Effect of 80 kDa protein of porcine follicular fluid on gonadotropin stimulated progesterone production in rat granulosa cells in vitro.

We reported the presence of a 80 kDa polypeptide in porcine follicular fluid that inhibited the binding of 125I-radiolabelled hFSH as well as hCG to the rat ovarian gonadotropin receptors. In the present study, the biological activity of the receptor binding inhibitor is determined using an in vitro bioassay procedure. Granulosa cells isolated from PMSG primed immature rat ovaries respond to exogenously added gonadotropins in terms of progesterone production. Addition of fractions containing the gonadotropin receptor binding inhibitory activity inhibited progesterone production stimulated by the gonadotropins in a dose-dependent fashion. The receptor binding inhibitory activity was also capable of inhibiting progesterone production stimulated by PMSG, which has both FSH- and LH-like activities in rats. In contrast, progesterone production stimulated by dbcAMP was not inhibited by the receptor binding inhibitor. This result indicates that the site of action of the inhibitor is proximal to the formation of the cAMP. The above observations point out to a possible role for this factor in modulating gonadotropin activity at the ovarian level.     

Ovarian sympathectomy in the guinea pig

The influence of ovarian adrenergic nerves on follicular growth during the estrous cycle in the adult guinea pig was ascertained by comparing follicular development in control and chemically sympathectomized ovaries from the same animal. Selective ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian membranous sac (bursa) on day 2 of the cycle (day 1 = day of estrus). The contralateral surgically closed ovarian bursa was injected with solvent used for 6-hydroxydopamine. Animals were laparotomized on days 5, 10 and 14 of the cycle. Blood from the utero-ovarian vein was collected bilaterally for measurement of progesterone and androstenedione. The ovaries were processed for histologic examination, and the number of follicles in each ovary was analyzed morphometrically. Sympathectomy on day 2 caused a decrease in healthy preovulatory follicles (greater than 700 micron diameter) on day 10 of the cycle. There were no differences in ovarian weights or the total number of follicles per ovary at this time. On days 5 and 14 of the cycle, there were no differences in ovarian weights, total number of follicles per ovary or follicles in any size classification. Sympathectomy did not alter progesterone levels in the utero- ovarian vein as compared to contralateral control levels. From control ovaries, there was a significant increase in progesterone in the blood of the utero-ovarian vein on day 10 but venous levels of progesterone from sympathectomized ovaries were not significantly different at any day of the cycle. In the venous effluent from sympathectomized ovaries, androstenedione was elevated at day 5 compared to days 10 and 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laparoscopic evaluation of the reproductive organs and abdominal cavity content of the lowland gorilla

Laparoscopy was used to examine the abdominal cavity and evaluate reproductive competence in six adult female gorillas which had not demonstrated copulatory activity within 12 months of examination. Blood samples were obtained on the day of laparoscopy and analyzed for serum estradiol-17β and progesterone. The uterus and oviducts of each gorilla appeared morphologically normal and free of lesions. The ovaries of three animals contained anatomical evidence of activity as indicated by the presence of luteal scars and a prominent corpus hemorrhagicum, corpus luteum, or developing follicles. No visible luteal or follicular tissue was observed on the ovaries of three gorillas. In the eldest female (age, 22 years) the ovaries were flaccid in texture and demonstrated irregularities in ovarian surface integrity, similar to that observed in women during reproductive senescence. Serum concentrations of estradiol-17β and progesterone on the day of laparoscopy corresponded with observed ovarian activity. All six females had extensive adhesions in the abdominal cavity exclusive of the reproductive organs. Numerous gut, omentum, and liver to peritoneum adhesions were observed. The results indicated that laparoscopy was a safe and effective method for evaluating anatomical competence in the gorilla. Uterine and ovarian integrity appeared morphologically normal, but only three of the six gorillas showed evidence of ongoing ovarian activity.     

Antigonadotropic effects of the bovine ovarian gonadotropin-releasing hormone-binding inhibitor/histone H2A in rat luteal and granulosal cells

