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Day-6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately 50% in the presence of 1.5 x 10(-4) M atropine (an accepted antagonist for ligands which bind to muscarinic cholinergic receptors). The accumulation of QNB was sensitive to temperature and was apparently saturable. In the presence of 2 nM QNB, Day-6 blastocysts accumulated 30.3 +/- 2.0 fmoles per blastocyst. When the cellular elements alone were examined, lesser amounts of specific binding were detected. Owing to the complexity of this multicompartmental system, Scatchard analysis did not provide meaningful results. This accumulation appears higher than that reported for other tissues such as rabbit heart homogenates or rabbit uterine endometrial cells. This muscarinic cholinergic accumulation may have some roll in blastocyst-maternal recognition.

Stromal cells and epithelial glands were separated after enzymic digestion of specimens obtained from 27 women at hysterectomy or endometrial biopsy during the luteal phase, and then cultured to confluence in vitro. PGE release into the culture medium (mean +/- s.e.m.: ng/mg protein/24 h) from gland cell cultures was not changed by oestradiol (17.6 +/- 1.3 for control and 25.5 +/- 2.8 for oestradiol, respectively). However, in the presence of oestradiol, PAF (5 ng/ml) significantly elevated PGE release to 44.2 +/- 5.8. No stimulation was observed in the presence of progesterone. Stromal cell medium had no effect on PGE release in gland cell cultures. PGE release was always much lower in stromal cell cultures than in glands (control: 4.7 +/- 0.6). PAF stimulated PGE release in the presence of oestradiol in these cells also; gland cell medium was without effect. In co-cultures of glandular and stromal cells, PGE release was more similar to that seen in gland cell cultures, with PAF being stimulatory under the influence of oestradiol. PGF release into the medium from the same gland cell cultures was significantly elevated by hormonal treatment, being greatest (62.0 +/- 11.3) with oestradiol alone, and was strongly inhibited in all wells by addition of PAF and stromal cell medium. In stromal cell cultures without hormonal addition, PGF levels (15.0 +/- 2.4) were similar to those seen in glands (18.1 +/- 3.1), and no stimulation was achieved by oestradiol (29.6 +/- 5.9). PAF was inhibitory on PGF release, while gland cell medium was without effect. Co-cultures gave PGF values generally similar to those of stromal cells; oestradiol was again stimulatory (55.0 +/- 9.3). PAF was significantly inhibitory in the presence of oestradiol. PAF (mean +/- s.e.m.: pmol/mg protein/24 h using a platelet serotonin release assay) in stromal cells was significantly increased from control [M199 alone] (0.31 +/- 0.12) by progesterone (1.00 +/- 0.17). Addition of PGE-2 (7.5 ng/ml) to progesterone-treated wells further increased PAF concentration (5.34 +/- 0.09), but was without effect in wells receiving oestradiol alone. Wells exposed to both hormones exhibited an intermediate response. Similar results were obtained with addition of gland cell culture medium, presumably due to its endogenous PGE content. In co-cultures, PAF concentrations were significantly elevated by progesterone alone (4.78 +/- 0.78) or when combined with oestradiol (2.38 +/- 0.51), but not by oestradiol alone. Treatment with PGE-2 caused no additional stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

When one considers the endometrium as a target for contraception, it is necessary to survey briefly what has been achieved heretofore, and how new knowledge has expanded the potential for development of antifertility agents which could specifically disrupt the implantation process. Until recently our understanding of the events occurring in the endometrium and the signaling between the developing embryo and the endometrium was scanty. Consequently the discovery of leads that might permit identification of agents to disrupt the implantation process was limited to hormonal and antihormonal compounds. Although the various hormonal and antihormonal approaches have merit, and one can envisage combination treatments being even more effective, they all have the potential problem of lack of specificity. Now that more specific factors controlling the implantation process are being uncovered, a path to the development of more specific implantation inhibitors appears possible.

The effects of adrenergic drugs and prostaglandins (PCs) on rabbit oviductal isthmus motility were compared. Balloon-tipped fluid-filled catheters placed in the lumen of the isthmus were used to record muscular activity. The intravenous (iv) injection of E-series PGs and the �-adrenoceptor stimulator isoproterenol both caused a nearly complete suppression of spontaneous oviductal activity. Pretreatment with the �-adrenoceptor antagonist propranolol effectively blocked the response of the oviduct to isoproterenol but had no effect on the response to E- or F-series PGs. A maintained spasmodic contraction was induced by the iv injection of both norepinephrine, an a-adxenoceptor stimulator, and F-series PGs. Pretreatment with the n-adrenoceptor antagonist phenoxybenzamine blocked the response of the oviduct to norepinephrine without having any effect on the response to PGEs or PGFs. A mutual antagonism between the a-adrenoceptor stimulator and PGEs, and between the p-adrenoceptor stimulator and PGFs was also demonstrated. The relationship and similarity between adrenergic and PC responses indicate that both agents may be synergistically involved in the control of oviduct motility. The demonstration that adrenergic antagonists block the response of the oviduct to adrenergic stimulation, but have no effect on the response to PGs, suggests that adrenergic

Uteri from mature baboons at various stages of the menstrual cycle were collected at autopsy. Transverse slices of the uteri were incubated with [3H]prostaglandins in Tyrode's buffer. Bound and free ligands were separated by filtration. Prostaglandin (PG) accumulation by the tissue slices was evaluated as a function of incubation time, PG type and concentration, temperature, wet weight of tissue and stage of the menstrual cycle. There was no significant difference in PG accumulation in response to PG type (PGE2 or PGF2α) or stage of the menstrual cycle. These results from baboon uteri were compared with those using baboon oviducts and also rabbit uteri and oviducts. Unlike the rabbit tissues, the baboon oviducts and uterine tissues did not exhibit specific net accumulation of prostaglandins. J. Endocr. (1985) 106, 49–53

