Evidence for a role of the ovarian surface epithelium in the ovulatory mechanism of the sheep: secretion of urokinase-type plasminogen activator

The extent of dissolution of tissues within the apical wall of the preovulatory ovine follicle (formative site of rupture) is greater than that of the counterpart basal hemisphere. It has been hypothesized that proteolytic enzymes released from contiguous ovarian surface epithelial cells contribute to apical follicular weakening and ovulation. Ovulation occurs from the dominant ovarian follicle of proestrous ewes at approximately 24 h after administration of luteinizing hormone-releasing hormone (LHRH). Follicular rupture was inhibited in sheep in which the ovarian surface epithelium was surgically removed at 8 (but not at 16) h following LHRH. Plasminogen activator bioactivity was greater within the follicular apex compared to basal wall at 12 h; this difference was negated by prior removal of epithelium at 8 h after LHRH. A low M_r plasminogen activator of the urokinase-type (uPA) was secreted by epithelial cells recovered from the surface of preovulatory follicles (Western blot analysis). Ovarian epithelium, not associated with a preovulatory follicle, produced very little uPA. Finally, ovulation was suppressed by intrafollicular injection (8 h post-LHRH) of uPA antibodies. It is suggested that secretion of uPA by ovarian surface epithelium and consequent plasmin up-regulation within neighboring tunica albuginea and follicular theca is a contributing factor in the mechanism of ovulation.     

Prostaglandin-independent anovulatory mechanism of indomethacin action: inhibition of tumor necrosis factor alpha-induced sheep ovarian cell apoptosis

Indomethacin, a nonsteroidal anti-inflammatory agent, is a potent inhibitor of ovulation in vertebrates. The presumptive obligate anovulatory mode of indomethacin action is via suppression of ovarian prostaglandin production. We report that a very high systemic dose of indomethacin (800 mg i.m.) is required to block ovulation in gonadotropin-treated anestrous ewes. A lower dose of indomethacin (200 mg), which negated the preovulatory rise in follicular prostaglandin (PGF(2alpha)) biosynthesis, did not prevent ovulation. Endothelial secretion of tumor necrosis factor (TNF)-alpha within the apical follicular wall (prospective site of rupture) was not altered by indomethacin; notwithstanding, the apoptosis (DNA-fragmentation)-inducing effect of TNF-alpha (a determinant of ovulatory stigma formation) was attenuated by 800 (but not 200) mg indomethacin. A suprapharmacological concentration of indomethacin also was necessary to protect ovarian surface epithelial cells from a (prostaglandin-independent) cytotoxic effect of TNF-alpha in vitro. It is concluded that indomethacin inhibits ovulation by anti-apoptotic mechanisms that can be dissociated from the paradigm of prostanoid down-regulation.     

Hormonal control of urokinase plasminogen activator secretion by sheep ovarian surface epithelial cells

Secretion of urokinase plasminogen activator (uPA) by ovarian surface epithelium (OSE) adjacent to the preovulatory ovine follicle has been implicated in apical tissue degradation and follicular rupture. In vitro experiments were designed to test the hypothesis that uPA release by OSE is under direct hormonal control. Epithelial cells were isolated from the ovarian surface of sheep using a polytetrafluorethylene scraper designed to dislodge adherent cells from culture flasks. Amidolytic cleavage of a uPA-specific chromogen (carbobenzoxy-L-gamma-glutamyl [alpha-ot-but]-glycyl-arginine-p-nitroanilide monoacetate) was used as a measure of enzymatic bioactivity in OSE-conditioned incubation media. Secretion of uPA by OSE suspensions from proestrous ewes was stimulated by exposure (2 h) to a preovulatory surge-like concentration of LH. OSE cells obtained during the luteal phase or anestrus were not responsive to LH. Baseline rates of uPA secretion and expression of estradiol receptors (in situ immunofluorescence detection) were not affected by reproductive status. Induction of uPA secretion by anestrous OSE was attained after priming (6 h) with estradiol-17beta; responsiveness was attributed to gonadotropin receptor (ligand binding) up-regulation. Monolayers of OSE established on polyethylene membranes secreted uPA predominately in a basal (i.e., toward the substratum) direction. We suggest that OSE in juxtaposition with the (hyperemic) wall of the preovulatory follicle is perfused by surge levels of LH, invoking uPA release into underlying ovarian tissues.     

