Inhibitors of nitric oxide synthase influence oocyte maturation in rats.

OBJECTIVE: To examine the effect of inhibiting nitric oxide synthase (NOS) on the number of ovulated oocytes and on oocyte meiotic maturation. METHODS: Female Sprague-Dawley rats (25 days old) were superovulated with a subcutaneous injection of 10 U pregnant mare's serum gonadotropin, followed 52 hours later by a subcutaneous injection of 10 U human chorionic gonadotropin (hCG). Three hours before and 3 hours after hCG injection, the rats were treated orally with the vehicle (0.5% methylcellulose) as the control or with either of two NOS inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME) and L-N6-(1-iminoethyl)-lysine (L-NIL). The rats were killed 20 hours after hCG injection, and oocytes present in the oviduct were flushed, counted, and classified for stages of meiosis. In addition, ovarian oocytes (12 hours post-hCG) and ovulated oocytes were treated with an immunofluorescent stain for the presence of endothelial NOS (eNOS). RESULTS: Strong positive staining for eNOS was observed in the cytoplasm of ovarian and ovulated oocytes. Control rats ovulated an average 43.0 +/- 4.1 oocytes each, which was lowered with either L-NAME or L-NIL (23.8 +/- 4.3 and 23.5 +/- 4.0 oocytes per rat, respectively; P < .002). We observed that significantly fewer ovulated oocytes obtained from rats treated with NOS inhibitors were at metaphase II (P < .006), the normal stage of meiosis for unfertilized oocytes, and a significantly greater percentage of oocytes displayed atypical morphology as compared with control oocytes (P < .0001). CONCLUSION: Ovarian nitric oxide synthesis is required for maximal ovulation, and a lack of nitric oxide during the periovulatory period results in severe defects in oocyte maturation.

An unusual active hexose transport system in human and mouse preimplantation embryos.

In a metabolic study of human and mouse preimplantation embryos (preembryos), we measured glucose uptake and phosphorylation with nonradioactive 2-deoxyglucose (DG) as tracer. Initial experiments indicated an active hexose transport capacity, a property thought to be restricted in mammals to intestinal villi and kidney tubules [Baly, D. L. & Horuk, R. (1988) Biochim. Biophys. Acta 947, 571-590]. Significant findings are as follows: (i) During a 60-min incubation with a low level of DG, mouse blastocyst DG rose to levels up to 30 times that of the medium. (The intestinal active system does not transport DG [Crane, R. K. (1960) Physiol. Rev. 40, 789-825].) (ii) Active preembryo transport was not blocked (as it would have been in the intestine) by phlorizin [Alvarado, F. & Crane, R. K. (1962) Biochem. Biophys. Acta 56, 170-172 and Sacktor, B. (1989) Kidney Int. 36, 342-350] or by replacement of Na+ with choline+ or K+ [Crane (1960) and Sacktor (1989)]. (iii) Transport of DG was blocked by cytochalasin B (which is not true for the intestinal transporter). We conclude that a distinct active hexose transporter and at least one facilitated transporter are present in preembryos, perhaps appearing in tandem on different membranes during formation of the increasingly complex preembryo structure.

Fasting hyperglycemia in type I diabetes mellitus.

The differential diagnosis of fasting hyperglycemia in type I diabetes includes the Somogyi effect, the dawn phenomenon, and insufficient insulin administration. To determine the causes of fasting hyperglycemia and their effect on subsequent daytime blood glucose control, the authors retrospectively reviewed blood glucose profiles of 126 patients with type I diabetes. The Somogyi effect accounted for 12.6% of all instances of fasting hyperglycemia, the dawn phenomenon, 24.1%, and poor control, 63.3%. Measurement of 3 AM and 5 AM blood glucose values is the key to making a correct diagnosis. Once a patient's fasting hyperglycemia is placed in one of these groups, appropriate treatment can be started.

Evidence for a role of cysteinyl leukotrienes in mouse and human sperm function.

The in vitro fertilizing capacity of mouse spermatozoa was inhibited by FPL-55712, a receptor antagonist of the cysteinyl-containing leukotrienes LTC4, LTD4, and LTE4. Inhibition occurred whether the compound was present in the medium during the capacitation period or during the fertilization period. Additionally, FPL-55712 inhibited the penetration of human spermatozoa into zona-free hamster oocytes. Inhibition of fertilization did not appear to be caused by an effect on sperm motility or on the oocyte. These observations suggest that mouse and human spermatozoa require cysteinyl leukotriene activity for both fertilization and oocyte penetration.

High progesterone/estradiol ratio in follicular fluid at oocyte aspiration for in vitro fertilization as a predictor of possible pregnancy.