Membrane preparations (100 000 g pellet) of rabbit, baboon and tree shrew (Tupaia belangeri) uteri were studied for binding of [3H]prostaglandin E2 ([3H]PGE2). Unbound [3H]PGE2 was separated by filtration through Whatman GF/F filters. Nonspecific binding was determined by the amount of radioactivity associated with the filters in the presence of a 100-fold excess of radioinert PGE2. PGE2 bound to membranes could be displaced by some other prostaglandin (PG) molecules: PGE1, 16,16-dimethyl-PGE2, PGA1 and PGF2α, but not by 6-keto-PGF1α, PGD2 or arachidonic acid. No PGE2 binding was detected using either membrane ghosts from red blood cells or liposomes. Apparent equilibrium of the binding was reached by 60 min. There was no difference in dissociation constant (Kd) values between rabbits of different reproductive stages (mean range±s.e.m. was from 4·6±0·3 to 5·5 ± 1·0 nm), but pregnant baboons showed a significantly lower value (3·3±0·4 nm) than did cyclic animals (12·0 ± 2·0 nm). Binding capacity (Bmax) values, in contrast, were different only between oestrous rabbits and other reproductive stages. The small amounts of [ill] tissue only permitted estimates of the Kd and Bmax values to be made; these were 3·8 nm and 499 fmol/mg protein for oestrous animals and 5·4 nm and 674 fmol/mg protein for animals on day 7 of pregnancy, assuming only one class of sites. The present results demonstrate the presence of specific binding sites for PGE2 in uteri from several species.

Summary. Rats were treated with LH at 08 :00 h on the first day of dioestrus or on Days 1 and 2 of dioestrus. Peroxidase activity increased within 3 h in females injected with LH on Day 1 and was associated with a depletion of ascorbate that lasted until the afternoon of Day 1. Values of both substances then returned to basal values by the morning of Day 2. A second LH injection on Day 2 produced effects similar to those seen after LH on Day 1. Females treated with LH did not display greater \g=D\5-3\g=b\-HSD activity than did controls on Day 1 after one LH injection, but did on Day 2 after two LH injections. Nonetheless, the changes were only modest by comparison with those of peroxidase. The peroxidase\p=n-\ascorbatesystem appears to be involved in the mechanism responsible for the increased secretion of ovarian progesterone resulting from LH stimulation.

An increase in preload from 0.75 to 10 g caused an increase in the sensitivity of rabbit aortic strips to phenylephrine, serotonin, and KCl. Several eicosanoid synthesis inhibitors were utilized to determine whether production of endogenous eicosanoids contributed to the change in muscle sensitivity. Quinacrine (10 microM), indomethacin (10 and 50 micrograms/ml), meclofenamate (50 and 100 micrograms/ml), 5,8,11,14-eicosatetraynoic acid (ETA, 50 and 100 micrograms/ml), and nordihydroguaiaretic acid (NDGA, 10 microM) all shifted the concentration-response curves of various agonists to the right, indicating a decrease in the sensitivity of the muscle to these agents. Thromboxane synthesis inhibition by 1 microM (E)-3-[4-(1-imidazolylmethyl]phenyl-2-propenoate (OKY 046) exerted no effect on sensitivity. Indomethacin at both concentrations caused a parallel shift in the high and low preloaded strips but was unable to alter the influence of preload on sensitivity. A similar effect was observed with the lower concentrations of meclofenamate and ETA. NDGA and higher concentrations of meclofenamate and ETA not only shifted the sensitivity in both high and low preloaded strips but also eliminated the preload effect. These results indicate that cyclooxygenase and lipoxygenase metabolites both alter aortic smooth muscle sensitivity to contractile agents and suggest that lipoxygenase metabolites may play a role in the change in sensitivity seen with preload.

Metabolism of PGE-2 and PGF-2 alpha by cytosolic fractions (100 000 g supernatant) of rabbit uterus, oviduct and lung was measured in vitro. Metabolism of PGE-2 was greater than that of PGF-2 alpha for oviduct and uterus. After an ovulating injection of hCG metabolism of both PGE-2 and PGF-2 alpha by lung and uterus declined linearly up to 72 h (during the time of ovum transport). The amount of PG metabolism by the oviduct did not change significantly during this period, but the percentage changes of PGE-2 and PGF-2 alpha metabolism from oestrous values did differ, and perhaps indicated a change in the ratio of intracellular PGs. No change of metabolism of either PG by lung, uterus or oviduct occurred at 24 or 72 h after an injection of 250 micrograms oestradiol cyclopentylpropionate given concomitantly with the hCG (a treatment regimen which causes 'tube-locking' of ova). However, progesterone treatment, in a regimen known to cause accelerated transport of ova through the oviduct, caused significantly enhanced metabolism of both PGE-2 and PGF-2 alpha by uterus and oviduct, but not lung, 30 and 48 h later except for PGE-2 by uterus at 30 h. These results suggest that changes in metabolism of PGE-2 and PGF-2 alpha by the oviduct may be involved in the mechanisms controlling ovum transport.

