Mechanisms subserving the trophic actions of insulin on ovarian cells. In vitro studies using swine granulosa cells.

Direct actions of insulin on gonadal tissues have been difficult to demonstrate in vivo. We have developed an in vitro system in which swine ovarian cells remain highly responsive to trophic actions of insulin. Purified porcine insulin significantly augmented the biosynthesis and secretion of progesterone by cultured granulosa cells. These stimulatory actions of insulin were dose- and time-dependent and saturable. Under serum-restricted conditions, insulin also significantly amplified the capacity of estradiol and 8-bromo cyclic AMP to stimulate progesterone production. Inhibitors of protein and RNA synthesis (cycloheximide, actinomycin D, and alpha-amanatin) inhibited insulin action. The stimulation of progesterone production by insulin was attributable to increased biosynthesis of pregnenolone, rather than diminished catabolism of progesterone to its principal metabolite, 20alpha-hydroxypregn-4-en-3-one. Insulin also enhanced progesterone production in the presence of a soluble sterol substrate, 5-cholesten-3beta,25-diol, which readily gains access to the mitochondrial cholesterol side-chain cleavage system. Moreover, exposure of granulosa cells to insulin produced a three- to sevenfold increase in mitochondrial content of cytochrome P-450 measured by difference spectroscopy, with a corresponding increase in mitochondrial cholesterol side-chain cleavage activity. The capacity of insulin to facilitate progesterone biosynthesis by ovarian cells was mimicked by the insulinlike somatomedin, multiplication stimulating activity, but not by epidermal growth factor, fibroblast growth factor, or porcine relaxin. Insulin's augmentation of progesterone production reflected a selective action on progestin biosynthesis, since insulin significantly suppressed estrogen biosynthesis by granulosa cells.Thus, our investigations indicate that insulin acts on ovarian cells selectively to stimulate pregnenolone (but not estrogen) biosynthesis. The actions of insulin are exerted by processes that require protein and RNA synthesis, and by mechanisms that augment mitochondrial cytochrome P-450 content and facilitate the utilization of cholesterol in the side-chain cleavage reaction. The striking mimicry of insulin effect by multiplication stimulating activity suggests that insulin action may be mediated through somatomedin receptors. Moreover, in view of the high concentrations of somatomedin in ovarian follicles in vivo, our in vitro observations suggest that specific trophic actions of insulin or insulinlike growth factors are likely to significantly regulate the differentiated function of the Graafian follicle in vivo.

Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries.

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.

Metabolism of 25-hydroxycholesterol by rat luteal mitochondria and dispersed cells.

The metabolism of 25-hydroxycholesterol (25-OH-cholesterol) to progestins by mitochondria and dispersed cells prepared from ovaries of PMSG-hCG-primed rats was studied. Mitochondria converted [3H]25-OH-cholesterol into [3H]pregnenolone and [3H]progesterone. Unlabeled 25-OH-cholesterol also stimulated mitochondrial steroidogenesis in a dose-dependent, saturable fashion. A direct relationship between rates of steroid synthesis in the presence of 25-OH-cholesterol and mitochondrial cytochrome P-450 levels was found. Although steroid production and cytochrome P-450 content per milligram protein were higher in mitochrondia prepared from ovaries removed on day 8 post hCG than on either day 1 or day 14, steroid production per nanomole cytochrome P-450 was similar. Treatment of rats with hCG 1 h before killing significantly increased mitrochondrial steroid synthesis from endogenous substrate but had no effect on metabolism of 25-OH-cholesterol. Dispersed cells increased progestin production by 6-fold when incubated with 25-OH-cholesterol. The effects of 25-OH-cholesterol were dose dependent and saturable. While both LH and (Bu)2cAMP stimulated progestin synthesis from endogenous substrate, secretion of progestins with these agents reached levels only 60% of those observed in the presence of 25-OH-cholesterol. Neither LH nor (Bu)2cAMP altered the metabolism of the dydroxysterol by the cells nor did cycloheximide, which substantially inhibited progestin secretion in the absence of the hydroxysterol. However, animoglutethimide did block the stimulation of steroidogenesis by 25-OH-cholesterol. We conclude that 25-OH-cholesterol is an effective steroidogenic substrate for rat luteal tissue. With its use, information regarding the maximal capacity of luteal tissue to produce progestins in vitro can be obtained.

Regulation of ovarian cholesterol metabolism: control of 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase.

