Embryonic abnormality caused by male pheromonal effect in pregnancy block in mice.

BALB/cA females mated with BALB/cA males and exposed to DDK males showed 100% pregnancy block when exposure started at Days 0-3 of pregnancy. When the exposure to DDK males started at Days 4, 5, 6, and 7, the pregnancy rate was 33.3%, 50%, 90%, and 100%, respectively. (Hereafter, the BALB/cA females exposed to DDK males are referred to as the experimental females, and those exposed to other BALB/cA males as the control females.) Cell number of embryos at Day 3 of pregnancy from the experimental and control females after exposure starting from Day 0 was, respectively, 27.0 +/- 1.2 (mean +/- SEM) vs. 48.6 +/- 1.0 (p < 0.05) for the morulae and 34.8 +/- 1.3 vs. 64.1 +/- 2.0 (p < 0.05) for the blastocysts. Embryos from the experimental and control females after exposure starting from Day 3 or Day 4 were histologically examined after 24-h exposure. Poor development of trophoblast and inner cell mass was observed at Day 4 as general defects of the experimental embryos. In addition, about one third of the embryos were unexpectedly at the more advanced stages than the controls (abnormal overdevelopment). At Day 5, disintegration of the trophoblast layer and primitive endoderm was observed as common abnormalities of experimental embryos, but overdevelopment was not observed at this stage. Disturbance in trophoblast differentiation or failure of its maintenance may be the primary developmental defect displayed by embryos developing in a deteriorated uterine environment under the influence of the odor of alien males.

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts.

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.

Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.

Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.

A factor(s) from a rat trophoblast cell line inhibits prolactin secretion in vitro and in vivo.

The purpose of the present study was to measure the inhibitory action of secretions from trophoblast cells on prolactin (PRL) secretion in cycling and pregnant rats, and to determine whether factor(s) from trophoblast cells act directly on anterior pituitary cells. A rat choriocarcinoma cell line (Rcho)--a line consisting of trophoblast cells, including differentiated giant cells that secrete members of the placental PRL family--was used. When Rcho cells (1 x 10(6) cells) were transplanted under the kidney capsule of cycling rats, tumors developed and the rats went into constant diestrus. Eight days after cell injection, plasma progesterone was significantly increased in treated rats compared to controls, whereas plasma and pituitary PRL and pituitary PRL mRNA levels were significantly decreased. Similar PRL results were seen on Day 9 of pregnancy after injection of Rcho cells on Day 0 or Day 1 of pregnancy. To determine whether secretions from Rcho cells had a direct effect on anterior pituitary cells to inhibit PRL release, anterior pituitary cells were enzymatically dispersed and cultured for 4 days. Conditioned medium was obtained from 9-day Rcho cell cultures and concentrated by ultrafiltration. A fraction containing substances with molecular weights greater than 10,000 suppressed PRL release from the pituitary culture after 3 and 24 h. Conditioned medium containing substances with molecular weights between 1000 and 10,000 had no effect on PRL release, nor did conditioned medium from a placental cell line designated HRP-1. HRP-1 also contains trophoblast cells but does not contain the differentiated giant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin-induced decrease in hypothalamic tyrosine hydroxylase activity and corresponding increase in prolactin release are abolished at midpregnancy and by transplants of rat choriocarcinoma cells.

