicles following ACTH treatment in the sow

Three milliliters of blood from the present commercially produced heartwater infective blood vaccine (Ball3 stock) was experimentally tested in sheep and cattle for infectivity and efficacy. Results obtained for this vaccine dose were statistically not different from results for the prescribed 5 ml vaccine dose.     

Intrafollicular insulin-like growth factor-binding protein levels in equine ovarian follicles during preovulatory maturation and regression

The profiles of insulin-like growth factor-binding proteins (IGFBPs) in follicular fluid have been characterized in a number of mammals (rats, pigs, sheep, cattle, humans) and are good indicators of follicular status. We studied the IGFBP profiles of equine serum and ovarian follicular fluid recovered at various stages of the follicular phase. The levels of IGFBPs were related to the morphology and the steroidogenic activity of the follicles. Follicular fluids were recovered by ultrasound-guided follicular aspiration. In the first experiment, the dominant follicles of 10 mares were partly punctured (aspiration of 0.5-2.2 ml of fluid) once at the early dominant stage (22-25 mm in diameter) and a second time at the preovulatory stage (PO), 34 h after induction of ovulation. Among these 10 PO follicles, 5 were classified as healthy whereas the other 5 were classified as hemorrhagic, as assessed by ultrasonic morphology and subsequent ovulation or not. In another group of mares (n = 5), the largest follicle was punctured once at the late dominant stage (33-35 mm in diameter) and then at the PO stage, 34 h after induction of ovulation. Serum was prepared at each puncture session. In the second experiment, follicular fluid was recovered from the dominant and contemporary cohort subordinate follicles (n = 5 mares). Samples were individually assayed for estradiol-17beta and progesterone content by RIA, and IGFBPs were studied by using Western ligand blotting and densitometry. Equine serum and follicular fluid displayed IGFBP at 42-44 kDa (likely corresponding to IGFBP-3), 28-32 kDa (likely corresponding to IGFBP-5), 24 kDa (likely corresponding to IGFBP-4), and 35 kDa, identified as IGFBP-2 by immunoblotting, plus one band at 120 kDa. IGFBP were clearly more abundant in serum than in fluid from healthy follicles. In the follicular fluid, 42-44-kDa IGFBP was the major binding protein, and its level was almost constant at the various physiological statuses studied. Follicular development of the dominant follicle in each mare was characterized by a decrease in intrafollicular IGFBP-2 and 28-32-kDa IGFBP levels before LH stimulation and by an increase in IGFBP-2 after LH stimulation. Follicular regression of large follicles, as well as subordinate ones, was characterized by a low level of intrafollicular estradiol-17beta and was associated with an increase in IGFBP-2, 24-kDa IGFBP, and 28-32-kDa IGFBP intrafollicular levels. Taking these results together, we have demonstrated clear correlations between the intrafollicular levels of estradiol-17beta and IGFBP-2 and 28-32-kDa IGFBP. Therefore, follicular growth and regression in the mare are associated with specific changes in IGFBP levels. These changes could be of crucial importance for follicular development in ovulation or atresia.     

Immunohistochemical localization of epidermal growth factor receptors, epidermal-growth-factor-like and transforming-growth-factor-alpha-like peptides in chicken ovarian follicles

The purpose of this study was to determine the presence of epidermal growth factor receptor and its potential ligands epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in the tissues of the maturing follicles in the ovary of laying ISA-Brown hens using peptide-specific immunohistochemical methods. Cryostat sections, 6-8 microns thick, were made from fresh-frozen tissues of F1-F4 (largest to fourth largest) and large white follicles and they were immunostained for epidermal growth factor receptor, epidermal growth factor or transforming growth factor alpha using specific polyclonal antibodies. The EGF receptor and both ligands were detected in the granulosa, theca interna and theca externa layers of the follicles. The EGF receptor was localized both in the plasma membrane and cytoplasm of all cell types. EGF was predominantly cytosolic, whereas TGF-alpha was found in the plasma membranes and perinuclear areas of all cell types. The concentration of the receptor and both ligands decreased with follicular maturation. This observation is consistent with our previous observation that the response to EGF and TGF-alpha decreases as follicles mature, and thus provides further evidence that the receptor or the ligands may have a regulatory role in avian ovarian function.