Progesterone and oestrogen receptors in the female genital tract throughout pregnancy in tammar wallabies

The tammar, Macropus eugenii, is a monovular macropodid marsupial which has a post-partum oestrus and an 11 month embryonic diapause. Progesterone and oestradiol cytosol receptors were measured by Scatchard analyses and single point analysis in the lateral vagina, endometrium and myometrium of the gravid and contralateral non-gravid uterus throughout pregnancy, immediately after parturition and during seasonal reproductive quiescence. In endometrial tissues, both progesterone and oestradiol receptors doubled in concentration in both gravid and non-gravid uteri between day 0 and day 5 of pregnancy, coinciding with previously described peak values in peripheral plasma progesterone and oestrogen. Receptor concentrations in endometrial tissue during seasonal quiescence were not significantly different from those immediately after reactivation. After day 12 of pregnancy, downregulation of both progesterone and oestradiol cytosolic receptors occurred concomitant with the increase in progesterone in the peripheral plasma. However, there was a unilateral increase in oestradiol receptor concentrations in endometrium obtained from the non-gravid uterus between day 25 of the 26.5 day gestation and immediately after parturition. Myometrial receptor concentrations mirrored those of the endometrium but were lower. Concentrations of progesterone receptor in the lateral vaginae were at the lower limit of detection, while the oestradiol cytosol receptor concentrations were even lower in this tissue. Thus, the steroid receptor concentrations provide another example of local unilateral endocrine responses in the reproductive tract of the tammar. These results also indicate that the downregulation of progesterone and oestradiol receptors that occurs in both uteri in mid- and late-pregnancy is selectively and locally reversed before parturition in the non-gravid endometrium in response to the local effects of follicular oestradiol from the ipsilateral ovary.     

Effects of oestradiol-17 beta on peripheral plasma concentrations of LH and FSH in ovariectomized tammars (Macropus eugenii)

Two experiments, each using 8 animals, were conducted in the non-breeding and breeding seasons, respectively, and each animal was injected with 4 different doses of oestradiol benzoate over 4 trials. The resulting physiological concentrations of plasma oestradiol caused depression of both LH and FSH values. The highest dose elicited a biphasic response in LH with a pulse-like surge at 24 h after injection. There was no significant difference between the response of either hormone at the two times of the year and it is concluded that, in tammars, there is no seasonal difference in the responsiveness of the hypothalamus/pituitary to the negative feedback effect of oestradiol.     

Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

Effects of corpus luteum removal on progesterone, estradiol-17 beta and LH in early pregnancy of the tammar wallaby, Macropus eugenii

The quiescent corpus luteum of female tammars was reactivated by removal of the pouch young (RPY). The reactivated corpus luteum was ablated 3 days after RPY. Plasma progesterone and oestradiol concentrations were measured by radioimmunoassay in these and in sham-operated controls. Excision of the CL abolished the rise in progesterone seen at Day 5-6 in the sham-operated animals (130.7 +/- 56.6 vs 452.4 +/- 176.0 pg/ml, mean +/- s.d.). By contrast, oestradiol-17 beta values increased within 6-16 h of CL excision to 16.3 +/- 6.9 pg/ml and remained high for 1-3 days while in the sham-operated animals there were less sustained and more variable peaks of 10-20 pg/ml between Days 3 and 5 (mean 12.0 +/- 3.6 pg/ml at Day 4-5). We conclude that the early transient increase in peripheral plasma of progesterone is of luteal origin but the source of the oestradiol remains unknown.     