Differential effects of indomethacin on the sheep ovary: Prostaglandin biosynthesis, intracellular calcium, apoptosis, and ovulation

Cells of the apical wall of the dominant follicle and contiguous ovarian surface epithelium become apoptotic with the approach of ovulation in the sheep. It was hypothesized that indomethacin, an established inhibitor of prostaglandin biosynthesis and ovulation, would protect apical ovarian cells from programmed death. The anovulatory potencies of two systemic doses of indomethacin (200 and 800 mg) were tested in gonadotropin-stimulated ewes. A complete blockade of ovulation occurred at the higher dose of indomethacin. Ovulation was not inhibited by 200 mg indomethacin. Both doses of drug suppressed follicular prostaglandin production below pregonadotropin levels. Immunofluorescence detection of digoxigenin end-labeled (fragmented) DNA was used as a marker of apoptosis among ovarian surface epithelial and granulosa cells recovered from the apical hemisphere of preovulatory ovine follicles. Cellular DNA fragmentation was averted in animals given 800 mg indomethacin, whereas apoptosis ensued after 200 mg. A sustained increase in cytosolic calcium is generally a prerequisite to apoptotic DNA fragmentation and cell death. Indeed, intracellular calcium, detected by fluorescence of fura-2, was elevated in ovarian cells of animals destined to ovulate (controls, 200 mg indomethacin) in comparison to (safeguarded) cells of anovulatory ewes (800 mg indomethacin). These observations provide circumstantial evidence that apical ovarian cell degeneration by calcium-mediated apoptosis is a determinant of follicular instability and rupture, but that these events are unrelated to the gonadotropin-induced rise in prostanoid production characteristic of preovulatory follicles.     

In-vitro Production of Meiosis Inducing Substance (MIS) by Isolated Amphibian (Rana pipiens) Follicle Cells*

Previous studies indicated that pituitary hormone induced oocyte maturation in preovulatory amphibian ovarian follicles is mediated by somatic elements of the follicle. In this study procedures were developed for isolating and culturing follicle cells and their ability to produce meiosis inducing substance (MIS) was assessed. Defolliculated oocytes surrounded by a single layer of follicle cells but not denuded oocytes matured in response to frog pituitary hormone (FPH) stimulation. Cultured follicle cells secreted MIS following stimulation with FPH. The amount of MIS activity produced was related to the number of follicle cells cultured and the dose of FPH utilized. Radioimmunoassay (RIA) analysis of medium from follicle cell cultures demonstrated that FPH stimulated steroid (progesterone) secretion from these cells. Addition of cAMP to follicle cell cultures enhanced FPH stimulated steroid production. The results indicate that follicle cells retain FPH responsiveness when uncoupled from the immature oocyte and exhibit both MIS and steroid secretory functions.     

The preovulatory rise of ovarian ornithine decarboxylase is required for progesterone secretion by the corpus luteum

Ovarian progesterone secretion during the diestrus stage of the estrous cycle is produced by luteal cells derived from granulosa and thecal cells after the differentiation process that follows ovulation. Our results show that blockade of the preovulatory rise of ovarian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by treatment with the specific inhibitor alpha-difluoromethylornithine (DFMO) leads to a significant decrease in the ovarian progesterone content and a dramatic fall in the plasma levels of this hormone during the following diestrus. The same inhibition was produced in spite of the fact that both luteinizing and follicle stimulating hormones were given concomitantly with DFMO. On the other hand, the acute rise in the plasma progesterone levels observed after administration of human chorionic gonadotropin to mice at different periods of the estrous cycle was not affected by DFMO administration. Our results indicate that although elevated levels of ODC are not required for acute ovarian steroidogenesis, the preovulatory peak of ovarian ODC activity observed in the evening of proestrus may be critical for the establishment of a constitutive steroidogenic pathway and progesterone secretion by the corpus luteum during the diestrus stage of the murine estrous cycle.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Sequence of apoptosis and inflammatory necrosis within the formative ovulatory site of sheep follicles