Activities of enzymes involved in cholesterol metabolism were measured in ovaries of PMS-hCG-primed immature rats. Microsomal 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in sterol synthesis, was low during the period of maximal steroid production and cholesteryl ester storage. Acyl CoA:cholesterol acyltransferase (ACAT), the microsomal enzyme catalyzing sterol esterification and cytosolic cholesteryl ester hydrolase and mitochondrial cholesterol side chain cleavage activities, paralleled storage of sterol esters and levels of plasma progesterone, being highest on day 7 post hCG. Further experiments demonstrated that HMG-CoA reductase and ACAT are regulated by different mechanisms than the sterol esterase and cholesterol side chain cleavage system. When blood sterol levels were lowered by 4-aminopyrazolo (3,4-d)pyrimidine, plasma progestin concentrations fell and ovaries failed to accumulate sterol esters. HMG-CoA reductase increased more than 10-fold as a result of this treatment, while ACAT was 15% of that measured in controls. In contrast, cholesteryl esterase and the cholesterol side chain cleavage system were not affected. Intravenous infusion of human lipoproteins reversed the effects of 4-aminopyrazolo (3,4-d)pyrimidine. Raising blood levels up to 6-fold with either cholesterol-supplemented diets or intravenous injections of rat lipoproteins had no effect upon ovarian sterol metabolism, suggesting that the process by which blood cholesterol is utilized is saturated at normal blood sterol levels. Intravenous injection of LH on day 7 post hCG increased HMG-CoA reductase and decreased ACAT activity in addition to depleting cholesteryl esters. Prior treatment of rats with aminoglutethimide prevented the effects of LH on both HMG-CoA reductase and ACAT, indicating that changes in these enzymes were a consequence of the steroidogenic stimulus imposed by LH. Treatment with aminoglutethimide alone on day 7 post hCG did not further depress the already low HMG-CoA reductase activity, but ACAT and cholesterol ester storage were stimulated while sterol esterase activity was not altered. Aminoglutethimide also produced elevated ACAT activity and sterol ester storage in animals treated on day 2 post hCG, a time when there is normally little sterol ester accumulation. We conclude that HMG-CoA reductase and ACAT are regulated, in a reciprocal fashion, by ovarian cholesterol balance. When sufficient cholesterol is available, HMG-CoA reductase is suppressed and ACAT increases to facilitate esterification of sterol in excess of cell needs. When exogenous sterol supplies cannot meet ovarian demands, HMG-CoA reductase rises, and there is a concomitant decline in ACAT. Thus, gonadotropic control over these enzymes may be exerted, in part, through modulation of the supply of cholesterol to the ovary and/or through regulation of the rate of sterol utilization for hormone synthesis.

Regulation of cytochrome P-450scc in immature porcine granulosa cells by FSH and estradiol.

FSH, estradiol, or a combination of FSH and estradiol enhanced secretion of progesterone by primary cultures of immature porcine granulosa cells; they also increased the mitochondrial content of cytochrome P-450 and mitochondrial cholesterol side-chain cleavage activity. FSH or estradiol alone increased mitochondrial cytochrome P-450 levels and cholesterol side-chain cleavage activity to a similar extent. The combination of FSH and estradiol further increased cytochrome P-450 and side-chain cleavage activity. Our results demonstrate that FSH, estrogen and the combination of the hormones stimulate progestin synthesis by granulosa cells, at least in part, by increasing cholesterol side-chain cleavage enzyme levels. These observations suggest a dual hormonal regulation of the enzyme catalyzing the rate-limiting step in the biosynthetic pathway of steroid hormones.

Diagnosis of impending late abortion.

PIP: During the years 1966 to 1972, 19 pregnant women, all but 1 in the midtrimester, reported to the outpatient clinic because of urinary symptoms. 13 were multigravidas, 6 were secundigravidas. Their chief complaint was urinary urgency and frequency of 2 or 3 days duration only. There was no dysuria or fever. 11 also complained of a pressure sensation in the lower abdomen. 3 reported a pink watery vaginal discharge. Their urinary symptoms were found to be related to the passive dilation of the cervix without uterine contractions. 16 were not too advanced to be treated by emergency cerclage using an Ethicon Mersileve band in a McDonald type procedure. Patients were confined to bed for 2-3 days and oral antibiotics given. Symptoms subsided and all but 1 of these women so treated had vaginal deliveries at term. When urinary tract symptoms appear in the second trimester of pregnancy without evidence of urinary infection, a high index of suspicion and careful attention to the meager symptoms is necessary to make early diagnosis of a dilating cervix. Timely surgical treatment can result in fetal salvage. The single most important traumatic factor in cervical incompetence is thought to be the forceful dilation of the cervix when articial abortion is performed. In 10 of these women curettage had artificially terminated the preceeding pregnancy. In 2 others artificial abortion had been followed by 1 or 2 premature deliveries. In only 4 of the 19 cases were there no previous traumas to the uterus. It is assumed therefore that cervial incompetence, late spontaneous abortion, and premature delivery will become more common in those countries which recently liberalized their abortion laws.^ieng

Communicating septate uterus with double cervix: a rare malformation.

The class of uterine malformations known as communicating uteri is characterized by two separate uterocervical cavities connected by an isthmic communication. Nine types have been described. We report the second proven case of a septate communicating uterus with double cervix. Investigation of secondary infertility in a woman with a history of second-trimester spontaneous abortion revealed two cervices on a speculum examination. A work-up for uterine anomaly began with endovaginal sonography, which demonstrated a normal fundal contour. A septum symmetrically dividing the endometrial cavity and two cervical canals were seen. The separated endometrial echoes converged at the isthmus, indicating a communicating uterus. Hysterosalpingography confirmed the diagnosis; injection of each cervix resulted in opacification of both hemicavities via the isthmic defect. Laparoscopy confirmed the normal fundal contour. It is believed that the etiology of this malformation involves failure of fusion of the distal müllerian ducts and arrested septal resorption above the isthmus.

