Rat Ovarian Follicles Research Articles

Gel filtration and sedimentation behaviour of receptor-gonadotrophin complexes from rat ovary.

Membrane particles of luteinized rat ovaries and immature granulosa cells of rat ovarian follicles were labelled with [125I]human chorionic gonadotrophin (hCG), membranes of mature granulosa cells were also labelled with [125I]ovine follitropin (oFSH). Hormone-receptor complexes were solubilized with Triton X-100 and their physical properties were characterized by sedimentation and gel filtration. No difference in the macroscopic physical properties of the specific receptor-hCG-complex was observed between different developmental stages of granulosa cells. The Strokes radius of the specific receptor-[125I]hCG-complex was 58 A and the sedimentation coefficient 6.7 +/- 0.3 S. The physical properties of free LH/hCG-receptor of luteinized ovaries were also characterized. The Strokes radius was found to be 57-59 A and the sedimentation coefficient 4.8-5.6 S. Both [125I]hCG and [125I]oFSH formed non-specific complexes with ovarian as well as with non-gonadal membrane components. The sedimentation coefficient of the non-specific complex was 1.3 +/- 0.5 S and the Strokes radius 49 A. These results show that the macroscopic physical properties of the receptor-hCG-complex do not change during the maturation and subsequent luteinization of the granulosa cells. The methods used were not enough discriminating to reveal the existing structural differences between receptor-oFSH-complex and receptor-hCG-complex.

Hormonal modulation of gap junctions in rat ovarian follicles.

Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.

Quantitative and qualitative cytochemical analysis of changes in polysaccharides-proteins in rat ovulable and atretic ovarian follicles during the estrous cycle.

The glycosaminoglycan (periodic acid — Schiff, PAS) and hyaluronic acid (alcian blue) content of the membrana granulosa, zona pellucida and antrum of rat ovarian follicles was analyzed qualitatively and quantitatively during the estrous cycle in three types of follicles: ovulable, early atretic and late atretic. The qualitative analysis consisted of the conjunctive localization of PAS-reactive, fluorescent granules within the membrana granulosa. The quantitative analysis consisted of microdensitometric measurements of PAS and alcian blue staining within the zona pellucida and antrum of the ovulable and atretic follicles. For the localization of PAS granules within the granulosa cells, ovaries were removed on the day of proestrus, fixed in 6% paraformaldehyde, embedded in methacrylate and sectioned. Following the examination of the cells for fluorescence, the same section was stained with PAS and lead-hematoxylin. In ovulable follicles there was no fluorescence in the membrana granulosa while PAS granules occurred exclusively within the cells of the cumulus and corona radiata. In late atretic follicles, fluorescent-PAS reactive granules were located in the granulosa cells at the periphery of the follicle. During early atresia no fluorescence and very few PAS granules were observed in the granulosa cells. Since fluorescence is a marker for some lysosomes, these observations suggest that the PAS granules in the ovulable follicles may not be a type of lysosome. The amount of stain in the zona pellucida and antrum of the three follicular types was quantified using a scanning and integrating microdensitometer. On all days of the estrous cycle, PAS intensity was higher in the zona pellucida than in the antrum of the three follicular types. PAS staining in the respective antra was the same on all days of the estrous cycle. Intrafollicular PAS staining in the zonae pellucidae differed during the cycle. With respect to the zonae pellucidae, staining intensity in the three follicles was identical on estrus. On diestrus-1, staining intensity was the same in the ovulable and early atretic follicles and less in the late atretic follicle. By diestrus-2 and on proestrus, PAS intensity was highest in the zona pellucida of the ovulable follicle and less in the zona pellucida of both types of atretic follicle. In contrast to this pattern of staining, alcian blue staining intensity was identical in the zona pellucida of all follicles throughout the cycle. There was no difference in intra-antral alcian blue staining intensity on estrus and diestrus-2. On diestrus-1 and proestrus, staining intensity was greater in the antrum of the late atretic follicle than in the antra of the other follicular types. These studies indicate that glycosaminoglycan content is greater in the zona pellucida of the ovulable follicle of the rat on the last two days preceding ovulation than in the zona pellucida of either the early or late atretic follicles. In contrast, hyaluronic acid content remains constant in the zona pellucida of the three follicular types throughout the estrous cycle. These studies also give the first indication that, in the rat, the localization of PAS granules exclusively in the cumulus oophorus and corona radiata may be used to identify ovulable follicles.