The aim of this study was to define the temporal and spatial patterns of apoptosis, necrosis and inflammation within preovulatory ovine follicles. A gonadotrophin surge was induced in pro-oestrous ewes by GnRH, and isolated follicles were hemisected into apical and basal segments at 0, 10, 18 and 22 h (the time of ovulatory stigma development) after GnRH. Ovarian surface epithelial and granulosa cells were isolated and assessed by fluorescence microscopy for membrane phosphatidylserine translocation-annexin V (early-stage apoptosis), oligonucleosomal DNA nick endlabelling (advanced apoptosis), and nuclear propidium iodide incorporation (necrotic membrane disruption). Thecal shells were analysed for interstitial blood cells. Preovulatory follicles were also hemisected and subjected to electrophoretic DNA degradation analysis. Annexin V binding and in situ DNA fragmentation among ovarian surface epithelial and granulosa cells along the follicular apex were high 18 and 22 h after GnRH. Propidium iodide staining of apical ovarian surface and granulosa cells was apparent at 22 h. There was a coincident increase within the apical theca as the time of ovulation approached in extravasated leucocytes (18 and 22 h) and erythrocytes (22 h). Apoptotic DNA laddering and necrotic DNA smears within the follicular apex were evident on agarose gels at 18 and 22 h, respectively. In contrast, ovarian surface epithelium not associated with the ovulation site and the basal follicular wall were largely unafflicted. It is suggested that both modalities of cellular death, apoptosis and necrosis (with acute inflammation and vascular injury), contribute progressively to follicular stigma formation and ovarian rupture.     

Tumor necrosis factor alpha inhibits gonadotropin hormonal action in nontransformed ovarian granulosa cells. A modulatory noncytotoxic property

It has been suggested that resident ovarian macrophages may play a role in the regulation of ovarian function through local paracrine secretion of regulatory molecule(s). It is the objective of the in vitro studies reported herein to evaluate the potential ovarian relevance of one such macrophage product, tumor necrosis factor alpha (TNF-alpha). To this end, use was made of a primary culture system of rat ovarian granulosa cells, the functional status of which was monitored by the acquisition of estrogen, progestin, and proteoglycan biosynthetic capacity. Whereas treatment with the gonadotropin follicle-stimulating hormone (FSH), a potent functional regulator, resulted in a substantial increase in the extent of aromatization (conversion of androgenic steroid precursors to estrogens), concomitant exposure to TNF-alpha (10 ng/ml) produced significant (p less than 0.05), yet reversible inhibition (71 +/- 7%) of this FSH effect. This specific activity of TNF-alpha was characterized by a projected minimal effective dose of less than 0.1 ng/ml, an apparent median inhibitory dose of 0.56 +/- 0.14 ng/ml, and a minimal time requirement of 48 h. Significantly, the direct effect of TNF-alpha could not be accounted for by a decrease in cellular viability or plating efficiency, nor by a decrease in the number of cells or their DNA content. Instead, TNF-alpha inhibited FSH hormonal action at the level of stimulatable adenylate cyclase activity, exerting no apparent effect either at the level of the FSH receptor or at site(s) of action distal to cAMP generation. The effect of TNF-alpha was not limited to the attenuation of estrogen biosynthesis, exerting qualitatively similar effects on FSH-supported progestin and proteoglycan biosynthetic capacity. As such, these findings are in keeping with the notion that subnanomolar concentrations of TNF-alpha, possibly of ovarian macrophage origin, may comprise the signal of a paracrine loop designed to attenuate gonadotropin action thereby playing a potential role in the development and/or demise of the ovarian follicle.     

Seasonal adiposity and delayed ovulation in a vespertilionid bat,Scotophilus heathi: role of tumor necrosis factor-alpha

The aim of this article was to evaluate the physiological significance of tumor necrosis factor-alpha (TNF-alpha) in seasonal accumulation of adipose tissue, hyperinsulinemia, and anovulation in Scotophilus heathi. The result showed seasonal variations in the circulating TNF-alpha level. A higher level of circulating TNF-alpha was observed during quiescence and recrudescence, whereas a lower level of TNF-alpha was observed during winter dormancy and the preovulatory period. An increased circulating TNF-alpha level coincided closely with accumulation of adipose tissue and hyperinsulinemia. Immunocytochemical localization of TNF-alpha in the ovary showed immunoreactivity mainly in the oocytes and theca-interstitial cells. The oocytes of small and medium-sized follicles showed strong TNF-alpha immunostaining, whereas weak immunoreactivity was observed in the large antral follicles. The atretic follicles showed mild TNF-alpha immunostaining. TNF-alpha immunoreactivity in the ovary was slightly higher during the quiescence and preovulatory periods compared with the periods of recrudescence and winter dormancy. TNF-alpha alone significantly increased androstenedione and estradiol production by the ovary in vitro but did not augment the luteinizing hormone (LH)-induced androstenedione production. However, TNF-alpha did augment LH-induced estradiol production. The results of this study suggest the involvement of TNF-alpha in the interaction among adipose tissue accumulation, insulin resistance, and ovarian activity in S. heathi.     