Arachidonic acid (AA) is often utilized in in vitro studies to label cellular pools of AA or to elicit cellular responses dependent on eicosanoid production. Because of the hydrophobic nature of AA, organic diluents such as ethanol or dimethyl sulfoxide are utilized in preparing concentrated solutions. The fate of AA when added to aqueous medium is not generally considered because of the dilution of the AA, although some investigators utilize bovine serum albumin (BSA) to solubilize as well as to trap AA and its hydrophobic metabolites. These experiments demonstrate a rapid and progressive decline in AA concentration when added to aqueous media in tissue baths and in glass test tubes. The extent of the decline was greater in the tissue baths than in the test tubes. The calcium ionophore A23187, which is used to stimulate AA metabolism, is also hydrophobic, and its concentration also decreased when added to aqueous media. The decline in the concentration of both AA and A23187 was due to adsorption to the container walls. The presence of 1% BSA in the aqueous solution attenuated and even eliminated the decline in the concentration, indicating binding of the two agents to the protein. However, the presence of BSA in culture medium inhibited the A23187-induced stimulation of AA metabolites in baboon aortic smooth muscle cells. These results underscore the complexities arising from the in vitro use of hydrophobic substances in biological systems.

Summary. Thirty-one virgin female rats in pro-oestrus were mated with fertile males (Day 0). Groups of four to six animals were dosed by mouth with Compound I.C.I. 33,828 (100 mg/kg) on 3 successive days starting, in different groups, on Days \p=m-\1,0, 1, 2, 3 and 4, respectively. Twelve control rats were given the vehicle only. In all groups treatment with I.C.I. 33,828 reduced the number of rats found pregnant at laparotomy, but in the treated animals found pregnant at laparotomy the mean number of implantation sites was not significantly different from that in the controls. In only one group, that dosed on Days 2 to 4, were no implantation swellings found in any animals. The results at autopsy showed that treatment resulted in heavy mortality of foetuses after implantation in those rats in which implantation occurred. In the control rats, 6\m=.\6%of the eggs shed were lost before and 6\m=.\6% after implantation. The corresponding figures for all the treated animals were 63\m=.\7and 30\m=.\2%respectively. Moreover, in the treated rats the development of the foetuses which survived to Day 19 was retarded, as judged by the size of the swellings on Day 8 or 11, and by the weight of the foetuses which at autopsy amounted on average to only two-thirds of that of the controls. In an experiment in which pseudopregnant rats were dosed with I.C.I. 33,828 either before or before and after one horn of the uterus had been traumatized, it was found that the compound prevented the occurrence of a full deciduomal reaction. The effect was more marked when it was administered on Days 2 to 4 than on Days 4 to 6 of pseudopregnancy. From these results it was deduced that the compound might produce its antifertility effects by inhibiting pituitary function, thus upsetting the endocrine balance necessary for the maintenance of pregnancy.

Interference with pregnancy has androstenedione before implantation. The failure of pregnancy was attributed to the regression of the corpora lutea of pregnancy. The effects of androstenedione on pregnancy were dose dependent and could be prevented by concomitant administration of cortisol acetate. Adrenalectomy on day 1 of pregnancy did not alter the effects ofandrostenedione on pregnancy, and only the effects of the lower doses of androstenedione could be prevented by cortisol in adrenalectomized animals. Progesterone was able to protect intact hamsters against all the effects of androstenedione except the decrease in ovarian weight. It is suggested that sufficient androstenedione is metabolized in vivo to estrogen to produce the observed effects. {Endocrinology 80: 1152, 1967)

(0 A new technique is described in which 120-μ anionic resin spheres, impregnated with the radioactive gold isotope 198Au, are employed to simulate rabbit eggs. By their use, a study has been made of the pattern of egg transport in the Fallopian tube of the rabbit in vivo. (2) It has been shown that, in the first 8 hr after ovulation, the spheres have travelled 49% of the length of the tube. During the next 40 hr, movement down the tube is very slow until, at 48 hr after ovulation, the spheres are concentrated above the utero-tubal junction, 79% of the distance down the tube. (3) It has been possible to study the passage of the spheres into the uterus both at 56 and 64 hr after ovulation. Of the total number of spheres studied, 55% had passed into the uterus at the earlier and 90% at the later time. (4) Evidence is also included to show that the movement of the spheres reproduces the normal movement of eggs fairly closely. Their mean rate of progress is some 4% slower than the eggs, and this is due to a small proportion of the spheres lagging or

SUMMARY The technique of tracing the movement of 'artificial eggs' (radioactive spheres) by autoradiography was used to study the effects of progesterone and oestrogen either alone, or in combination, on egg movement through the reproductive tract of ovariectomized rabbits. Ovariectomy was performed 7 days before the transport experiments, except in one treatment group when it was done 35 days previously. The animals were autopsied at 8, 24, 48 or 56 hr. after insertion of the spheres into the ampullae of the Fallopian tubes. There were five or more animals in each group except one. The effects of various hormonal replacements were studied. It was found that removal of the ovaries, whether 7 or 35 days before the transport experiments commenced, caused extremely irregular movement of the spheres through the Fallopian tube. Marked differences were recorded between the behaviour of the spheres in animals ovariectomized for the shorter as compared with the longer period. None of the hormonal treatments produced normal movement of the spheres through the Fallopian tube. Progesterone delayed movement of the spheres through the ampulla very markedly, but if the spheres reached the isthmus they passed into the uterus on all occasions. Neither the isthmus nor the utero-tubal junction in animals so treated proved a barrier to the movement of the spheres into the uterus. Movement of the spheres through the uterus of these animals was inhibited, and most of the spheres recovered were found in the half of the uterus nearest to the utero-tubal junction. Oestradiol benzoate, in the dose administered, had an accelerating effect on the passage of the spheres through the tube, and also through the uterus. Some spheres were found to have entered the vagina in three out of the four time-groups studied. It was observed that despite the accelerating effect of oestrogen on the movement of the spheres through the Fallopian tube a certain percentage, even after the longest interval studied, was trapped at the junction of the ampulla and the isthmus. The two groups treated with oestrogen and progesterone in combination tended to produce results similar to those observed after treatment with either progesterone or oestrogen alone, depending on which hormone was dominant. It is concluded that the amount of oestrogen present is critical in ensuring rapid transport through the ampulla, but that a proper balance of oestrogen and progesterone must be maintained to ensure retention of the spheres or eggs in the isthmus until the correct time for entry into the uterus. It is clear that this pattern of egg movement through the Fallopian tube cannot be re-created by fixed daily doses of progesterone and oestrogen.