Diagnostic imaging in puerperal febrile morbidity.

This retrospective study was conducted to assess the value of imaging in patients with refractory puerperal febrile morbidity. During a 36-month period, 31 patients were referred for ultrasound and/or computed tomography or magnetic resonance imaging because of postpartum fever unresponsive to broad-spectrum antibiotic therapy of at least 72 hours' duration. Hematomas were identified in 11 women. Abscesses were diagnosed in seven patients, ovarian venous thrombosis in two, vesicouterine fistula in one, small-bowel obstruction in one, and a subcutaneous seroma in one. Twenty-one women had endomyometritis, 13 of whom also had other extrauterine abnormalities (abscess in six, hematoma in four, and ovarian venous thrombosis, vesicouterine fistula, and small-bowel obstruction in one each). Retained placental tissue was found in two women with endomyometritis. Only two subjects had negative imaging studies. In most patients, imaging led to definitive diagnosis and specific therapeutic measures resulting in resolution of the febrile morbidity. Our experience suggests that these imaging techniques may be helpful in evaluating puerperal fever.

Communicating uteri: review and classification with introduction of two previously unreported types.

Four cases of communicating uteri, representing two previously unreported types, are presented. Forty-nine reported cases of communicating uteri are reviewed and reclassified into nine types. Clinical and embryologic aspects are discussed.

Role of androgenic hyperactivity in anovulation.

In the course of an investigation of 60 patients with clomiphene-resistant anovulation, 35 cases of androgenic hyperacitvity were detected. Fractionation of urinary 17-ketosteroids (17-KS) by a rapid method of chromatography proved to be both practical and reliable for the detection and classification of androgenic disorders of adrenal, ovarian, or mixed origin. In contrast to the total 17-KS values, the fractionated 17-KS values were elevated in all but one of these cases. Following dexamethasone suppression, individual 17-KS showed significant decreases in both adrenal and mixed adrenal-ovarian cases, in contrast to ovarian cases in which no significant change was detected. Human chorionic gonadotropin (HCG) stimulation combined with dexamethasone suppression did not cause any significant change in individual 17-KS values in the adrenal group, whereas both the mixed adrenal-ovarian and ovarian cases showed significant increases. Of 34 treated patients, 22 conceived, 21 had normal deliveries, and 1 aborted. Twelve became ovulatory. Eleven patients were treated with dexamethasone, nineteen with combined dexamethasone and clomiphene, two with dexamethasone and HCG, and two with HCG only.

Controlled ovarian hyperstimulation and transvaginal intratubal insemination as an alternative to gamete intrafallopian transfer.

OBJECTIVE: To evaluate the efficacy of controlled ovarian hyperstimulation (COH) followed by intratubal insemination in the treatment of infertility. DESIGN: Retrospective analysis of 179 intratubal insemination trials in 78 women over a 48-month period. SETTING: Reproductive endocrinology practice. PATIENTS: Seventy-eight women, 26 to 44 years old (34 +/- 4.3 years; mean +/- SD), classified into subgroups according to diagnosis and age (< 40 or > or = 40 years). INTERVENTIONS: Patients underwent COH and intratubal insemination. MAIN OUTCOME MEASURES: The rates of pregnancy, delivery, spontaneous abortion, ectopic and multiple gestation, and complications were studied and analyzed statistically. RESULTS: Overall, there were 36 pregnancies (20.1% of 179 trials), 29 deliveries (16.2%), 5 of 36 first trimester abortions (13.9%), 2 of 36 ectopic pregnancies (5.6%), and 4 twin gestations. There were no serious complications. In 43 women < 40 years without male factor the first trial resulted in 15 pregnancies (34.9%); in 27 women < 40 years with male factor the first trial resulted in 4 pregnancies (14.8%). Male factor reduced significantly the probability of conception. In 19 women < 40 years of age with either ovarian dysfunction or infertility of unknown cause, the first trial resulted in 9 pregnancies (47.4%), which was significantly better than the 10 pregnancies achieved in the first trial in the remaining 51 women < 40 years of age. Peak serum E2 levels and number of mature follicles had a significant effect on pregnancy rates. CONCLUSIONS: Intratubal insemination yielded pregnancy rates comparable to those published for GIFT at reduced cost and with fewer complications.

Hepatolenticular degeneration (Wilson's disease) and pregnancy. A review and report of a case.

This article presents a case report of Wilson's disease in pregnancy and a review of this entity during gestation. Biochemical and pathological data are reviewed and current treatment is discussed. Pertinent questions of interest to the obstetrician are indicated with reference to Wilson's disease.