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating PRL levels in two physiological models: 1) pregnant rats expressing (day 8) or not expressing (days 11 and 16) PRL surges, and 2) ovariectomized rats transplanted with rat choriocarcinoma cells, which secrete functional placental lactogen-I. Over a 4-min period between 0900 and 1400 h, rats were administered either vehicle or 5-HT (20 micrograms/6 microliters) through lateral ventricular cannulae. Plasma PRL levels were determined by RIA. NSD 1015 (25 mg/kg intraarterial), a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, was injected 20 min after initiation of ventricular infusion. Ten min later, the stalk-median eminence (SME) was dissected. The rate of DOPA accumulation, determined by measuring DOPA levels in the SME by HPLC, was used as an index of tyrosine hydroxylase catalytic activity, indicating tubero infundibular dopamine neuronal activity. In day-8 pregnant rats 5-HT reduced DOPA accumulation to 57% of vehicle-injected controls and increased circulating PRL levels 13-fold. In contrast, on days 11 and 16 of pregnancy 5-HT did not alter DOPA accumulation in the SME or plasma PRL levels. In nonpregnant rats ovariectomized for 24 h, 5-HT decreased DOPA accumulation in the SME to 43% of vehicle-infused controls and increased PRL levels approximately 26-fold. However, in nonpregnant rats with rat choriocarcinoma cells, 5-HT produced no changes in either DOPA accumulation in the SME or in circulating PRL levels. The inability of 5-HT to reduce tyrosine hydroxylase activity after mid-pregnancy may account for the lack of a PRL response. Placental lactogens secreted at midpregnancy, particularly placental lactogen-1, may induce this loss of 5-HT effect.

A trophoblast-specific factor(s) suppresses circulating prolactin levels and increases tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.

Rat choriocarcinoma (Rcho) cells, which are morphologically similar to trophoblast giant cells of the normal placenta and produce placental lactogen-I in vivo, were used to investigate placental feedback on PRL secretion and tuberoinfundibular dopaminergic neuronal activity. Rcho cells were injected into female rats either intracerebroventricularly 60-65 h before use or under the kidney capsule 10-14 days before use. The following endocrine conditions were used: 1) ovariectomized rats with or without bromocriptine treatment, 2) immature (40-44 days old) rats, 3) adult cycling (diestrous) rats, and 4) pregnant rats. Serum PRL levels in ovariectomized, diestrous, and immature female rats were suppressed to less than 20% of control levels by secretions from the Rcho cells. Tyrosine hydroxylase (TH) activity in the stalk-median eminence (SME) was increased 2-fold above control activity in Rcho-treated ovariectomized and immature female rats. When TH activity was reduced to 40% of control levels by 50 h of bromocriptine treatment, secretions from Rcho cells increased TH activity 3.5-fold to levels similar to those for Rcho alone. Even though Rcho treatment suppressed PRL levels, TH activity in the SME of cycling (diestrous) rats was not altered after either central (65 h) or peripheral (12 days) administration of cells. TH mRNA levels in the arcuate nuclei were unaltered by Rcho cells in immature female and adult cycling rats. TH mRNA levels in ovariectomized rats were markedly reduced 75% by 50 h of bromocriptine treatment and modestly reduced 33% 65 h after injection of Rcho cells. However, Rcho cells partially reversed the bromocriptine-induced decline in TH mRNA to levels seen for Rcho cells alone. On day 7 of pregnancy, secretions from Rcho cells abolished the nocturnal and diurnal PRL surges characteristic of early pregnancy and suppressed circulating PRL levels throughout the day to less than 20% of intersurge PRL levels. Rcho cells eliminated the semicircadian rhythm in TH activity in the SME, which was out of phase with the twice daily PRL surges of early pregnancy. TH activity was increased by Rcho factor(s) at 0330 h (nocturnal surge) and 1800 h (diurnal surge), but not at 1000 h (intersurge). MMQ cells, pituitary-derived clonal PRL-secreting cells, similarly terminated the biphasic rhythm of PRL release and tuberoinfundibular dopaminergic neuronal activity during early pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Late pregnancy and rat choriocarcinoma cells inhibit nocturnal prolactin surges and serotonin-induced prolactin release.