A gonadotropin-releasing hormone (GnRH)-binding inhibitor (GnRH-BI) was purified from bovine ovaries and identified as histone H2A. In the present studies, the biological effects of partially purified and purified ovarian GnRH-BI, as well as calf thymus histone H2A, were examined in rat ovarian cells. Since GnRH has direct antigonadotropic actions on these cells, the effects on luteinizing hormone-stimulated cAMP accumulation in luteal cells and follicle stimulating hormone-induced cAMP and progesterone production in granulosal cells were evaluated. Antigonadotropic activity in both luteal and granulosal cells coeluted directly with GnRH-BI activity during purification from bovine ovaries, and the antigonadotropic effects were dose dependent and reversible. In contrast to GnRH, GnRH-BI maximally inhibited gonadotropin responses and the effects of GnRH-BI were not blocked by a GnRH antagonist. The purified ovarian GnRH-BI and calf thymus histone H2A had identical antigonadotropic properties, and the half-maximal concentrations for inhibiting the gonadotropin responses of granulosal and luteal cells was 2 and 5 microM, respectively. In conclusion, the ovarian GnRH-binding inhibitor, identified as histone H2A, not only inhibits the high affinity binding of GnRH to rat ovarian membranes but also evokes GnRH-like antigonadotropic responses in rat ovarian cells that do not appear to be mediated by binding to GnRH receptors.     

Effect of cadmium chloride on the ovulation and structure of ovary in the inbred KP and CBA mice strains

A single dose of cadmium chloride (2.2 mumol/100 g of body weight) brought about in the sensitive KP strain alterations of the ovarian structure and disturbances in the ovarian cycle manifested by a prolongation of diestrus. Following cadmium administration and increase in the amount of atretic follicles, especially those showing stages 1I and 2II of degeneration, was observed in the ovaries of KP mice. The corpora lutea contained numerous degenerated cells and were infiltrated by abundant connective tissue cells. The used dose of cadmium chloride showed no influences upon progesterone level. The absence of changes in the ovarian morphology and in the duration of the oestrus cycle in CBA females suggest that this strain is resistant to cadmium chloride.     

Hepatic lipase deficiency attenuates mouse ovarian progesterone production leading to decreased ovulation and reduced litter size

The lipolytic enzyme hepatic lipase (HL) may facilitate mobilization of cholesterol substrate for ovarian steroidogenesis. We investigated whether HL was necessary for optimum reproduction in the female mouse by analyzing breeding performance and ovarian responses to gonadotropins in HL-/- mice. HL-/- female mice bred with HL-/- males had the same pregnancy success rate and pup survival rate as did wild-type (WT) mice but had significantly smaller litters, producing 1.7 fewer pups per litter. Mice were primed with eCG/hCG, and at 6 h post-hCG the HL-/- mice had smaller ovaries than did the WT mice. HL deficiency specifically affected ovarian weight; adrenal gland weights did not differ between WT and HL-/- mice. HL-/- mice weighed more than age-matched WT mice. Between the two mouse genotypes, uterine weights were the same, indicating that estrogen production was equivalent. However, the HL-/- ovaries produced significantly less progesterone than did the WT ovaries within 6 h of hCG stimulation. HL-/- ovaries had the same number of large antral follicles as did the WT ovaries but had fewer hemorrhagic sites, which represent ovulations, fewer corpora lutea, and more oocytes trapped in corpora lutea. We suggest that reduced progesterone synthesis following hCG stimulation attenuated the final maturation of preovulatory follicles, resulting in smaller ovaries. Furthermore, reduced progesterone production limited the expression of proteolytic enzymes needed for tissue remodeling, resulting in fewer ovulations with a corresponding increase in trapped or unovulated oocytes and providing a possible explanation for the smaller litter size observed in spontaneously ovulating HL-/- mice.     

Progesterone receptor in the rat ovary: further characterization and localization in the granulosa cell

We have recently described a progesterone receptor in the cytosol of ovaries of hypophysectomized, estrogen-primed, immature rats. This progesterone receptor was shown to be a thermolabile, saturable protein, which is specific for progestins (R5020 and progesterone), and elutes at the void volume of a Sephadex G-200 column. In the present study, we performed a more detailed analysis of the biochemical properties of this receptor and examined its cellular localization within the ovary. Treatment of the ovary cytosol with protamine sulfate and N-ethyl maleimide abolishes the specific binding of 3H-R5020, indicating that the receptor is an acidic protein containing cysteine residues necessary for binding. Gel exclusion chromatography shows the progesterone receptor to have a mean Stokes radius of 86 A and a molecular weight of approximately 300,000 daltons. Kinetic analysis indicates that the receptor--R5020 complex dissociates very rapidly, with a t1/2 of 10 minutes. The cytosol of isolated granulosa cells bind 3H-R5020 specifically, demonstrating that the ovarian progesterone receptor is present in the granulosa cell.     