Hormonal regulation of cyclic AMP binding to specific receptor proteins in rat ovarian follicles. Characterization by photoaffinity labeling.

Hormonal regulation of cyclic AMP binding to specific receptor proteins in rat ovarian follicles. Characterization by photoaffinity labeling.

Effect of exogenous hormones on the activity of Δ5,3 β-hydroxysteroid dehydrogenase in cultured granulosa cells from proestrus and preovulatory rat ovarian follicles

Activity of delta 5,3 beta-hydroxysteroid dehydrogenase (delta 5,3 beta-OH SD) in cultured granulosa cells isolated from rat ovarian follicles at various hours of proestrus was histochemically investigated. Control cultures as well as cultures with addition of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, and estradiol were carried on. In control cultures activity of the enzyme followed more or less the pattern observed in vivo. Exogenous LH added during preovulatory LH surge showed additive effect on enzyme activity. Other hormones stimulated enzyme activity on the morning of proestrus and at the time close to ovulation, when prolactin exerted most stimulatory effect.

Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex.

Cell-to-cell communication was characterized in cumulus-oocyte complexes from rat ovarian follicles before and after ovulation. Numerous, small gap junctional contacts were present between cumulus cells and oocytes before ovulation. The gap junction are formed on the oocyte surface by cumulus cell processes that transverse the zona pellucida and contact the oolemma. The entire cumulus mass was also connected by gap junctions via cumulus-cumulus interactions. In the hours preceding ovulation, the frequency of gap junctional contacts between cumulus cells and the oocyte was reduced, and the cumulus was disorganized. Electrophysiological measurements indicated that bidirectional ionic coupling was present between the cumulus and oocyte before ovulation. In addition, iontophoretically injected fluorescein dye was tranferred between the oocyte and cumulus cells. Examination of the extent of ionic coupling in cumulus-oocyte specimens before and after ovulation revealed that ionic coupling between the cumulus and oocyte progressively decreased as the time of ovulation approached. In postovulatory specimens, no coupling was detected. Although some proteolytic mechanism may be involved in the disintegration of the cumulus-oocyte complex, neither the cumulus cells nor the oocyte produced detectable levels of plasminogen activator, a protease which is synthesized by membrana granulosa cells. In summary, cell communication is a characterisitc feature of the cumulus-oocyte complex, and this communication is terminated near the time of ovulation. This temporal pattern of the termination of communication between the cumulus and the oocyte may indicate that communication provides a mechanism for regulating the maturation of the oocyte during follicular development before ovulation.

PRE-OVULATORY CHANGES IN RAT FOLLICULAR CYCLIC AMP AND SENSITIVITY TO GONADOTROPHINS

ABSTRACT Stimulation of ovarian follicles by high concentrations of gonadotrophins has been reported to induce a refractory state in the follicles to further stimulation by the same agent. The present study was undertaken to determine whether a physiological gonadotrophic stimulus would induce a similar desensitization. The levels of cyclic AMP (cAMP) were measured in rat pre-ovulatory follicles before and after the estimated period of gonadotrophin release. The PMSG injected pre-pubertal rat was used as a model of spontaneous ovulation as previously described. Follicles were explanted before and after the period of gonadotrophin release on the day before ovulation (day 30 in the present model) and cAMP was measured either directly or after 1–2 h of incubation. The follicles explanted in the evening of day 30 showed higher levels of cAMP than those isolated in the same morning irrespective of their being incubated or not. Follicular lactic acid production and progesterone synthesis as measured in the incubation medium showed the same pattern. The increase in all three parameters in "evening follicles" could be prevented by giving the animals pentobarbitone before the estimated gonadotrophin release. When the follicles were extirpated in the evening of day 30 and incubated in the presence of LH or FSH, significantly higher concentrations of the two hormones were needed to elicit a further increase in cAMP as compared to follicles isolated in the morning of the same day or from rats treated with pentobarbitone. It is concluded that the pre-ovulatory surge of gonadotrophins induces a desensitization of the cAMP system of rat ovarian follicles toward further action by LH or FSH. This refractory state, however, is not complete but relative under physiological conditions. In the rat, the duration of this phenomenon seems to coincide with the slight but significant elevation of follicular cAMP and to last from a few hours after the gonadotrophin surge up to ovulation. Possible mechanisms of this effect as well as its purpose are discussed.

