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This report addresses the following question: Can ovulation be blocked through a combined action of two drugs, which individually have no effect on ovulation. Ovulation blockade in hamsters was achieved through synergism of two drugs that individually had no effect on ovulation. Treatments were given at 18:00 h on pro-estrus. The keystone finding was that cycloheximide (CX), in a dose too small to affect ovulation, can be made to block ovulation by pretreating hamsters 15 min earlier with pentobarbital. Ovulation blockade was also achieved with phenobarbital + CX, ketamine + CX, and ether + CX. When given alone, none of the four anesthetics interfered with ovulation. The mechanism by which the anesthetic/CX synergism works is not known. The pituitary can be excluded as a target, because it is not necessary for ovulation after 15:30 on pro-estrus. It is speculated that CX reduces the synthesis of ovarian proteins needed for follicle rupture, and the anesthetics augment this process through action on either the ovary, the brain, the liver, or a combination thereof.

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This is a theoretical paper. Climacteric in women has two basic consequences: (i) Sterility, which is interpreted here as a positive feature, because it protects the older woman from the hardships of pregnancy. (ii) Hormonal deficiency, which is interpreted here as a negative feature, because it leads to deleterious changes in various parts of the body. Can these negative features be regarded as evolutionary defects? The answer is no, if the following points are considered. Through human innovations, women's natural lifespan of less than 30 years has been extended to almost 80 years. Had the lifespan not been manipulated, most women would not reach climacteric, which normally does not begin before the fourth decade of life. When interpreting climacteric as a phenomenon precipitated by man rather then by Nature, it follows that the negative features of climacteric should not be regarded as evolutionary defects.

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The activities of phosphofructokinase, pyruvate kinase, citrate synthase and creatine kinase were determined in blastocysts from rabbits at 144 h post coitum and in similar blastocysts cultured for 24 h with or without oestradiol-17beta (1 microgrm/ml). There was a significant increase in all the enzymes during the 24-h culture period but oestradiol had no effect.

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Concentrations of both nucleotides were significantly higher in Day-6 than in Day-5 blastocysts but the ratio of cAMP to cGMP changed from 0.5 to 1.5.

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4- to 8-cell mouse PIE's (preimplantation embryos) were cultured in vitro for 48 hr in media containing various concentrations of the anti-estrogen CI-628. Graded effects of the various concentrations were observed with the highest concentration (1.5 microng/ml) being 100% effective in blocking development to the blastocyst stage. The deleterious effects of the drug were prevented to some extent by including estradiol-17beta in the medium. Our interpretation is that the effect of CI-628 was due to its anti-estrogenic properties. If this interpretation is correct, then the results support our hypothesis that the development of mouse PIE's depends upon estrogen which originates in the PIE.

PIP: The hypothesis that mouse preimplantation embryos (PIEs) can synthesize estrogens and that PIE estrogen is a controlling factor during preimplantation embryogenesis was tested. Pregnant female mice of the Carworth strain (CFW1) were used. They were killed on the morning of either Day 3 or 4 of pregnancy. Mouse PIEs of 4-8 cells were exposed to an antiestrogen, CI-628, for 48 hours in media containing several concentrations of the antiestrogen. The concentration of 1.5 mcg/ml, the highest used, was 100% effective in blocking development of the blastocyst stage. The deleterious effects of the CI-628 were partly prevented by including estradiol-17beta in the medium. PIEs which did not develop appeared degenerating. CI-628, previously designated CN-55,945-27, is a nonsteroidal antiestrogen which acts by displacing estrogen from estrogen receptor sites. Its effect was thought to be due to its antiestrogenic properties. If so, results support the hypothesis that the development of mouse PIEs depends on estrogen which originates in the PIEs.

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Fertilized eggs were incubated for 2 hours in a medium containing estradiol-17beta and then transferred into the uteri of day 5 pseudopregnant rats. These eggs, but not estrogen-free control eggs, induced a local increase in capillary permeability. We suggest that the blastocyst factor which induces the local increase in capillary permeability during early pregnancy is estrogen synthesized by the blastocyst.

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It has been shown by others that treatment with only progesterone is sufficient to induce implantation and maintain pregnancy in ovariectomized-adrenalectomized rabbits. Based on these results, it was deduced that induction of implantation in the rabbit does not require estrogen. In making this deduction, ""blastocyst estrogen'' was not taken into account. In the present study, blastocyst development and implantation were prevented by instilling the anti-estrogen CI-628 into the uterine lumina of pregnant, ovariectomized, progesterone-treated rabbits. Our interpretation of these results is that the CI-628 PREVENTED THE ACTION OF ""BLASTOCYST ESTROGEN''. However, on the basis of the present results, we cannot determine whether the specific target of ""blastocyst estrogen'' is the blastocyst, the uterus, or both.

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Rabbit blastocysts recovered at 144 hours post coitum contained the prostaglandins F and E-A. We suggest that one or more of these prostaglandins act as mediators in blastocyst steroidogenesis. (In another study we have demonstrated steroidogenesis in rabbit blastocysts).

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Rabbit preimplantation embryos were flushed from the reproductive tract at 24 hr (1- to 2-cell stage), 48 hr (morula), 72 hr (morula), 96 hr (blastocyst), 120 hr (blastocyst), and 144 hr (blastocyst) post coitum. At 168 hr (early postimplantation period), gestation sacs were excised, frozen, and sectioned in a cryostat. Delta5-3beta-Hydroxysteroid dehydrogenase [3(or 17)beta-hydroxysteroid:NAD(P) oxidoreductase, EC 1.1.1.51] activity was determined histochemically in whole preimplantation embryos and in sectioned postimplantation embryos. 3beta-Hydroxysteroid dehydrogenase activity began at 48 hr and was sustained through the late blastocyst stage (144 hr), with the exception of a brief drop, possibly cessation, of activity at 72 hr. There was no activity at 168 hr. Since 3 beta-hydroxysteroid dehydrogenase is a key enzyme in the metabolism of steroid hormones, its presence is strong evidence for steroidogenesis. Only 144-hr preimplantation embryos were used to determine 17 beta-hydroxysteroid dehydrogenase (estradiol-17beta:NAD 17-oxidoreductase, EC 1.1.1.62) activity, which was present, suggesting synthesis of estrogen. By means of radioimmunoassay, 144-hr preimplantation embryos were found to contain estradiol-17beta. Other authors have shown that rabbit blastocysts contain progesterone and other steroids, and these embryos can synthesize steroids from non-steroid and steroid precursors. Therefore, our results plus those of others prove that rabbit preimplantation embryos synthesize steroid hormones. Our present and previous results (with rats, hamsters, and mice) suggest that the steroid hormones synthesized by the embryo are critical for preimplantation embryogenesis and for implantation of the lbastocyst.

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