Studies of oviduct motility in rabbits have been made using indwelling balloon-ended fluid-ifiled catheters. Daily recordings were made on 12 rabbits for periods up to 21 days. Oviduct motility was characterized by bursts of activity which lasted for 2-3 mm followed by silent periods ranging up to 20 mm. In some cases contractile activity continued without intermission for many minutes. There was no correlation for bursts of activity in left and right oviducts of the same animal. Daily variations of muscular activity were seen in 10 of 12 rabbits, and in 6 of these 10 cyclical peaks of activity occurring every 4-7 days were observed. Each oviduct exhibited its own pattern of cyclical variation. In 4 of the 10 rabbits. exhibiting variations in muscular activity of the oviduct, ovulation was induced by an intravenous injection of 100 IU of human chorionic gonadotrophin. Following injection little increase in muscular activity of the oviduct was seen for 4 hr, by which time bursts of contraction occurred every 15 mm. There was then a rapid increase in the mean amplitude of contractions until 10 hr, followed by a further slower increase up to 24 hr after injection. At this latter time bursts of activity occurred every 5-8 mm. During the next 2 days there was a sharp fall in the amplitude of muscular contractions to preinjection levels.

SUMMARY The effects of chlormadinone (6-chloro-Δ6-17α-acetoxyprogesterone), an orally active progestational agent without significant oestrogenic activity, on the response of the ovaries of intact and hypophysectomized immature female rats to exogenous gonadotrophin have been examined. Administration of the steroid whether starting on the same day as, or 4 days before treatment with gonadotrophin, did not depress the ovarian response in intact rats. In hypophysectomized animals, pretreated with the progestagen, the ovarian response to gonadotrophin was depressed. In intact rats, treatment with the steroid and pregnant mare serum gonadotrophin (PMSG) resulted in ovulation, whereas in similar animals given PMSG alone no corpora lutea were found. Corpora lutea were seen in all groups given PMSG and human chorionic gonadotrophin (HCG) but ovulation occurred earlier when, in addition, treatment with the steroid was included. In only one experiment with intact rats did administration of the steroid alone cause a significant increase in uterine weight compared with controls. In neither experiment on hypophysectomized animals did such an increase occur, and significant decreases were recorded.

Photomicrographs of the cytological changes of the sperm head and of the female chromatin in the rabbit egg, from the time of sperm penetration and activation of the second meiotic metaphase up to the formation of pronuclei and syngamy, are presented. During a study on factors affecting sperm penetration of rabbit eggs, some anomalous eggs were observed. One unfertilized egg possessed two second meiotic metaphases and associated first polar bodies, while another similar egg had two first and two second polar bodies, two sets of female chromatin, and one fertilizing sperm. In this latter egg fertilization was apparently proceeding normally. Two eggs within a single zona pellucida were also seen; one of these was a primary oocyte, while the other was a normally penetrated egg. Evidence is also presented which indicates that the incidence of polyspermy (usually dispermy) is increased, if eggs enter the oviduct at 14 hours or later after mating. This is thought to be due to more fully capacitated sperm present at the site of fertilization at these later times, and thus there is a greater chance of two or more sperm entering the vitellus at the same time.

Ten groups, five to ten animals per group were used in demonstrating that progesterone plus cortisol acetate can induce implantation and maintain embryonic growth in hamsters subjected to ovariectomy and adrenalectomy on Day 1 or on Day 4 of pregnancy, Fetal weights on Day 14 were, however, significantly lower than in sham-operated control animals, although they were not different from those in animals only ovariectomized and given progesterone. It is concluded, therefore, that it is unnecessary to invoke secretion of estrogen either from the ovaries or from the adrenals for induction of implantation in hamsters.

When pseudopregnant hamsters were ovariectomized and their uteri traumatized on day 4, deciduomal growth (not significantly different from that seen in intact controls) could be maintained by daily injections of 1, 2 or 4 mg of progesterone either alone or in combination with 1, 2 or 5 μg of estrone. When ovariectomy was performed on day 1 of pseudopregnancy and uterine traumatization on day 4, induction and maintenance of a deciduomal response could be achieved with 1 or 2 mg/day of progesterone alone (or in some cases in combination with 1 μg/day of estrone) from days 1 to 3 followed by 1 or 2 mg/day of progesterone in combination with 1, 2 or 5 μg/day of estrone from days 4 to 7. The best results were obtained with progesterone alone (2 mg/day) before trauma and estrone (2 μg/day) and progesterone (2 mg/day) after trauma.

SUMMARY Implantation was largely prevented in rats given a single dose of I.C.I. 46,474 on day 4 of pregnancy. Aminoguanidine bicarbonate given on days 2–5 inclusively prevented this effect in intact, but not in adrenalectomized rats. Neither histamine dihydrochloride nor dl-histidine prevented the effect of I.C.I. 46,474 on implantation. Histidine, but not histamine, prevented the action of aminoguanidine in intact pregnant rats given I.C.I. 46,474. The failure of aminoguanidine to reverse the effects of I.C.I. 46,474 in adrenalectomized rats indicates that I.C.I. 46,474 does not prevent implantation by virtue of its anti-histaminic activity.