Ovarian dysfunction in streptozotocin-induced diabetic rats

The effect of streptozotocin diabetes on some ovarian functions in adult rats was examined. Diabetic diestrus animals showed reduced ovary weight and lower circulating levels of progesterone. Scatchard plots of binding data derived from ovarian particulate fractions of normal and streptozotocin diabetic rats revealed the presence of one class of binding sites with high affinity for 125I-hCG. The apparent association constant of the hCG receptors of diabetic ovaries was comparable to that of normal gonads. However, a marked decrease (42%) in the number of hCG binding sites was found in diabetic animals. With isolated luteal cells similar results were obtained, and the administration of insulin to streptozotocin diabetic rats restored to normality the number of hCG binding sites. The maximal response of progesterone production by luteal cells from control ovaries was obtained with 10(-10) M hCG. A 100-fold higher concentration of hCG was required for the maximum stimulation of cAMP synthesis. The cAMP response of cells from diabetic rats was significantly higher than that of control cells. However, luteal cells from diabetic rats showed some loss of sensitivity in the synthesis of progesterone during incubation with hCG. Most of the alterations seen in diabetic female rats could be restored with insulin therapy, indicating that insulin plays an important role in the regulation and maintenance of normal reproductive functions. It is suggested that the diminution of the LH receptor population causes the disruption of normal luteal cell function. This fact could be responsible for some of the reproductive alterations in the diabetic female rat.     

Biochemical and hormonal characterization of follicles from follicular and luteal phase ovaries of goat and sheep.

Follicles from goat and sheep ovaries were characterized for their biochemical and hormonal parameters to investigate the effect of developmental stage of follicles on ovarian steroidogenesis. The follicles were isolated mechanically from follicular and luteal phase ovaries and divided in 6 morphologically different groups (small, medium and large follicular and small, medium and large luteal). Follicles were characterized for their contents of protein, DNA, estradiol-17 beta and progesterone and the activity of 3 beta-hydroxysteroid dehydrogenase. There was a progressive increase in the contents of all these biomolecules and activity of the enzyme as size of follicles increased in both the follicular and luteal phase ovaries. Follicles from follicular phase ovaries exhibited higher estradiol-17 beta content than those shown by luteal phase follicles. The reverse pattern was obtained for progesterone content. The results provide the basic data on biochemical and hormonal entities at different stages of follicular development in small ruminants which may be useful for in vitro studies on regulation of follicular development and steroidogenesis.     

Concentration of unconjugated adrenogenic hormones and their precursors in normal and polycystic ovaries.

Dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone concentration was determined by our sensitive gas-liquid chromatographic method in ovarian tissues obtained from surgery of patients without hirsutism and with Stein-Leventhal syndrome. The steroids, except testosterone, were detectable in all ovaries studied. Dehydroepiandrosterone and androstenedione, regarded as preandrogens, were present in an increased amount in almost all patients with polycycstic ovaries. Gas chromatographic evidence was obtained for the presence of testosterone in two of the cases. The delta4/3betaOH ratio reflecting 3beta-hydroxysteroid dehydrogenase activity was decreased only in same patients with the Stein-Leventhal syndrome suggesting that the impaired function of this enzyme is not an obligatory feature of polycystic ovaries. Concentration of pregnenolone and progesterone measured in a part of cases varied in a great range although the determination was caried out before luteal phase. Simultaneous determination of hormones in both ovarian tissues revealed an active and an inactive period of the gland in the given time, since a great difference of hormone concentration in bilateral ovarian tissues were observed. A comparison of hormone content in ovaries and the urinary excretion of metabolites showed poor correlation between the two parameters of hormone production.     

Pattern of arterial supply to the ovaries in Nili-Ravi buffaloes: Relationship with the distribution of ovarian structures

Reproductive tracts of 12 heifers and 18 adult buffaloes were collected from a slaughter house to study gross ovarian morphology and the distribution of follicles and corpora lutea on the surface of the ovary relative to the pattern of the arterial supply. Mean weight and volume of the ovaries were 3.8 ± 0.3 g and 4.08 ± 0.07 cm³, respectively, and were higher in adult buffaloes, but the difference was a function of body weight. Seventy percent of the ovaries were supplied by a single artery while the remaining 30% received two branches of the ovarian artery. Regardless of the position of entry of a single branch, ovarian follicles were located more frequently in the central (41.1%) than in medial (30.7%) or lateral portions of the ovary (28.2%). In ovaries supplied by two branches of the ovarian artery, follicles were distributed more uniformly on the ovarian surface. Number of follicles on the ovarian surface tended to increase when one of these branches was medial in position. Corpora lutea were observed more frequently in the part(s) of the ovary adjacent to the point of arterial entry regardless of the number of branches. Presence of a corpus luteum on the ovary had a negative effect on follicular development. The data suggested a possible relationship of the ovarian activity of the buffaloes with the pattern of arterial supply to the ovaries. Pattern of arterial supply might be contributing to the growth of the follicles, possibly by either saving them from atresia or by stimulating their growth.     