Uterine and embryonic metabolism after diapause in the tammar wallaby, Macropus eugenii

Pouch young were removed from lactating tammars to terminate embryonic diapause. Uterine metabolism was assessed at periods afterwards by incubating endometrial explants with [3H]leucine, and measuring the incorporation into acid-soluble material. Blastocysts were incubated with [3H]uridine to assess uptake and incorporation into acid-soluble material. Uterine reactivation, shown by an increase in the rate of leucine incorporation into secreted protein, was evident by Day 4 after removal of pouch young and was significantly more in both secreted and tissue protein by Day 6. Both continued to increase in gravid and non-gravid uteri up to Day 12. By the end of pregnancy (Day 26) uterine metabolism in the gravid uterus produced 2-3 times more secreted protein than in the non-gravid uterus, demonstrating a local feto-placental influence on the uterus. Tissue incorporation had declined in endometrium of gravid and non-gravid uteri by Day 26. Day 5 embryos were metabolically more active than in quiescence, although expansion of the embryos was not seen until Day 9. The early reactivation of the uterus and embryo from diapause suggests that it is not triggered by the previously described peaks of progesterone and oestradiol in plasma at Day 5, although there may be an earlier, increased sensitivity to these steroids which allows uterine reactivation to precede changes in peripheral plasma concentration.     

Effects of removal in late pregnancy of the corpus luteum, graafian follicle or ovaries on plasma progesterone, oestradiol, LH, parturition and post-partum oestrus in the tammar wallaby, Macropus eugenii

Concentrations of oestradiol-17 beta, progesterone, and luteinizing hormone (LH) were measured in plasma collected at 6- to 12-h intervals from tammars around the time of parturition and post-partum oestrus. Parturition occurred on Day 26 or 27 after reactivation of lactation-delayed pregnancy and coincided with a precipitous decline in progesterone levels. A sharp rise in oestradiol, from basal concentrations of less than 10 pg/ml to a peak of 13 to 32 pg/ml, as well as oestrus, followed the drop in progesterone by 8.3 and 9.8 h, respectively. The LH surge was dependent on the oestradiol rise and followed it by 7 h. Ovulation followed mating by about 30 h and the LH surge by 24 h. Removal of the ovary with the large Graafian follicle prevented the oestradiol rise, oestrus and the LH surge, but not parturition. Peripartum changes in peripheral oestradiol do not appear to be involved in initiation of parturition but the oestradiol rise and associated change in the oestradiol:progesterone ratio are important signals for post-partum oestrus and the LH surge.     

Fetal role in the control of parturition in the tammar, Macropus eugenii

When female tammars carrying dormant blastocysts were injected with progesterone at the time of removal of their pouch young the development of the fetus was advanced and parturition occurred 5 days earlier than in the control tammars. In these tammars the prolactin pulse was also advanced by 5 days but the usually concomitant fall in progesterone was not. In non-pregnant tammars similar injections of progesterone did not advance the subsequent fall in progesterone, oestrus, or the LH pulse. In non-pregnant tammars injected with ovine prolactin on Day 26, to mimic the prolactin pulse, plasma progesterone was reduced to basal levels within 12 h, significantly earlier than in controls. Conversely, in 5 pregnant and 1 non-pregnant tammar injected with ovine prolactin on Day 23, to mimic the condition induced by advancing the time of parturition with progesterone, the decline in plasma progesterone was not advanced and the endogenous prolactin pulse, parturition, post-partum ovulation and the LH pulse all occurred after intervals similar to those of controls. The results support the view that the fetus is associated with the pre-partum prolactin pulse in maternal plasma and that a prolactin pulse at this stage is luteolytic in non-pregnant tammars.     

Patterns of plasma progesterone, androgen and oestrogen concentrations and in-vitro ovarian steroidogenesis during embryonic diapause and implantation in the mink (Mustela vison)