The purpose of the present study was to determine the effect of hormonal secretion by trophoblast cells on serotonin-induced release of PRL in both pregnant and nonpregnant rats. In the first experiment, three compounds that effectively lead to stimulation of serotonin receptors were injected ip or intraarterially between 0900-1200 h on day 8 or 16 of pregnancy. These included the serotonin precursor 5-hydroxytryptophan (20 mg/kg BW); a releasor of serotonin, fenfluramine (10 mg/kg BW); and a serotonin S2 receptor agonist, DOI (2,5-dimethoxy-4-iodophenyl-2-aminopropane-HCl; 500 micrograms/kg BW). When injected on day 8, these treatments significantly (P less than 0.01) increased the level of plasma PRL within 30 min after the injection. However, on day 16 the same treatments could not induce any change in the plasma PRL level. In the second experiment, rat choriocarcinoma (Rcho) cells, which secrete placental lactogen I in vivo, were injected beneath the kidney capsule on day 1 of pregnancy. Control pregnant rats injected with the cell culture medium RPMI-1640 containing 20% FBS continued to have a nocturnal PRL surges on days 7, 8, and 9, with the peak value of plasma PRL occurring at 0400 h. Rats injected with the cells had Rcho tumors at the site of injection when analyzed on day 9. These rats also had significantly (P less than 0.05) reduced nocturnal PRL surges on days 7 and 8 of pregnancy compared to the control animals, and on day 9, the PRL surge was completely blocked. In another group of day 9 pregnant rats containing Rcho tumors, DOI-induced PRL release was blocked by Rcho cells, whereas in controls, plasma PRL increased from 5 to 47 ng/ml. The final experiment tested whether the presence of Rcho cells affected serotonergic- or TRH (1 microgram/rat)-induced PRL release in cyclic rats that were ovariectomized 1 day before drug injection. Injection of Rcho cells 8 days earlier completely inhibited 5-hydroxytryptophan- or DOI-induced PRL release, but did not affect TRH-induced PRL release. These results indicate that the absence of PRL surges after midpregnancy may be due in part to the inability of serotonin to stimulate PRL at this time compared to early pregnancy. Secretion of placental lactogens or other PRL-like peptides from the placenta in the pregnant rat may be antagonistic to the normal stimuli that cause the PRL surges of early pregnancy, resulting in a loss of surges.

Effects of progesterone on concentrations of monoamines in hypothalamic areas and plasma prolactin levels in rats.

The role of progesterone to increase prolactin (PRL) secretion on the first estrous day in pubertal rats was compared with its role in adult cyclic rats. The first estrus was induced by the administration of pregnant mare serum gonadotrophin (5 IU) at 28 days of age. A subcutaneous administration of 2.5 or 7.5 mg of progesterone/100 g body wt significantly increased the concentration of plasma PRL in pubertal rats within 4 hr. The PRL level obtained after progesterone administration was greater than that in similarly treated adult rats. The concentration of dopamine in the arcuate nucleus-median eminence (ARC-ME) in pubertal rats significantly decreased after a lower dosage of progesterone was administered, but no change was found in the preoptic area concentration. In adult estrous rats, the concentration of dopamine in the ARC-ME showed a tendency to decrease after the administration of a larger dose of progesterone (7.5 mg/100 g body wt). No change was observed in the concentrations of indoleamines in the preoptic area and ARC-ME after the administration of progesterone in both pubertal and adult rats. The concentrations of dopamine in the preoptic area and ARC-ME were lower in pubertal rats than in adults. The concentration of 5-hydroxyindole acetic acid and the ratio of 5-hydroxyindole acetic acid to 5-hydroxytryptamine in the ARC-ME were higher in pubertal rats than in adults. These results indicate that progesterone causes a greater increase in tonic PRL secretion in pubertal rats than in adult rats and that a lower hypothalamic dopamine activity and a higher serotonin activity in pubertal rats may account for these differences.

Plasma progesterone concentrations and length of the first spontaneous oestrous cycle in pubertal rats.