Administration of DHA (2.5, 5 or 10 mg/day sc) on days 2–5 interfered with pregnancy in rats. This effect could be produced in intact and adrenalectomized rats and was potentiated in partially hepatectomized ones, total failure of implantation being produced by daily doses of 10, 10 and 5 mg, respectively. Although doses of 2.5–10 mg/day of DHA greatly reduced the deciduomal response of the uterus to trauma in pseudopregnant rats, implantation failure was due not to this, but to expulsion of the eggs from the reproductive tract by day 5 of pregnancy. This effect was similar to that produced by administration of estrogens. Cortisol acetate did not influence the effect of DHA on pregnancy. In intact or adrenalectomized immature rats, the uterus responded to administration of DHA with a dose dependent increase in weight and histological changes similar to those produced by estradiol. In ovariectomized and ovariectomized-adrenalectomized animals smaller responses to similar doses of DHA were produced. In inta...

SUMMARY Administration of chlormadinone, an orally active progestational agent without significant oestrogenic activity, to intact immature female rats did not affect either ovarian or uterine weight significantly compared with controls. A single injection of human chorionic gonadotrophin (HCG) caused a 73 % increase in uterine weight in 24 hr. over the control value. This dose significantly increased ovarian weight and although it caused some stimulation of follicular development, ovulation during this time did not occur. When animals were treated with chlormadinone for 8 days, and received HCG on the 8th day, uterine weight was 170% greater than in the controls and 56% greater than with HCG alone. The uterine weight produced was similar to that found in animals treated with mestranol, a potent oestrogen, and HCG. In ovariectomized animals HCG did not affect uterine weight, while the small increase produced by chlormadinone was unaltered when HCG also was given. Mechanisms are discussed by which this augmentation of the uterine response to HCG might be produced. It seems most likely that chlormadinone administration causes storage of endogenous gonadotrophin in the pituitary, and that the exogenous gonadotrophin acts as the 'trigger' for the release of stored hormone, probably by a direct action on the hypothalamus.

Administration of androstenedione (4 or 8 mg/rat/day subcutaneously) on days 2–5 interfered with pregnancy in rats. This effect could be produced in intact, in adrenalectomized or in partially hepatectomized animals. These doses of androstenedione greatly reduced the deciduomal response of the uterus to trauma in pseudopregnant rats. Implantation was prevented, however, not because of the failure of the deciduomal response, but because androstenedione caused expulsion of the developing eggs from the reproductive tract by Day 5 of pregnancy (i.e., before implantation). These effects appear similar to those produced by estrogens, and normal implantation occurred when progesterone was given with androstenedione. Cortisol acetate also tended to prevent the effects of androstenedione on pregnancy in intact and in adrenalectomized, but not in partially hepatectomized rats. This suggests that the protective effect of cortisol is mediated by the liver. In intact or adrenalectomized immature rats, the uterus respo...

Ovariectomy of hamsters on day 1 or day 4 of pregnancy without steroid replacement resulted in complete failure of implantation. This was, however, induced by daily subcutaneous (s. c.) injection of either 1 or 2 mg progesterone. Although implantation could occur in the absence of oestrogen, greater numbers of eggs implanted in its presence after ovariectomy on day 1. Oestrogen administration (1 μg/hamster/day s. c. of oestrone) also increased the proportion of embryonic development to day 14 in the ovariectomized, progesterone-treated animal.

chorionic gonadotrophin, with iproniazid (25 mg/kg/day sc) or reserpine (0.25 or 1.0 mg/ kg/day sc). The animals were then killed at 26, 50 or 90 hr after insemination and the ovulating injection. The oviducts were divided into three portions-comprising the ampulla, distal and proximal isthmi. These portions and the uteri were flushed for recovery of eggs and then processed, along with the ovaries, for determination of the content and concentration of norepinephrine (NE). The levels of NE were substantially increased in all these tissues of all groups over those of untreated animals by the monoamine oxidase inhibitor and decreased to near zero by reserpine. The most significant changes in content and concentration of NE occurred in the distal isthmus, and to a lesser degree in the proximal isthmus. There was some indication of retarded egg transport associated with decreased NE in the isthmus of the reserpine-treated groups and accelerated transport with elevated NE in the isthmus of iproniazid-treated groups. These alterations in the speed of egg transport were, however, only marginal when compared to the effects of progesterone (accelerated) and estrogen treatment (retarded transport) in previous experiments. Furthermore, these hormonal treatments had both significantly elevated NE levels in the isthmus (Bodkhe and Harper, 1971). In the present experiments total NE in the tissue was measured, and no information on changes of NE free in the neurons was obtained. Thus egg transport through the oviduct may be controlled mainly by small changes in free NE or hormones may exert their effects in ways other than by changes in noradrenergic activity.

Newly ovulated eggs in cumulus, stained with toluidine blue, were transferred to the ampullae of the oviducts of (a) 5 rabbits at the time of ovulation, and thereafter at intervals of 24 hr for the 3 succeeding days, and (b) 21 does in estrus and at various times during induced pseudopregnancy. The mean times for the eggs to traverse the ampulla were 7.5 ± 0.7, 8.4±0.8 and 16.0±3.8 min in estrus, at ovulation, and on day 7 of pseudopregnancy, respectively. During this period the mean time of transport increased by 1.2 ± 0.28 min/day. This increase was highly significant. Owing to differences in oviduct lengths between rabbits, the rate of transport along the ampulla (mm/min) was felt to be a more valid measurement. The mean rates of transport were 11.4 ± 0.9, 8.9 ± 1.5 and 5.7 ± 0.8 mm/min in estrus, at ovulation and on day 2 1/2 of pseudopregnancy, respectively. During this period the mean rate of transport decreased by 2.36 ± 0.58 mm/min/day, a highly significant decrease. No further change was noted be...