Ultrasonographic and laparoscopic evaluation of the reproductive tract of the captive female African lion (Panthera leo)

The use of transabdominal ultrasonography to assess the oestrous cycle has not been previously described in the African lion (Panthera leo). Twelve sexually mature lionesses and five female cubs had their reproductive organs assessed by transabdominal ultrasound. Ovarian findings were compared to laparoscopic findings while performing laparoscopic ovariectomy or salpingectomy. Vaginal cytology was performed and serum progesterone levels were determined. By combining all data the oestrous cycle stage of each lion was determined. One lion was far pregnant and was not operated on. In adults a uterine body could be seen ultrasonographically in 67% of lions while mural structures could be distinguished in 44% of lions. Five uterine horns could be seen in 3 lions. In 12 adults 10 ovaries were found of which eight had discernable follicles or luteal structures. During laparoscopy 12 active ovaries were seen with luteal structures seen in 11 ovaries and follicles in 2 ovaries. Using laparoscopy as the gold standard, ultrasonography had a sensitivity of 66% and specificity of 83% to detect ovarian reproductive activity. Two uterine cysts and a cluster of periovarian cysts were seen in three different lions. Three lions were pregnant, two were in oestrus, three in a luteal phase (dioestrus), and four were in anoestrus. Transabdominal ultrasound in combination with serum progesterone levels and vaginal cytology can be used to assess ovarian cyclical activity with reasonable accuracy in captive bred lions.     

Serotoninergic mechanisms in ovarian cycle (author's transl)]

Influence of 5-HT on the rat follicle rupture mechanism was studied by the administration of p-clorophenyl-alanine (p-CPA) (300 mg/kg) at different stages of the ovarian cycle. The ovarian cycle of treated rats was subject of investigation, as well as the histology of tubes and ovaries. Administration of p-CPA at 10 hours of metestrus phase induces an inhibition of ovulation. Predominant diestrus phases in the ovarian cycle and luteinitation of ovaries suggested an increase in progesterone secretion probably owed to a correspondingly greater prolactine secretion. Adequate 5-HT levels in central structures proved to necessary for normal ovulation.     

Presence of bacteria in porcine follicular fluid and their ability to generate an inhibitor of follicle-stimulating hormone binding to receptor

Follicular fluid obtained from porcine ovaries collected at slaughter and distributed by the National Institutes of Health was contaminated by bacteria which appeared to be of intestinal origin. This follicular fluid showed increased follicle-stimulating hormone binding inhibition (FSH-BI) activity following incubation under conditions which facilitated bacterial growth. No such increase in FSH-BI activity was observed following incubation of follicular fluid from which bacteria were removed by repeated filtration. Our data suggest that bacteria found in the follicular fluid were capable of generating a substance with FSH-BI activity. This substance has an apparent molecular weight greater than 6000, based on membrane diafiltration studies. The possible presence of bacteria in follicular fluid and their ability to generate a substance which interferes with FSH binding to receptor should be considered in studies on factors in follicular fluid that are considered to regulate ovarian function or development.     

Assay,properties, and regulation of rat ovarian δ-aminolevulinic acid synthetase activity

δ-Aminolevulinic acid synthetase (EC 2.3.1.37) has been detected in homogenates of rat ovaries. Optimal substrate and coenzyme concentrations, and parameters for assay of ovarian δ-aminolevulinic acid synthetase have been determined. Subcellular fractionation studies have shown that enzyme activity is predominantly localized in the mitochondrial fraction. Fasting, which is known to increase enzyme activity in the adrenal and to have no effect on activity in the testis, had no effect on enzyme activity in the ovary. Administration of the hepatic inducer allylisopropylacetamide or the hormone progesterone failed to alter activity of the ovarian enzyme. The activity of the enzyme was significantly increased during the diestrus-1 phase of the estrus cycle, during pregnancy, and by human chorionic gonadotropin at 24 and 48 h, suggesting that ovarian δ-aminolevulinic acid synthetase and the synthesis of heme may be under hormonal control.     