The length of the first spontaneous oestrous cycle in pubertal Wistar-Imamichi strain rats determined by vaginal smears varied from 5 to 18 days. The variation was ascribed to the period (3-16 days) of the stage of vaginal smears consisting of leucocytic cells (L stage). Plasma progesterone concentration and the decidual reaction in the uterus were used as indicators of the function of the corpus luteum and the L stage period was categorized as short, lasting for 3-6 days (average 4 days) with non-functional corpora lutea, or long, lasting 9-16 days (average 12 days) and with functional corpora lutea. Rats with the long L stage showed nocturnal and diurnal prolactin surges, but no daily changes in prolactin values were observed in rats with a short L stage. Daily changes in prolactin concentrations were maintained by the administration of progesterone in rats ovariectomized on Day 6 of the L stage. Plasma progesterone values on Day 6 of the L stage decreased with ergocornine treatment on Days 4 and 5 of the L stage and administration of bovine prolactin restored the level. These results indicate that the L stage observed in the first oestrous cycle is maintained by a positive feedback relation between progesterone and prolactin secretions.

Positive feedback relationship between progesterone and daily change of prolactin secretion that develops functional corpora lutea in PMSG-induced first oestrous cycle in prepubertal rats.

1. Mechanisms regulating the appearance of the pseudopregnancy (PSP)-like dioestrus induced by pregnant mare serum gonadotrophin (PMSG, 5 i.u.) treatment in prepubertal rats were studied. 2. The appearance rate of PSP-like dioestrus in the PMSG-induced first oestrous cycle was low in rats treated at 22 days of age. All rats treated at 28 or 31 days of age showed PSP-like dioestrus. 3. The daily change in plasma prolactin levels observed in rats treated at 28 days of age disappeared after ovariectomy. The implantation of Silastic tubings containing progesterone at ovariectomy maintained the change. 4. These results indicate that the PSP-like dioestrus can be induced by the positive feedback relationship which is established between progesterone and prolactin by 28 days of age.

Development of progesterone dependency in the appearance of the nocturnal prolactin surge in immature rats.

Basal concentrations of plasma prolactin in immature, Wistar-Imamichi strain rats at 25, 28 and 31 days of age were 5-12 ng/ml and no prolactin surges were observed in intact immature rats. Plasma progesterone values ranged from 5 to 9 ng/ml, while plasma oestradiol concentrations increased from 11 to 27 pg/ml between 25 and 31 days of age. When oestradiol was administered to ovariectomized 25- or 28-day-old rats by s.c. insertion of an implant, plasma prolactin concentrations at 05:00 and 12:00 h were similarly elevated 3 days after the operation. Oestradiol did not induce a nocturnal prolactin surge. The progesterone implants in ovariectomized rats at 28 days of age or on the first day of oestrus increased plasma prolactin values at 05:00 h. The magnitude of the progesterone-induced prolactin surge was greater when progesterone was given closer to the time of the first ovulation (about 34 days old). Pretreatment with oestradiol amplified the progesterone-induced prolactin surge. Mechanisms causing nocturnal prolactin surges are more sensitive to, and respond over a longer time period, to progesterone in pubertal rats than in adult animals. The results suggest that progesterone initiates the nocturnal surge of prolactin release and that oestradiol can amplify the effects of progesterone.

Enhancement of the response of hen granulosa cells to LH with norepinephrine in vitro.

1. The effect of norepinephrine (NE) on progesterone production in hen granulosa cells was examined. After 4 days of the culture, the progesterone production was stimulated by chicken LH but not by NE during a 4 hr incubation. 2. The LH-stimulation progesterone production was not affected by the presence of NE when given together with LH, but was significantly increased when the cells were precultured with NE for 2 days. 3. The effect of NE was inhibited by phentolamine, but not by propranolol. 4. The results indicate that NE may enhance the responsiveness of the hen granulosa cell to LH through alpha-adrenergic receptors.

Changes in catecholamine contents in ovarian follicular theca, progesterone contents in granulosa, and LH concentrations in peripheral plasma during the ovulatory cycle in the hen.