SUMMARY By means of the technique of tracing the movement of 'artificial eggs' (radioactive spheres) by autoradiography (Harper, Bennett, Boursnell & Rowson, 1960) a study has been made of the effects of a decreasing oestrogen and various increasing progesterone doses on egg movement through the reproductive tract of ovariectomized rabbits. Five or more animals were autopsied at 8, 24, 48 and 56 hr. after insertion of the spheres into the ampulla of the Fallopian tube. It was hoped that such treatments would produce hormonal conditions similar to those in intact rabbits after ovulation, and that therefore normal tubal transport would occur. The treatments used produced normal movement of the spheres through the ampulla and the isthmus, and most of them were retained in the Fallopian tube up to 48 hr. after insertion. However, by 48 hr. one or more spheres were observed to have entered the vagina in all treatment groups. None of the treatments caused most of the spheres to enter the uterus between 48 and 56 hr. after insertion, as would be the case in intact animals at a similar time after ovulation. It was concluded, however, that such treatments could produce tubal transport normal up to 48 hr. in ovariectomized rabbits, and that the presence of oestrogen appeared to be the controlling factor.

Unfertilized eggs recovered fronu donor ral)bits shortly after ovulation induced by injection of HCG were transferred to the oviducts of mated recipients or recipients inseminated with 200 nuillion spermatozoa and injected with HCG at known times. It was found that although only 407r of such eggs were penetrated in 1.5 h following transfer to mated recipients at 9-9.75 h after mating, 58 and 66� were penetrated during the same period if transfers were delayed until 13.75-14.25 and 19.5-20.0 h after mating, respectively. By 24 h after nuating the percentage of eggs penetrated had fallen again to 43%. It was shown by ligation of the oviduct at the uterotubal junction that adequate sperm were in the oviduct by 9.25-9.75 h after inselllinatiOn to ensure this enhanced fertilization following transfer at 14 or 17 h hut not 20 lu after insemination. Tiuese results implied that although spernu continued to migrate from the utenis into the oviduct, more sperm in the oviduct at later times was not responsible for this plsenomellon. Further experiments in which ligation was done at the anipullar-isthmic junction or the middle of the isthnuls showed that sperm were retaiiued in the proxilnal isthmus just above the uterotuhal junction for several hours. When whole ovidncts were fixed and serially sectioned and the number .‘.nd distribution of sperm determined, it waS fnund that at 9 h after insemination few sperm were at the site of fertilization. In the oviducts of animals killed at 14 or 20 h after insemination, the spernu in tlue oviduct (irrespective of whether tile uterotubal junction luad been ligated or not) had become redistributed with many more found at the site of fertilization. It is concluded that more eggs are penetrated in a short period of time (1.5 h) followed late transfers because there is tile ilnmediate posSii)ility of egg and sperm Ineeting.

I.C.I. 46,474, the trans-isomer of 1-(p-\g=b\-dimethylaminoeth\x=req-\ oxyphenyl-1,2-diphenylbut-1-ene, is very effective in terminating early pregnancy by preventing implantation when given during the first 4 days after insemination to female rats. In this species, the compound is weakly and atypically oestrogenic and is also anti-oestrogenic, as indicated by its inhibitory effect on the response to exogenous oestrogens of the vaginal epithelium (cornification) and uterus (weight-increase). It is suggested that it can prevent implantation in rats by virtue of its anti-oestrogenic activity, i.e. by counteracting the oestrogen-release from the ovaries which is believed to occur on the 4th day and upon which implantation appears to depend. The corresponding cis-isomer (I.C.I. 47,699) is very different in its properties and behaves in all respects like a conventional oestrogen (orally active\p=m-\e.g.dienoestrol). In rats it is very much more potent as

Hemi-ovariectomy on days 1, 2 and 3 after the appearance of leukocytes in vaginal smears in rats (made pseudopregnant by reserpine injections at estrus) caused a return of proestrus between 2 and 3 days after operation in all animals. There was compensatory ovulation at the first or second postoperative proestrus, as shown by counting healthy implantation sites at laparotomy following a successful mating. Progesterone replacement immediately after hemiovariectomy successfully maintained the persistent leukocytic vaginal smears of pseudopregnancy in all animals so treated. Sham operation was without effect. (Endocrinology 87:173,1970) THERE IS good evidence to indicate that the central nervous system chronically inhibits the secretion of prolactin in the rat (1–4). It has been postulated that the factor which stimulates luteotrophin secretion does so by suppressing this neural inhibition, and that, during pseudopregnancy, progesterone may maintain the secretion of luteotrophin by a positive feedback (3).

Shelesnyak was first to suggest an essential role for histamine in the process of implantation of the fertilized ovum in the rat uterus (for references see Shelesnyak, 1957). He showed (1954) that antihistaminic drugs, instilled into the uterine lumen in pseudopregnant rats, would suppress the decidual response to endometrial trauma, and demonstrated further (Shelesnyak, 1957) that pyrathiazine HCl (Pyrrolazote), which is very active in this sense, would prevent implantation in the injected uterine horns of pregnant rats if applied in the same way during the first few days post coitum. However, when given subcutaneously, Pyrrolazote\s=r\had little effect on implantation, the interference with pregnancy being greatest when the drug was administered on Days 8 to 10 after insemination (Shelesnyak & Davies, 1955). The relative inefficacy of the drug given systemically was attributed to its failure to reach the uterus in sufficient concentration\p=m-\aconclusion based on tissue-distribution studies with other antihistamines. In mice, Shelesnyak & Davies obtained a similar response to subcutaneous diphenhydramine (Benadryl\s=r\).A dose of 1 mg/kg/day for 6 or 10 days affected pregnancy only after implantation. Goldstein & Hazel (1955) found that 50 mg/kg/day ofpyranisamine maleate (Neo-Antergan\s=r\),given subcutaneously to mice for 7 days, did not affect the number becoming pregnant. Sulman (1955) observed no adverse effects on pregnancy from Benadryl\s=r\,Pyribenzamine\s=r\ (tripelennamine), Histadyl\s=r\(methapyrilene), Trimeton\s=r\(pheniramine), Tagathen\s=r\(chlorothen) or Decapryn\s=r\(doxylamine), given in doses of 100 \g=m\g/mousetwice daily for 6 weeks. In our laboratories (Walpole, unpublished) no interference with the decidual response to uterine trauma was observed in groups ofpseudopregnant rats given phenoxybenzamine (Dibenzyline\s=r\),promethazine (Phenergan\s=r\),pyranisamine