Androgen Production by Theca and Granulosa Isolated from Proestrous Rat Follicles1

Theca and granulosa isolated from proestrous rat ovarian follicles were cultured for 72 h in the presence or absence of highly purified luteinizing hormone (LH, 0.1 microng/ml) and/or follicle-stimulating hormone (FSH, 0.1 microng/ml). Medium was collected and replaced at 3, 6, 12, 24, 48, and 72 h of culture and measured for testosterone by radioimmunoassay. Pieces of isolated theca secreted androgen; androgen production was greatest during the first 12 h of culture. Addition of highly purified LH to the culture medium produced a significant increase (P less than 0.001) in the thecal androgen secretion, while addition of highly purified FSH had no significant effect. Addition of LH + FSH to culture medium produced the same effect as addition of LH alone. The response of the theca to LH was dose-dependent with doses of 0.01 microng/ml or greater eliciting a maximum response. The addition of eoxgenous progesterone (5 x 10(-7)M) to culture medium had no effect on thecal androgen production. Thecal androgen secretion was the same in the presence or absence of fetal calf serum. Since the testosterone antibody used was not entirely specific for testosterone, testosterone and the cross-reacting androgen, 17beta-hydroxy-5alpha-androstan-3-one (DHT), were chromatographically isolated from samples and assayed separately. The androgen measured in culture medium was found to consist primarily of testosterone; DHT was present in much lower concentrations. Granulosa cells isolated from the same follicles as the theca and grown in monolayer culture produced negligible amounts of androgen. It is concluded that the theca is the site of follicular androgen production and that LH regulates androgen secretion by rat ovarian follicles. The results suggest that the theca provides the androgen precursor needed for follicular estradiol-17beta synthesis.

The distribution of collagenous connective tissue in rat ovarian follicles.

The layers of thecah tissue in the ovarian follicles of mammals normally contain collagen. During the past year, it has been reported that the follicle wall of the rat uterus is an exception. The present report demonstrates that the rat follicle does contain collagen in abundance. This thecal collagen is very conspicuous when the tissue has been stained with phosphotungstic acid. However, collagen is not obvious in ultrathin sections that have been stained with the lead solutions which are commonly used in electron microscopy. These results maintain the current hypothesis that the mechanism of ovulation requires the disruption of collagenous connective tissue in the follicle wall.

Absence of neutral proteinase activity in rat ovarian follicle walls at ovulation.

Neutral proteinase activity was undetectable in extracted rat ovarian follicle walls at ovulation with fluorescein-bovine-gamma-globulin as substrate. Ovulation was induced by 25 IU pregnant mare serum and 40 IU human chorionic gonadotropin. Follicle walls were extracted into Hanks salt solution by freezing and thawing homogenizing grinding and Triton detergent. Although the enzyme was not assayed at saturating substrate concentration positive controls were run with extracted human leucocytes yielding 5.3-6.6 ng fluorescein/mg protein/5 hours. Follicle walls exhibited 8.8-21.7 ng/mg protein/5 hours of acid proteinase activity (pH 4.0) but less than .04 units of activity (the limit of sensitivity of the assay) at pH 7.4. Therefore the ovarian follicle does not rupture because of proteinase activity.