A scanning electron microscopic study of the luteo-follicular complex. II. Events leading to ovulation.

Morphological changes on the ovarian surface of different mammals both before and during ovulation have been examined by scanning electron microscopy. Preovulatory follicles were blisterlike structures that protruded markedly from the ovarian surface. Basal areas of preovulatory follicles were covered with polyhedral cells containing numerous microvilli, whereas on the lateral surfaces, superficial cells were elongated and possessed few microvilli. At the apex of the follicle, cells were very flattened and possessed few microvilli, which were present only in regions of intercellular contact. In some apical areas, cells appeared to be degenerating, whereas in other regions, groups of cells had "sloughed off." In addition, a fluidlike material was observed to exude from intercellular spaces of the superficial epithelium and to cover some apical cells. By transmission electron microscopy, the same fluidlike material was observed to (1) infiltrate the connective tissue of the tunica albuginea, (2) accumulate under the basal lamina, and (3) distend intercellular spaces of the superficial epithelium. Just prior to ovulation, large, irregular areas of the apex were ruptured and the oocyte, covered with a large amount of fluid, appeared to emerge from the follicle. At ovulation, the oocyte was not completely covered with follicle cells and the zona pellucida was clearly evident. The surface of the zona was quite irregular and contained numerous infoldings, channels and crypts. Follicle cells had polyhedral or star shapes and possessed large cytoplasmic evaginations that obliquely penetrated the zona. Both the zona pellucida and corona cells were covered with a fine layer of granular material. The SEM results and parallel TEM observations suggest that a local increase of fluids (edema) may be an important factor in the final decomposition of the distended and weakened apex of the preovulatory follicle. In addition, the participation of follicle cells, smooth muscle cells and the oviduct in the escape of the oocyte from the ruptured follicle is discussed.     

Influence of 5 alpha-reduced androgens on oestradiol secretion by granulosa cells of pregnant mare serum gonadotrophin treated immature rats

The effect of aromatizable androgens (testosterone and androstenedione) and naturally occurring 5 alpha-androstane, 3 alpha 17 beta-diol and 5 alpha-androstane, -3 beta, 17 beta-diol on oestradiol secretion by granulosa cells isolated from preovulatory follicles of PMSG-primed immature rats was investigated. The amount of oestradiol secreted by granulosa cells in the absence of exogenous aromatizable androgen in a 4h incubation was negligible. However, the addition of testosterone or androstenedione resulted in concentration dependent increases in oestradiol secretion. The 5 alpha-reduced androgens inhibit oestradiol and oFSH-stimulated oestradiol secretion by the granulosa cells in the presence of exogenous testosterone. The least potent of the androgens tested in causing this effect being the 5 alpha-androstane-3 alpha, 17 beta-diol. This result suggests that the naturally occurring 5 alpha-reduced androgens have a direct effect on androgen-aromatizing enzymes. The effect of these androgens may have an important connotation with respect to the control of the onset of puberty and regulation of ovarian oestradiol secretion within the microenvironment of an ovarian follicle.     

Interleukin 1beta mediates the effect of high D-glucose on the secretion of TNF-alpha by mouse uterine epithelial cells.

Previous observations have shown that tumour necrosis factor alpha (TNF-alpha) synthesis is increased in the uterus of diabetic rats and that the epithelial layer lining the uterine lumen is the major site of TNF-alpha over-production. In the present study, TNF-alpha secretion was found to be stimulated by high D-glucose levels in primary cultures of mouse uterine luminal cells but not in cultures of the mouse uterine epithelial WEG-1 cell line. Experiments were performed to investigate the possibility that non-epithelial cells may mediate the influence of high D-glucose on TNF-alpha production by uterine epithelial cells. Immunocytochemical analysis revealed the reproducible presence of a small proportion of macrophages in primary cultures. Macrophages of the RAW 264.7 cell line were found to secrete more interleukin (IL)-1beta (but not TNF-alpha) when cultured in high D-glucose. TNF-alpha production in WEG-1 cells was increased upon exposure to IL-1beta and both protein kinase-C and tyrosine kinase pathways appeared to be involved in TNF-alpha stimulation. Addition of IL-1 receptor antagonist to primary cultures partially abrogated the effect of high D-glucose. Since WEG-1 cells do not produce IL-1beta, the data lend support to the hypothesis that uterine epithelial cells synthesize high levels of TNF-alpha in response to hyperglycaemia via an increase in IL-1beta secretion by stromal macrophages.     

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus

The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.     