The purpose of this study was to determine whether endogenous prostaglandins are involved in the regulation of sexual steroid receptor concentration and cytosol to nucleus translocation in the rabbit uterus. The animals were injected s.c. with indomethacin (10 mg/kg) twice daily on days 5 and 6 and on the morning of day 7 of pregnancy or pseudopregnancy and killed 2 h after the last injection. The indomethacin treatment did not change progesterone and oestradiol serum concentrations compared with animals injected with vehicle only. No differences were observed either in progesterone receptor or oestradiol (pregnant animals only) receptor concentration and intracellular distribution in endometria of indomethacin-treated or control animals. In an extended experiment indomethacin given to oestrogen-pretreated ovariectomized rabbits did not inhibit the nuclear progesterone receptor accumulation induced by a single progesterone injection. These results suggest that there is no direct relationship between prostaglandins and sexual steroid receptors in the rabbit uterus. J. Endocr. (1985) 107, 197–203

Platelet-activating factor (PAF) concentrations were low in the non-pregnant, oestrous uterus (mean +/- s.e.m.: 2.2 +/- 1.2 pmol/g, n = 3). However, uterine PAF increased dramatically during pregnancy to a maximum of 37.8 +/- 4.90 pmol/g (n = 7) on Day 5. By Day 7, PAF concentrations in the uteri of pregnant rabbits had returned to levels similar to those found at oestrus. In contrast, uterine PAF in pseudopregnant rabbits peaked at 30.6 +/- 2.8 pmol/g (n = 8) on Day 4, declined to 20.5 +/- 2.4 pmol/g (n = 8) on Day 5 and then remained at that concentration through Day 7. Uterine PAF co-migrated with synthetic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-phosphocholine) in both thin-layer and normal-phase high-performance liquid chromatography. PAF activity in the uterus during pregnancy and pseudopregnancy was found almost exclusively in the endometrium; little or no PAF was found in myometrium, uterine flushings or blastocysts. While no PAF was detected in blastocysts on Days 5 and 6 of pregnancy, the presence of the embryo appears to modulate biosynthesis and/or degradation of PAF by the uterus, since PAF decreased significantly in uterine tissue apposed to the implanting embryo (but not in similar areas between such attachment sites). Increased concentrations of PAF in the preimplantation rabbit uterus followed by a dramatic decrease on the day of blastocyst attachment suggest that this potent inflammatory autacoid may play a vital role in implantation.

Blastocysts recovered from oil- or indomethacin-treated donor rabbits between 5 1/2 and 6 days after insemination and hCG injection were transferred to oil- or indomethacin-treated recipients between 135 and 147 h after hCG injection. Indomethacin treatment of donor rabbits (10 mg/kg s.c.) given every 6 h during the day before transfer had no effect on subsequent implantation of the blastocysts. However, indomethacin treatment of the recipients (10 mg/kg s.c. every 6 h from 120 to 168 h after hCG) prevented implantation of all transferred blastocysts, although 6 of the 8 rabbits died between Days 9 and 16 of (pseudo)pregnancy. Restriction of the indomethacin treatment of the recipients to only 3 injections of 10 mg/kg s.c. between 128 and 140 h after hCG injection had no effect on the implantation of the transferred blastocysts. It is concluded that indomethacin exerts its inhibitory influence on implantation via an action on the endometrium rather than on the blastocyst.

Endotoxin derived from Salmonella enteritidis (Boivin) completely interrupted pregnancy in the rabbit when given as a single dose (10 or 20 microgram/kg) intravenously on Day 4 or Day 8 of pregnancy. Indomethacin (2 or 10 mg/kg) was unable to prevent this effect.

Chemical modifications to the LHRH structure have resulted in highly active agonists and several such compounds, e.g. [d-Ser(tBu)6,Pro9 -NHEt]LHRH, [d-Trp6,Pro9 -NHEt]LHRH and [d-Nal(2)6]6]LHRH have been studied clinically. Highly active antagonists were discovered more recently, e.g. [Ac_d-pC1_Phe1,d-pC1_Phe2,d-Trp3,d-Arg6,d-Ala10]LHRH and [N-Ac-d- Nal(2)1,d-pF-Phe2,d-Trp3,d-Arg6]LHRH, but these are only just reaching the stage of clinical trial. There are now available sufficiently potent analogs of both types for adequate testing as contraceptive agents. There is still scope for improvement in design, e.g. increased resistance to metabolism and hence prolonged in vivo bioactivity may be possible. More potent compounds may be discovered, but their mode of action probably will be the same as those already described. LHRH analogs have proved non-toxic, despite their interference with endocrinological control mechanisms. It is possible to evaluate the potential of these compounds for contraception. Already certain trends are clear. Although there was hope that LHRH analogs might be contraceptive by several mechanisms (e.g. luteolytic, abortifacient and defective luteal phase), present evidence suggests that only inhibition of ovulation in the female and inhibition of spermatogenesis in the male will be effective. Even in the modalities where contraception may be achieved, it will probably be necessary to use steroids – progestins in the female and androgens in the male – as adjuncts to LHRH analog treatment.