Peripheral plasma progesterone concentrations exhibited an increase 10 days before implantation, coinciding with the resumption of blastocyst growth and with a decrease in plasma androgen values (DHA, androstenedione, testosterone). No definite pattern of oestrone was observed and oestradiol concentrations remained undetectable. The production of steroids by dispersed luteal cells showed that the growth of the corpora lutea paralleled that of blastocysts and resulted in hypertrophy followed by hyperplasia of the luteal cell. The production of progesterone in the medium increased with blastocyst size up to implantation; it was enhanced by mink charcoal-treated serum, but prolactin, LH, FSH or a combination of these hormones did not affect the progesterone production, whatever the stage of diapause. DHA and androstenedione secretion increased in the two last stages of blastocyst growth and was enhanced by LH. The conversion of androstenedione and testosterone into oestrone and oestradiol was observed at all stages of embryonic diapause, indicating that corpora lutea contain aromatase activity even at an early stage. The secretion of oestrone was higher than that of oestradiol. The non-luteal tissue contributed up to 50% of the steroid production; while progesterone and androgen production remained constant, that of oestradiol decreased at the end of the delay period. These results indicated a change in the size and the secretory capacity of the luteal cell related to blastocyst development and implantation. Although progesterone was the main product of the corpora lutea, androgens and oestrogens were also secreted.     

Effects of progesterone and oestradiol on RNA and protein metabolism in the genital tract and on survival of embryos in the ovariectomized ewe.

The hormonal regulation of metabolism in the genital tract and the development of embryos during early pregnancy in the ewe have been examined. Ovariectomized ewes received injections of maintenance progesterone, oestrous oestradiol and priming progesterone according to schedules designed to simulate endogenous ovarian secretion during early pregnancy, around the time of oestrus and during the luteal phase of the oestrous cycle immediately preceding oestrus. The survival and development of embryos was dependent upon the dose of maintenaince progesterone and the duration of treatment at the time of transfer, but changes in progesterone dose did not change endometrial protein or RNA metabolism on particular days. Both priming progesterone and oestrous oestradiol were required for normal embryo development. Priming progesterone and oestrous oestradiol each increased endometrial RNA/DNA ratios during early pregnancy. There were no interactions between priming progesterone and oestrous oestradiol, their effects being simply additive. Neither maintenance nor priming progesterone had any effect on protein and RNA metabolism in the oviduct. It is suggested that in the intact ewe oestrogen secreted at oestrus and progesterone secreted prior to oestrus play important roles in the establishment of a uterine environment suitable for the subsequent normal development of embryos.     

Effects of prostaglandin and prolactin on luteolysis and parturient behaviour in the non-pregnant tammar, Macropus eugenii

In Exp. 1 non-pregnant female tammars were injected, on Day 26 (the day parturition would normally occur) after removal of pouch young, with saline, 200 micrograms ovine prolactin or 5 mg PG and changes in plasma concentrations of progesterone, prolactin, PGF-2 alpha metabolite (PGFM), oestradiol-17 beta and LH were determined. Luteolysis occurred in females treated with prolactin alone, while treatment with PG first induced a rapid rise in prolactin and subsequently a significant decrease in plasma progesterone. After prolactin treatment the oestradiol peak, oestrus and the LH surge were advanced significantly compared to the saline-treated females. In Exp. 2 the effects of the same treatments as used in Exp. 1 were determined on Day 23 and again on Day 26 after removal of pouch young in non-pregnant females. On Day 23 both prolactin and PG induced significant elevations in plasma progesterone, but luteolysis did not occur. On Day 26 the treatments initially induced significant elevations in plasma progesterone but these were followed by luteolysis within 8-12 h after treatment. PG treatment induced parturient behaviour in the non-pregnant females within 3-21 min and this persisted during the period that plasma concentrations of PGFM were elevated. The results show that PG induces birth behaviour and the release of prolactin, while prolactin first induces an elevation of plasma progesterone concentrations and, in the mature CL on Day 26, subsequently induces luteolysis.     

Effects of oestradiol-17 beta and progesterone on the number of plasma cells in uteri of ovariectomized mice

Immunoglobulins A and G were localized by immunoperoxidase labelling in uteri of ovariectomized mice treated with oestradiol-17 beta and progesterone. The administration of oestradiol or progesterone alone to ovariectomized mice for 3 days increased the number of IgA plasma cells from about 1 to 14 per histological section. When the two hormones were administered simultaneously for 3 days the number of plasma cells per section was equal to or greater than with either hormone alone. Treatment with oestradiol followed by progesterone in a sequence that prepares the uterus for implantation resulted in about 31 IgA plasma cells per section. Counts of IgG plasma cells showed similar trends but the numbers were smaller. The results indicate that progesterone increases rather than decreases the number of plasma cells in the mouse uterus. This is consistent with observations on intact mice during oestrus and pregnancy and suggests that the marked increase in endometrial plasma cells at the time of implantation in mice is a response to progesterone acting on an oestrogen-primed uterus.     