Secretion of platelet-activating factor by periovulatory ovine follicles

Secretion of platelet-activating factor (PAF) in vitro by ovine follicles and ovarian interstitium obtained at various times before, during and after the endogenous preovulatory surge of luteinizing hormone (LH) and ovulation was quantified by radioimmunoassay. Release of PAF by the preovulatory follicle increased within 2 h after initiation of the surge of LH. Capacity for secretion of PAF was greatest at the time of ovulation, then declined thereafter. Production of PAF by ovarian interstitium throughout the periovulatory period was relatively low and did not change with time. It appears that PAF could act as an intrafollicular mediator in the mechanisms of ovulation and(or) luteinization.     

Interleukin-1β and tumor necrosis factor-α have opposite effects on fibroblasts and epithelial cells during basement membrane formation

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are typical proinflammatory cytokines that influence various cellular functions, including metabolism of the extracellular matrix. We examined the roles of IL-1beta and TNF-alpha in basement membrane formation in an in vitro model of alveolar epithelial tissue composed of alveolar epithelial cells and pulmonary fibroblasts. Formation of the basement membrane by immortalized rat alveolar type II epithelial (SV40-T2) cells, which ordinarily do not form a continuous basement membrane, was dose-dependently upregulated in the presence of 2 ng/ml IL-1beta or 5 ng/ml TNF-alpha. IL-1beta or TNF-alpha alone induced increased secretion of type IV collagen, laminin-1, and nidogen-1/entactin, all of which contributed to this upregulation. In contrast, while SV40-T2 cells cultured with a fibroblasts-embedded type I collagen gel were able to form a continuous basement membrane, they failed to form a continuous basement membrane in the presence of IL-1beta or TNF-alpha. Fibroblasts treated with IL-1beta or TNF-alpha secreted matrix metalloproteinase (MMP)-9 and MMP-2, and these MMPs inhibited basement membrane formation and degraded the basement membrane architecture. Neither IL-1beta- nor TNF-alpha-treated SV40-T2 cells increased the secretion of MMP-9 and MMP-2. These results suggest that IL-1beta participates in basement membrane formation in two ways. One is the induction of MMP-2 and MMP-9 secretion by fibroblasts, which inhibits basement membrane formation, and the other is induction of basement membrane component secretion from alveolar epithelial cells to enhance basement membrane formation.     

Ovarian activity in Booroola X Romney ewes which have a major gene influencing their ovulation rate

A marked difference in both the function and composition of individual ovarian follicles was noted in Booroola X Romney ewes (6-7 years of age) which had previously been segregated on at least one ovulation rate record of 3-4 (F + ewes, N = 21) or less than 3 (++ ewes, N = 21). Follicles in F + ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In F+ ewes (N = 3), the presumptive preovulatory follicles were 4.4 +/- 0.5 (s.e.m.) mm in diameter and contained 2.1 +/- 0.3 X 10(6) (s.e.m.) granulosa cells, whereas in ++ ewes (N = 3), such follicles were 7.3 +/- 0.3 mm in diameter and contained 6.5 +/- 0.8 X 10(6) cells. During a prostaglandin (PG)-induced follicular phase, the secretion rate of oestradiol from ovaries containing 3 presumptive preovulatory follicles in F + ewes was similar to that from ovaries with only one such follicle in ++ ewes. We suggest that the putative 'gene effect' in F + ewes is manifested during early follicular development and that it may be mediated via an enhanced sensitivity of granulosa cells to pituitary hormones. As a consequence, the development of 3 preovulatory follicles in F + ewes may be necessary to provide a cell mass capable of producing the same quantity of oestradiol as that from one preovulatory follicle in ++ ewes.     

Influence of follicular maturation on 3-hydroxy-methylglutaryl coenzyme A reductase activity in hen granulosa cells

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase: EC 1.1.1.34) was measured in a microsomal preparation of the granulosa of rapidly growing ovarian follicles of laying hens in the late preovulatory period (2-3 h before expected ovulation). The specific activity of the enzyme was measured in the five largest (F1-F5) preovulatory follicles, F1 being the follicle destined to ovulate first. Enzyme activity increased concomitantly with follicle size. The apparent Km of the enzyme decreased 60-80% from the smallest to the largest preovulatory follicle. There was no significant change in the Vmax during follicle development. Although our results have demonstrated the presence of HMG/CoA reductase in chicken granulosa cells and the progressive increase of its activity with follicular maturation, the quantitative significance of de-novo synthesized cholesterol as steroid hormone precursor remains to be ascertained.