Eighteen women undergoing in vitro fertilization (IVF) procedures were studied. All had optimal (900 to 1600 pg/ml) peak serum estradiol (E2) response to the same stimulation regimen with clomiphene citrate and menotropins; fertilization rate was above 64%; and two to four embryos in two to eight cell stages were replaced in each patient. All were considered to have optimal chances for conception. The authors compared progesterone (P), E2, and P/E2 ratio in serum and follicular fluid (FF) at the time of oocyte aspiration in eight patients who conceived (group I) and ten who did not (group II). Mean serum P and E2 levels and serum P/E2 ratio were not significantly different between the groups. In contrast, mean FF P concentrations (ng/ml) were significantly (P less than 0.05) higher in group I (9721 versus 5385), as was FF P/E2 ratio (19.0 versus 11.8; P less than 0.02). There was no significant difference in mean FF E2 concentrations between the groups. These data indicate that in IVF cycles with optimal serum E2 response to the stimulation protocol, FF P and P/E2 ratio at the time of oocyte aspiration may be predictive of subsequent implantation and pregnancy.

Cryopreservation of in vitro-fertilized human embryos: histologic and cytogenetic analysis of an ectopic conceptus.

In this study, 39 embryos from 17 patients were cryopreserved in a Planer R204 cell freezer using the protocol of Mohr et al. (J Vitro Fert Embryo Transfer 2:1-10, 1985). The procedure was modified by supplementing the cryoprotectant with 10% heat-inactivated and filtered (0.22 micron) maternal serum instead of fetal calf serum, and embryos were frozen in 500-microliter plastic straws instead of glass ampoules. After 12-25 weeks of storage in liquid nitrogen, 12 embryos from six patients were thawed at 8.0 degrees C min to room temperature, incubated in 75% maternal serum with Ham's F-10, and replaced in utero. One pregnancy occurred. The patient was a 34-year-old nulligravida with occluded fallopian tubes. A year prior, she conceived triplets from three embryos during an in vitro fertilization (IVF) cycle, but she delivered at 21 weeks and the infants did not survive. The second IVF attempt produced four embryos. Two were replaced during the IVF cycle, but they did not implant. Two were cryopreserved and replaced 25 weeks later. On day 28 after replacement, beta human chorionic gonadotropin (beta-hCG) was 4126 IU, but there was no gestational sac in utero on ultrasonographic examination. Laparoscopy disclosed a right tubal pregnancy which was removed with the fallopian tube. Histological examination demonstrated normal chorionic villi. The chromosomal pattern was 46 XX by direct analysis and cell culture.

Differential action of decidual luteotropin on luteal and follicular production of testosterone and estradiol.

Decidual tissue of the rat produces a hormone with physiological and biochemical characteristics similar to those of PRL. Because PRL affects both follicular and luteal production of testosterone and estradiol, it was of interest to determine whether decidual luteotropin affects basal and/or LH-stimulated ovarian secretion of steroids and whether it differentially affects follicular and luteal synthesis of testosterone and estradiol. The uteri of pseudopregnant adult rats were scratched on day 5 to induce decidual tissue formation. Pseudopregnant animals without decidua were used as controls. Rats were either hypophysectomized on day 8 or left intact. They were treated with 1.5 IU hCG/day or with vehicle between days 8-9. On day 9, blood was obtained from the ovarian vein, and both corpora lutea and large antral follicles were isolated and incubated in vitro. The presence of the decidua significantly suppressed both basal and hCG-stimulated ovarian secretion of estradiol, yet enhanced progesterone production. A similar inhibitory effect of decidual tissue on hCG stimulation of testosterone and estradiol was observed in the hypophysectomized rats. When the effect of decidua on follicles and corpora lutea was studied separately, it was found that follicles of rats with decidua produced significantly less testosterone and estradiol than follicles of rats without decidua. hCG administration to either intact or hypophysectomized rats markedly enhanced the follicular capacity to produce these two steroids. However, the degree of hCG stimulation of follicular steroidogenesis was significantly reduced by the presence of decidual tissue. In contrast, the decidua did not inhibit the in vitro steroidogenic capacity of corpora lutea. Luteal tissue of intact rats with or without decidua produced similar basal amounts of testosterone and estradiol and responded to a hCG challenge with comparable increases in the production of both steroids. After hypophysectomy, however, the responsiveness of corpora lutea to hCG stimulation differed in rats with or without decidual tissue. Whereas luteal cells of rats without decidual tissue gradually lost their responsiveness to hCG stimulation, luteal cells of rats with decidua remained highly responsive to hCG and produced high levels of testosterone and estradiol. In summary, the present investigation demonstrates that decidual luteotropin impairs ovarian secretion of estradiol and significantly inhibits the stimulatory effect of hCG on ovarian secretion of testosterone and estradiol.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and partial characterization of a prolactin-like hormone produced by rat decidual tissue.