The intraovarian progesterone modulation of follicle development in the rabbit ovary

Intraovarian progesterone levels were manipulated by surgically adjusting the number of corpora lutea (CL) present in rabbit ovaries and this model was used to study the local effect of luteal progesterone on growth of follicles. The results show that when a single CL or several CL were present, follicle growth was inhibited. However, when all CL on one ovary were removed, increased numbers of follicles grew even when a single CL was present in the contralateral ovary. These findings show that progesterone inhibits follicle growth and that at least part of its action is local, i.e., exerted within the ovary. Additionally, ovarian blood vessels and periovarian lymph ducts were cannulated, and samples were collected and analyzed for steroid and protein content. The results show that when CL were present, ovarian vein progesterone levels were elevated 10-30-fold over levels in ovaries without CL; this high concentration points to the blood vascular system as the principal carrier of the steroid within the ovary. Analysis of lymph showed that protein content was consistently high and that the progesterone concentration was not significantly altered with the presence of CL; these two findings show that ovarian capillaries are extremely permeable to proteins, but the unexpectedly low concentrations of progesterone in lymph may signal an intraovarian countercurrent mechanism by which it is returned to the blood.     

Cyclic AMP and steroidogenesis in superovulated rat ovaries

During the gonadotropin stimulated differentiation of ovarian follicles into corpora lutea, the concentrations of total cyclic AMP and protein bound cyclic AMP increased only marginally. In contrast, there was a 10 fold increase in progesterone production. After acute stimulation of luteinized ovaries with choriogonadotropin total cyclic AMP levels increase more than 6 times while bound cyclic AMP and plasma progesterone rose by 80 and 60% respectively. Our results question the role of cyclic AMP in the basal production of progesterone by the ovary, and suggest a relationship between bound cyclic AMP and progesterone synthesis only exists after acute gonadotropin stimulation.     

Effects of ovarian hormones on ovarian capillary blood flow in anoestrous ewes

The indicator fractionation technique with [86Rb]rubidium chloride as the indicator was used to determine the relative blood flow (RBF) as a measure of capillary blood flow in the ovaries of conscious, hormonally treated, anoestrous ewes. Treatment of ewes with either progesterone only or oestradiol only had no effect on ovarian RBF, but treatment with oestradiol subsequent to progesterone caused a significant increase (P less than 0.001). Consequently, it appears that progesterone-induced sensitivity of the ovarian vasculature to the vasodilatory effects of oestradiol may be responsible for increased ovarian blood flow around oestrus in cyclic ewes.     

Effect of cold and hot ambient temperatures on plasma progesterone concentrations in ewes with intact and denervated ovaries containing experimentally maintained corpora lutea

Twenty ewes in which maintained corpora lutea had been established were subject to 1 of 3 treatments: denervation of the ovaries by freezing, denervation of the ovaries using the chemical 6-hydroxydopamine, or control. The animals were exposed sequentially to normal (24.5 degrees C), cold (10.7 degrees C), normal (23.8 degrees C), hot (39.4 degrees C) and normal (24.6 degrees C) temperatures, each for 1 week. On the final 3 days of exposure rectal temperatures and heart rates were measured, and on the final day the body weights, respiratory rates, and blood glucose concentrations were measured and a series of 5 blood samples was collected from each ewe for determination of the progesterone concentrations. The progesterone concentration was greatest during the hot period in 8 of the 12 animals, particularly in the ewes with denervated ovaries (6 of the 7 animals). This suggests that high ambient temperatures increase progesterone concentrations non-specifically, and that denervated ovaries are more sensitive to the circulating catecholamines that presumably mediate this effect. The progesterone concentrations were lower (P less than 0.001) in the groups with freezing or chemically denervated ovaries (2.86 and 2.73 ng/ml respectively) than in the control group (3.38 ng/ml), suggesting that the ovarian innervation plays a physiological role in regulating progesterone secretion.     

Multiple inhibitory effects of a luteinizing hormone-releasing hormone agonist on hCG-dependent steroidogenesis and on FSH-dependent responses in ovarian cells in vivo and in vitro

[125I] labelled [D-Leu6, des-Gly-NH10(2)] LH-RH ethylamide (LH-RHa), when injected into immature female rats, bound specifically not only to the pituitary but also to the ovaries. LH-RHa inhibited hCG-stimulated progesterone production and ovarian weight augmentation in hypophysectomized immature female rats in vivo. FSH-induced ovarian hCG receptors and ovarian weight gain in diethylstilbestrol (DES)-treated hypophysectomized immature female rats were also suppressed by LH-RHa. Progesterone production by rat luteal cells in vitro was inhibited by LH-RHa. LH-RHa did not change the affinity or population of LH/hCG receptor in porcine granulosa cells in short term incubation. However, LH-RHa inhibited induction of LH/hCG receptor stimulated by FSH and insulin in long term culture of porcine granulosa cells. LH-RHa delayed hCG-stimulated cyclic AMP accumulation in porcine granulosa cells. These findings suggest that LH-RHa inhibits hCG-stimulated cyclic AMP accumulation and subsequent progesterone production as well as FSH-stimulated LH/hCG receptor induction by acting directly on ovarian cells.