Peripheral plasma levels of oestradiol-17 beta and progesterone in the bitch during the oestrous cycle, in normal pregnancy and after dexamethasone treatment.

Plasma oestradiol-17 beta concentrations in Labradors increased during pro-oestrus to an average maximal concentration of of 79-7 +/- 10-9 (S.D.) pg/ml, and then fell rapidly. In 6/7 bitches the peak occurred within 1 day of oestrus. No consistent changes in plasma oestradiol levels were observed during pregnancy and at parturition and the values were similar to those in late anoestrus. Plasma progesterone levels did not increase markedly during pro-oestrus. At oestrus, progesterone values rose and maximal concentrations, which varied from about 20 to about 55 ng/ml, were reached within a few days of the oestradiol peak. Plasma progesterone decreased in late pregnancy and in one of the three bitches studied in detail low or undetectable levels were reached 10 days before parturition. In the other two bitches an abrupt decrease in progesterone occurred just before parturition. Dexamethasone treatment (2 X 5 mg daily for 10 days) from Day 30 of pregnancy resulted in intrauterine death and resorption of the fetuses in the two bitches studied. Treatment from about Day 45 resulted in the birth of dead fetuses at Days 55 and 59 of pregnancy. The changes in plasma oestradiol levels were very small. No changes in plasma progesterone levels were seen when dexamethasone was given in late pregnancy, but an accelerated decline occurred after treatment in mid-pregnancy.     

Effect of indomethacin on plasma levels of vitamin D metabolites, oestradiol and progesterone in rabbits during early pregnancy

1. Inhibition of prostaglandin (PG) synthesis by indomethacin (Id) during early pregnancy in rabbits apparently disrupts the process of sex steroid production by the ovaries. 2. The role of PGs as mediators in steroidogenesis was tested by investigating the effect of Id alone or in combination with progesterone, with oestradiol and progesterone, or with a mixture of PGs, on plasma levels of 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) in rabbits at an early stage of pregnancy. 3. Id alone significantly reduced plasma levels of both 25(OH) D and 1,25(OH)2D. Treatment with Id in combination with either oestradiol and progesterone, or with a mixture of PGs, resulted in the restoration of plasma levels of both vitamin D metabolites as well as the restoration of plasma levels of progesterone, to their control values.     

Direct effects of varying doses of oestradiol on early embryonic development in in vitro culture of rat's two-cell embryos

Hyperstimulation in the rat using pregnant mares' serum gonadotrophin (PMSG) has been known to cause death in pre-implantation embryos, as well as enhancement of oestradiol production. This study examines the effect of oestradiol, in levels that are found in hyperstimulated pregnant rats, on pre-implantation embryonic development. Using a simplified in vitro system, 2-cell embryos retrieved from rats on the 2nd day of pregnancy were cultured in rat two-cell embryo culture medium (R2ECM) containing pharmacological doses of oestradiol for 96 h and scored daily in the morning. Three ngxmL(-1) oestradiol reduced the incidence of >8-cell embryos to morulae on the 5th day and blastocysts on the 6th day of development. Most embryos were retarded at the lower cell stages on the 5th day and degenerated by the 6th day. None of the blastocysts expanded on the last day of culture. Fifteen ngxmL(-1) oestradiol accelerated embryo development on the 3rd day but retarded development on the 4th day, and increased the incidence of degenerated embryos by the 5th and 6th day of development. These results suggest that the elevated oestradiol may constitute a mechanism by which PMSG induces death in pre-implantation rat embryos, possibly via a direct action on the embryos.     