Injections of reserpine (0.5 mg/kg) or chlorpromazine (30 mg/kg) from Day 2 through 6 after mating delayed implantation of blastocysts in all rats. This effect was reversed by concomitant administration of cortisone (2 mg/ rat/day) from Day 1, estrogen (0.1 5g/rat) on Days 3 and 4, or human chorionic gonadotropin (5 IU/rat) on Day 3 after mating. Adrenalectomy on Day 1 largely prevented the effect of chlorpromazine but not of reserpine on implantation. Treatment with reserpine or chlorpromazine from Day 6 through 13 of pregnancy caused a high incidence of abortion and/or embryonic resorption. Administration of cortisone concurrently with reserpine or chlorpromazine, or treatment with estrogen (0.2 5g/rat/day) prevented this effect. Exogenous ACTH (20 IU/ animal/day) produced effects similar to those of reserpine and chlorpromazine on the process of implantation and gestation. Its effect was also reversed by estrogen or HCG, but not by cortisone. (Endocrinology 87: 966, 1970)

The intention of this review is to consider how drug research has led to progress in the control of reproduction in women. Although regulation of reproduction in humans is of prime importance, the situation in domestic animals is also of economic significance. In general the principal object of therapy has been to limit fertility in humans and to increase it in animals of agricultural importance. There are of course notable exceptions to these generalities. For instance, it is clearly important to help childless couples conceive, and by contrast, decreasing fertility in farm animals in some circumstances can be no less necessary.

Unfertilized eggs recovered from donor rabbits at known times after injection of HCG were transferred to the oviducts of mated recipients (prevented from ovulating by having their ovaries covered with nail polish) at specific times after mating. When newly ovulated eggs (11.25–13.5 hours after HCG injection) were left in the oviducts of the recipients for 1, 1.5, 2, 2.5 and 3 hours, 15, 30, 44, 59 and 75% respectively were penetrated by spermatozoa. If the unfertilized eggs were allowed to age in the oviducts of the donor before transfer then fertilizability decreased: only 41 and 25% of eggs 15–15.5 hours and 17–17.5 hours old at time of transfer respectively were penetrated during 2.5 hours in the recipients' oviducts compared to 59% of 13–13.5 hour eggs. No beneficial effect from maturation in the donor of the unfertilized egg and its membranes was observed. When eggs with cumulus cells removed were transferred to recipients at 9–9.5 hours after mating, then a decrease in the percentage of eggs penetrated during 2.5 hours in the recipients' oviducts was found. Removal of both cumulus and corona cells had no greater effect than removal of cumulus alone. However, when eggs similarly treated were transferred to recipients at 14 hours after mating, neither removal of cumulus nor of cumulus and corona cells had any effect on the percentage of eggs penetrated during 2.5 hours. It is concluded that when conditions for fertilization are not optimal the cumulus is important for ensuring meeting of sperm and egg, but that when conditions are optimal then presence of these cellular layers is not important. Whether the products of ovulation play a role in the final stages of capacitation of sperm was tested by transferring eggs into the oviducts of rabbits unilaterally ovariectomized (more than 3 weeks previously) at 13.5–14.75 hours after mating. No difference in percentage of eggs penetrated was observed between oviducts on the ovariectomized and intact sides. The contact of the sperm with the products of ovulation in the one oviduct did not give them an advantage over the sperm in the other oviduct without such contact. It was found that although only 30% of newly ovulated eggs were penetrated in 1.5 hours following transfer to recipients at 9–9.5 hours after mating, 57 and 66% were penetrated during the same period if the transfers of similar eggs were delayed until 13.75–14.0 hours and 19.5–20.0 hours after mating of recipients respectively. It is suggested that these increases in percentages of eggs penetrated are a reflection of the presence of more sperm or of more fully capacitated sperm in the oviduct at these later times.

SUMMARY Trans-1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene (I.C.I. 46,474), given by mouth, was most effective in terminating early pregnancy in rats on the fourth day after mating. A single intravenous dose was effective on day 4 but even early on day 5 large doses had little effect. A single oral dose on day 1 or 2 sufficient to prevent implantation completely, caused accelerated tubal transport of the eggs and their premature expulsion from the uterus. This was partially blocked by progesterone and appears to be an oestrogenic effect. The corresponding dose on day 3 or 4 did not affect egg transport or development. After giving the compound on day 4, substantial but diminishing numbers of unimplanted eggs were recovered from the uterus up to day 11 of 'pregnancy'. Such blastocysts could be induced to implant in a proportion of rats by giving a single large injection of oestrogen at the same time as the compound (on day 4) or a progressively smaller dose up to day 11. These results support the hypothesis (Harper & Walpole, 1966) that I.C.I. 46,474 can prevent implantation in rats by counteracting the 'oestrogensurge' on day 4 necessary for its induction. They further suggest that the compound persists for some days in the uterus.

Newly ovulated eggs in cumulus, stained with toluidine blue, were transferred to the ampullae of the oviducts of (a) 36 mature rabbits 7 days after bilateral ovariectomy, and (b) 13 intact mature rabbits. These rabbits received various doses of progesterone and/or estradiol benzoate (EB), dissolved in cotton seed oil and injected subcutaneously. The mean times (min) for the eggs to move from the base of the infundibulum to the junction of the ampulla and the isthmus were recorded, and subsequently converted in relation to the length of the ampulla to the mean rates of transport (mm/min). Ovariectomy decreased the mean rate of transport to 4.5 ± 0.8 mm/min, and this was not significantly further decreased (2.8 ± 0.4 mm/min) by daily treatment with 2 mg progesterone. Treatment with 2 and 4 /xg EB/day restored the rate of transport (8.4 ± 0.9 and 10.0 ±0.6 mm/min, respectively) to the levels seen in estrous animals. Progesterone injections (2 mg), which were given 13 hr before the observations on transport t...