Histological Examination of the Rat Ovarian Follicle Wall Prior to Ovulation

The histological changes occurring in rat ovarian follicle walls prior to ovulation were studied by light and electron microscopy. Follicles were collected at 0, 5, 10, and 11 h after the administration of HCG to stimulate ovulation, and also at what was judged to be the instant before rupture from ovaries that were exteriorized in a plastic chamber for direct observation. The earliest morphological change detected was the leakage of erythrocytes from blood vessels, and thus their presence, presumably along with plasma, in the intercellular spaces of the follicle walls. Somewhat later, occasional theca cells died and degenerated, and slight signs of cytolysis appeared at the surface of all cell types. The tunica albuginea often became loose, with a large increase in the extent of extracellular space between cells. A conspicuous feature of follicle walls near the time of ovulation was the rounding up and detachment from the follicle wall of apparently healthy granulosa and theca cells; at ovulation the granulosa layer was absent from the stigma. Clumps of fibrin were commonly found in the extracellular space of the follicle wall and in follicular fluid, as was debris from degenerating cells. The final change noted before ovulation was the degeneration and loss of peritoneal epithelial cells. No collagen could be detected in follicle walls at any time. The early leakage of erythrocytes from blood vessels and the common occurrence of fibrin in the intercellular space and follicular fluid suggested that an acute inflammatory reaction may be involved in the formation and rupture of the stigma. The anti-inflammatory drugs aspirin, salicylate, and indomethacin inhibited ovulation in immature rats when administered in anti-inflammatory dosages shortly before the expected time of ovulation. The inhibition was observed in animals superovulated by exogenous PMS and HCG, as well as in animals ovulating spontaneously after a small dose of PMS. It is concluded that a process which involves prostaglandin synthesis is involved in follicle rupture, and that this step may possibly be the early vascular phase of an inflammatory response.

Effect of indomethacin and 7-oxa-13-prostynoic acid on luteinization of transplanted rat ovarian follicles induced by luteinizing hormone and prostaglandin E 2

The ability of prostaglandin E 2 (PGE 2) to initiate luteinization was demonstrated using a system of in vitro incubation of ovarian follicles followed by transplantation. Follicles from diestrous rats were incubated with 0.05 to 50 μg/ml PGE 2, 10 μg/ml luteinizing hormone (LH), or alone in Krebs-Ringer bicarbonate buffer plus glucose for 2 hr. Then follicles were transplanted under the kidney capsules of hypophysectomized recipients, with follicles exposed to PGE 2 on one side and those exposed to LH or buffer only on the other side. As determined at autopsy 4 days later and confirmed by histological examination, follicles exposed to PGE 2 at concentrations of 0.5 μg/ml or greater, or to LH, transformed into corpora lutea, but control follicles regressed. Incubation of follicles with LH in the presence of indomethacin, an inhibitor of prostaglandin synthesis, significantly reduced the incidence of luteinization. Prostaglandin E 2 (10 μg/ml) was able to override the inhibition of luteinization by indomethacin (150 μg/ml). The prostaglandin analogue 7-oxa-13-prostynoic acid (100 μg/ml) failed to prevent luteinization in response to either 5 μg/ml LH or 1 μg/ml PGE 2. Results with PGE 2 and with indomethacin suggest a role for prostaglandins in the luteinizing action of LH. We have reported previously that in vitro exposure of diestrous rat follicles to luteinizing hormone (LH) will result in transformation of the follicles to corpora lutea following transplantation under the kidney capsules of hypophysectomized rats. Dibutyryl cyclic AMP (DBC) mimics this effect of LH, and transplants produce progesterone in measurable amounts after both LH and DBC exposure when prolactin is administered in vivo to recipients. Kuehl et al. have suggested that prostaglandins may act as obligatory intermediates in the effect of LH on the ovary, acting between LH and adenylate cyclase. Preliminary results indicated that prostaglandin E 2 (PGE 2) could induce luteinization in our system. The extent of prostaglandin involvement in luteinization was further investigated in this work, using two reported antagonists of prostaglandin action, indomethacin and 7-oxa-13-prostynoic acid. Indomethacin has been found to inhibit synthesis of prostaglandins E 2 and F 2α; 7-oxa-13-prostynoic acid, which acts as a competitive antagonist of prostaglandins, prevented the effect of LH and prostaglandins E 1 and E 2 on cyclic AMP production in mouse ovaries.