By the use of high-performance liquid chromatography, the content of norepinephrine (NE) and epinephrine (E) in the theca of the largest and the second largest preovulatory follicle of the ovary of the hen was found to increase from 15 h (NE) or 18 h (E) to reach a peak 9 h (NE) or 9-6 h (E) before ovulation of the largest follicle. The content of progesterone in the granulosa of these follicles and the concentration of LH in the peripheral plasma of the same hen, both of which were measured by radioimmunoassays, showed a peak 6 h before ovulation. The results suggest that the catecholamines are involved in some events occurring in both follicles some several hours before ovulation of the largest follicle during the ovulatory cycle.

Changes in body weight, milk production, food and water consumptions and vaginal smears in rats during prolonged lactation.

Concurrent observations were made in the rat on the changes in the milk production, body and organ weight, food and water consumptions and in the ovarian function in two separate series of experiments in which the period of lactation was prolonged to 60 and 45 days, respectively, by replacing suckling pups by younger ones. In most of the variable examined, marked changes occured between day 20 and 30 of lactation, that is, at the stage correspondong to the end of the normal lactation period. Milk production rate during prolonged lactation expressed by daily gain in weight of litters decreased to 60% of the level before day 20, and was associated with a great reduction of nucleic acid contents of the mammary gland. Definite decreases in weight of the anterior pituitary and the adrenal glands were observed. The food intake remained constant from day 15 of lactation onwards, therefore, the feed efficiency for milk production declined gradually during the period of normal lactatation remaining at a low level thereafter.. After the first recurrence of vaginal estrus which also occured between day 20 and 30, the replacement of litters was followed by the appearance of estrus, whereas replacement before day 15 did not affect the ovarian function. In addtition to these changes, a depressing effect of estrus on the milk production was observed.

Duration of diestrous period and secretion of progestins during prolonged lactation in the rat.

The duration of the diestrous period in vaginal smears and the levels of progestins in ovarian venous blood were investigated in rats whose lactation period was prolonged by replacing pups at appropriate intervals. In prolonged lactating rats, the first diestrous period after the pospartum ovulation (21.7 +/- 0.8 days) was similar to that occurring in normal lactating rats. Subsequently proestrous or estrous vaginal smears reappeared at intervals during prolonged lactation. A total of 19 rats were studied during this period of prolonged lactation and 44 diestrous periods were observed. Forty-eight % of these periods ranged in length from 11 to 16 days (mean of 12.7). Very short diestrous periods lasting less than 3 days were observed 25% of the time. Diestrous periods lasting more than 17 days were observed only infrequently (7%). Progesterone concentration in the ovarian vein during the 2nd diestrous period reached a peak 4 days after the 1st estrus and the level was maintained until 11 days after the 1st estrus. The level of 20alpha-hydroxypregn-4-en-3-one remained fairly constant throughout the 2nd diestrous period.

Suppression of progesterone secretion in lactating rats by administration of ergocornine and the effect of prolactin replacement.

In order to assess the stimulatory effect of prolactin on the secretion of progesterone from corpora lutea in lactating rats, ergocornine meleate (ECO; 1 mg/day), an inhibitor of prolactin secretion, was administered subcutaneously on days 6 and 7 of lactation in primiparous rats. By day 8 of lactation, the concentration of progesterone in ovarian venous blood fell to a undetectable level in the ECO-treated animals, while the concentration in the control animals was very high at this stage of lactation. The level of 20-alpha-hydroxypregn-4-en-3-one was significantly higher on day 8 of lactation in ECO-treated than in control rats. Lactational dioestrus was interrupted by treatment with ECO and vaginal oestrus appeared 3-4 days after the start of treatment. Administration of ECO caused deleterious depression of milk production and of food intake of mother rats. In the pair-fed control animals, lactation continued almost normally throughout the experimental period. Prolactin (1 mg/day) administered simultaneously with ECO increased progesterone to levels even higher than those in control rats and restored 20-alpha-hydroxypregn-4-en-3-oen levels to those of the controls. The effect of the drug on milk production was alleviated. The results strongly suggest that prolactin is the most important factor in maintaining the function of corpora lutea in the lactating rats.