Previous studies have strongly, but indirectly, suggested that rat decidual tissue produces a prolactin-like hormone, decidual luteotropin, which markedly affects luteal cell function. However, it was also found that extracts of decidual tissue do not cross-react with antisera to either rat or ovine prolactin (PRL). The purpose of this study was to determine whether the decidual tissue contains a substance that binds to PRL receptors in rat luteal membranes and, if so, to identify, quantitate, and characterize this molecule with the use of an ovarian radioreceptor assay. Decidual tissue was induced in day 5 pseudopregnant rats by scratching the antimesometrial wall of the uterus; it was collected on day 9 and homogenized and extracted. Decidual tissue extracts bound specifically to ovarian PRL receptors. Graded dilutions of the extracts yielded curves that were parallel to the ovine PRL standard, indicating that decidual luteotropin competes for the same receptor sites on rat luteal membranes. To determine the levels of decidual luteotropin throughout pseudopregnancy, decidual tissue was obtained on each day between days 6-12. The PRL-like activity was detectable in decidual tissue as early as day 6, reached a maximum on day 9, and declined thereafter. The elution profile obtained from gel filtration of a day 9 decidual tissue extract displayed a major component of decidual luteotropin eluting at a Ve/Vo ratio of approximately equal to 2.0. Column chromatography indicated that decidual luteotropin corresponds to a protein with a molecular weight of 23,500. The hormone was heat labile, digestible by trypsin, and appears to contain disulfide linkages. In summary, this study reports the identification, quantitation, and partial characterization of a PRL-like hormone produced by the decidual tissue of the rat.

Endocrine role of the decidual tissue: decidual luteotropin regulation of luteal adenylyl cyclase activity, luteinizing hormone receptors, and steroidogenesis.

We have recently reported that the decidual tissue of pseudopregnant and pregnant rats produces a luteotropic substance, decidual luteotropin, which can sustain luteal function in the absence of PRL. However, the luteotropic activity of the decidua depends on the presence of LH in the circulation. The objective of the present investigation was to determine whether decidual luteotropin sustains luteal function by maintaining the luteal cell content of LH receptors and LH-responsive adenylyl cyclase. Pseudopregnant adult rats had their uteri scratched on day 5 to induce decidual tissue formation. Both day 5 hysterectomized and intact pseudopregnant animals were used as controls. On day 7, rats were injected with 0.4 mg 2-bromo-ergocryptine (CB-154) to suppress PRL secretion. Within 48 h, CB-154 administration to either control group not possessing decidual tissue induced a dramatic decrease in progesterone production, LH receptor content, and LH-stimulated adenylyl cyclase in luteal cells. In contrast, suppression of PRL secretion in rats with decidualized uteri had no effect on progesterone secretion, luteal cell LH receptor content, or LH-stimulated adenylyl cyclase. Removal of the decidual tissue immediately after CB-154 treatment caused a significant decline in LH receptors, whereas administration of PRL to control rats without decidual tissue reversed the detrimental effect of CB-154 on both receptor content and LH-stimulated adenylyl cyclase. Examination of the time course (0, 24, and 48 h) of CB-154 action in rats lacking decidual tissue indicated that the decrease in serum progesterone preceded any detectable loss in luteal LH receptor content and LH-stimulated adenylyl cyclase activity. The results also suggest that the decline in LH-stimulated cyclase activity is largely due to the decrease in LH-binding sites and not to an alteration in the cyclase system. PRL withdrawal does not decrease but, rather, enhances epinephrine-stimulated cyclase activity. In summary, results of these investigations have revealed that decidual luteotropin can maintain the luteal content of LH receptors and LH-responsive cyclase and can sustain luteal cell production of progesterone in the absence of PRL; the synergism between decidual luteotropin (or PRL) and LH on luteal steroidogenesis involves more than the maintenance of LH receptors; and PRL withdrawal has opposite effects on LH- and catecholamine-responsive adenylyl cyclases, decreasing the former and stimulating the latter.

Role of decidual luteotropin and prolactin in the control of luteal cell receptors of estradiol.

In pseudopregnant rats, the luteotropic effect of estradiol depends on the presence of prolactin or decidual luteotropin. To determine if prolactin and decidual luteotropin regulate the functions of estradiol by maintaining luteal cell availability of estradiol receptors, decidual tissue was induced on Day 5 of pseudopregnancy. Control rats were hysterectomized on the same day. On Day 8, the rats were injected with 0.4 mg of ergocryptine (ECO) to inhibit prolactin secretion. Control rats were administered the vehicle only. On Day 9, in ECO-treated hysterectomized rats, serum progesterone declined from a control value of 50 +/- 7 to 7 +/- 2 ng/ml. In addition, luteal cell content of cytosolic and nuclear estradiol receptors was reduced by 95% and 48%, respectively. However, serum estradiol levels remained unchanged (vehicle treated: 23 +/- 7 pg/ml; ECO treated: 23 +/- 6 pg/ml). Administration of prolactin (250 micrograms/day, Days 7-9) to hysterectomized rats completely reversed the effect of ECO on serum progesterone levels and estradiol receptor content in both luteal compartments and had no effect on serum estradiol levels. In contrast, suppression of prolactin secretion in decidua-bearing rats had no effect on progesterone synthesis (vehicle treated: 54 +/- 6 ng/ml; ECO treated: 66 +/- 5 ng/ml) nor did it significantly decrease cytoplasmic and nuclear estradiol receptor content in luteal cells. These results suggest that 1) the availability of cytoplasmic receptors limits the biological responses of estradiol and 2) the synergistic luteotropic effect of prolactin or decidual luteotropin and estradiol involves the maintenance of luteal cell estradiol receptors by either prolactin or decidual luteotropin.