Inhibition of luteinization of transplanted rat ovarian follicles by polyphloretin phosphate.

Using an incubation-transplantation technique, we have tested the ability of polyphloretin phosphate (PPP) to inhibit luteinization induced by LH, prostaglandin E2 (PGE2), and dibutyryl cyclic AMP (dbcAMP). Follicles from diestrous rats were first exposed to PPP (0.5 mg/ml) or a control incubation medium for 30 min, then LH (10 µg/ml, 30 min), PGE2 (10 µg/ml, 1½ hr), or dbcAMP (7 mM, 2½ hr) was added. Finally, follicles exposed to LH were transferred to a medium containing LH antiserum (40 min) to remove residual LH. After incubation, follicles exposed to PPP plus LH, PGE2, or dbc-AMP were transplanted under one kidney capsule, and those exposed only to LH, PGE2, or dbcAMP were transplanted under the other capsule of hypophysectomized female recipients. Histological examination showed that luteinization was prevented by incubation of follicles with PPP and any of the three luteinizing agents. The postincubation with LH antiserum and PPP was necessary for PPP to prevent luteinization, indicating that LH bo...

The fine structure of granulosa cell nexuses in rat ovarian follicles.

AbstractAn intercellular junction known as the nexus has been identified in developing granulosa tissue of rat ovaries. Nexuses are common in Graafian follicles of mature cycling rats and in similar follicles of immature animals stimulated by exogenous gonadotrophins. The angle at which the plane of section intersects a nexus significantly governs its internal appearance. Within obliquely cut nexuses there is often a cross‐striated pattern of uniform lines in specimens prepared by conventional fixation and staining procedures. These lines could be significant because they may represent points of continuity between communicating cells.Two forms of nexuses are observed. The abutment form is found at cell surfaces, is continuous with plasma membranes, and is frequently associated with cell processes. The other form of nexus is ring‐shaped and appears free within the cytoplasm. These circular profiles probably represent transverse views through nexus‐girdled cell processes as well as nexus‐bound spheres which may have pinched off the processes. Since the profiles from the two sources are ordinarily indistinguishable, both are designated “annular nexuses” even though some may be cell inclusions. Lanthanum tracer can percolate into the central region of abutment nexuses but is absent from the large majority of annular nexuses, suggesting that many of them are isolated from the extracellular space. Reconstruction of serial sections reveals that some annuli are parts of cell inclusions. Sphere formation could be a means of removing nexuses from cell surfaces.

Luteinization and steroidogenic activity of rat ovarian follicles cultured in vitro.

Ovarian follicles were isolated from estrous rats and cultured in vitro for 1–6 days. After one day of culture there were no changes in the morphology of the follicles. In about 40% of the follicles cultured for 2 days, oocytes showed signs of degeneration. This was followed by the appearance of luteinized nests of cells in the theca and/or granulosa layers. On the 3rd day of culture all follicles had degenerated oocytes, and luteinization became more intense. Progestin was synthesized at a steady rate throughout the 3–day culture period. In contrast, estrogen was synthesized only from days 0–2 of incubation and showed a consistent decline on day 3 in all cultures. Very significant increases in progestin syntheses were obtained by the addition of 10 ng of LH to the culture medium. One hundred ng produced a significant but erratic increase in progestin syntheses. The addition of 10 ng of LH produced a minimal increase in estrogen synthesis while the 100 ng caused a very significant rise during days one and...

CALCIFICATION OF THE OVARIAN FOLLICLE IN RATS.

SummaryThe induction of calcification in rat ovarian follicles by administration of parathyroid extract is reported. Calcium deposition is limited to the membrane granulosa and thecae interna of follicles in the early stages of antrum formation. It is suggested that the “calcifiability” of these tissues may be due in part to an action of endogenous estrogens.