Control of endometrial oxytocin receptor and uterine response to oxytocin by progesterone and oestradiol in the ewe

The effects of administration of progesterone and oestradiol on ovine endometrial oxytocin receptor concentrations and plasma concentrations of 13,14-dihydro-15-keto prostaglandin F-2 alpha (PGFM) after oxytocin treatment were determined in ovariectomized ewes. Ewes received progestagen pre-treatment, progesterone and/or oestradiol in 11 different treatment schedules. Progestagen pre-treatment decreased oxytocin receptor concentrations in endometrium from ewes treated subsequently with either progesterone for 5 days or progesterone for 5 days plus oestradiol on Days 4 and 5 of progesterone treatment. Oestradiol increased endometrial oxytocin receptor concentrations when administered on Days 4 and 5 of 5 days progesterone treatment. Progestagen pre-treatment followed by progesterone treatment for 12 days caused a large increase in oxytocin receptors and no further increase occurred when ewes were given oestradiol on Days 11 and 12, or when progesterone was withdrawn on Days 11 and 12, or these two treatments were combined. Oxytocin administration caused an increase in plasma PGFM concentrations in ewes which did not receive progestagen pre-treatment, and subsequently received progesterone treatment for 5 days and oestradiol treatment on Days 4 and 5 of progesterone treatment. Similarly treated ewes which received progestagen pre-treatment did not respond to oxytocin. Oxytocin administration also increased plasma PGFM concentrations in ewes which received progestagen pre-treatment followed by progesterone treatment for 12 days, progesterone treatment for 12 days plus oestradiol on Day 11 and 12 of progesterone treatment, progesterone withdrawal on Day 11 and 12, or progesterone withdrawal and oestradiol treatment combined. The results indicate that (1) progesterone pre-treatment affects oxytocin receptor concentrations in the endometrium and uterine responsiveness to oxytocin and (2) progesterone treatment alone for 12 days after a treatment which mimics a previous luteal phase and oestrus is sufficient to induce oxytocin receptors and increase oxytocin-induced PGF release. These results emphasize the importance of progesterone and provide information which can be used to form an hypothesis for control of luteolysis and oestrous cycle length in the ewe.     

Peripheral plasma levels of progesterone, oestradiol-17 beta, oestrone, testosterone, androstenedione and chorionic gonadotrophin during pregnancy in the marmoset monkey, Callithrix jacchus.

Concentrations of LH/CG, androstenedione and testosterone rose in early pregnancy to maximum values at 6--10 weeks. Thereafter LH/CG levels declined and androstenedione and testosterone levels remained at plateau values or declined until term. Progesterone, oestradiol-17 beta and oestrone increased after ovulation and remained high throughout pregnancy. At 12 weeks, when LH/CG levels were falling, progesterone and oestradiol rose well above the luteal-phase levels which were maintained for the first 12 weeks. Progesterone declined in the 2 weeks before birth, while oestradiol and oestrone remained high. Pregnancies of an unknown stage were dated by reference to a graph of uterine diameter, measured by abdominal palpation, in animals at known times after conception. Measurement of progesterone concentrations during the conception cycle gave more accurate dating and showed that the gestation length was 144 days.     

Variability in the response of the rabbit uterus to progesterone as influenced by prolactin

Ovariectomized rabbits from different breeders were treated at different times of the year with prolactin alone or with progesterone and the production of uteroglobin by the uterus was studied. There were seasonal, strain and dose variables in the uterine response to prolactin and progesterone. Treatment with prolactin (at 1 mg/day) plus progesterone generally induced higher levels of uteroglobin production than did treatment with progesterone alone. The differences were greatest in the winter for Tennessee animals and in the spring for animals from the New Mexico and North Carolina colonies. Ovariectomy produced a decrease (P less than 0.01) in the concentration of cytosolic oestrogen and progesterone receptors, and prolactin treatment restored the concentration to oestrous control values. However, there were no seasonally dependent changes in the concentration of the receptors for any of the treatment groups. Increased doses of prolactin (2 mg/day) induced high levels of uteroglobin production and new proteins to appear in uterine secretions of long-term ovariectomized rabbits but much lower levels (10-11%) when given to pregnant does. Additional ovulations were also noted plus adverse effects on the embryos.     

Effects of medroxyprogesterone acetate on gestation in mink

Mink ovariectomized 14 days after the first of two matings received injections of 2 mg MPA, the same MPA treatment + an oestradiol-17 beta implant or no replacement therapy. Some mink were ovariectomized after implantation and given a single dose of 2 mg MPA or no replacement therapy. MPA persisted in the serum at detectable levels for 13 or more days in all mink treated. MPA and MPA + oestradiol induced uterine growth but neither treatment was capable of inducing embryo implantation. More embryos were retained in mink treated with MPA alone and these appeared to be viable. Implanted embryos persisted for a longer period in animals that were ovariectomized and treated with MPA. MPA neither supported pregnancy nor permitted parturition. Serum LH was elevated by 1 week after ovariectomy and elevations persisted for a further 20 or more days. While MPA alone had no apparent negative feedback effects on LH, animals that received MPA + oestradiol did not display any elevation of LH, suggesting that oestradiol or a combination of MPA and oestradiol has a potent negative feedback in mink.     

Plasma oestradiol and progesterone during early pregnancy in the cow and the effects of treatment with buserelin

We have monitored plasma concentrations of oestradiol and progesterone after insemination in dairy cows, and investigated the effects of injection with 10 μg of the gonadotrophin-releasing hormone (GnRH) analogue buserelin, a treatment known to result in an improvement in conception rate. Animals were injected intramuscularly on Day 12 after insemination with 2.5 ml of either control saline (n = 29) or buserelin (n = 26). Blood samples were collected from Day 8 to Day 17 and milk samples from the day of insemination until Day 30. On the basis of milk progesterone profiles, in the control group 15 cows remained pregnant (control pregnant) and 11 underwent luteolysis (control not pregnant), whereas in the treated group 13 cows remained pregnant (treated pregnant) and 13 underwent luteolysis (treated not pregnant). Three cows were excluded from the control group owing to high progesterone at insemination or failed ovulation. In both the control and treated groups mean plasma progesterone concentration was significantly (P < 0.05) lower in non-pregnant cows than in pregnant ones from Day 12 post-insemination. However, no significant effect of buserelin treatment on plasma progesterone concentration was detected. In the control group the plasma oestradiol concentration was similar in the pregnant and non-pregnant cows. In the treated group, plasma oestradiol concentration in the non-pregnant cows was similar to that in the control group, whereas in the treated pregnant cows the plasma oestradiol concentration showed a significant (P < 0.05) decline after treatment. As oestradiol is known to stimulate the development of the luteolytic mechanism at this time, we suggest that buserelin is acting to reduce the strength of the luteolytic drive in some cows, thus improving the chance of the embryo being able to prevent luteolysis.     

Effect of passive immunization against progesterone on its distribution in circulation and tissues of mice

Anti-progesterone monoclonal antibody, injected into mice 32 h after mating at a dose that blocks the establishment of pregnancy, produced a significant reduction in the concentration of progesterone in the ovary and uterus within 6 h after treatment. Uterine concentrations remained lower in treated compared with control animals for at least 24 h after injection. There was an associated transient increase in plasma LH and FSH concentrations, but there was no change in plasma prolactin values. The percentage of total progesterone in the circulation that was unbound was reduced after treatment, but the concentration of unbound progesterone was increased. Studies of antibody binding of steroid in the presence of uterine progesterone receptor protein showed that there was a stoichiometric relationship in the distribution of ligand between the two binders. The present findings suggest that the effects of passive immunization against progesterone are associated with perturbation of tissue concentrations of steroid in the target organ as a result of high antibody concentrations in the circulation.