Pituitary response of prepuberal lambs to oestradiol-17 beta.

In two experiments 48 prepuberal Merino ewe lambs were injected with oestradiol-17 beta (E2) or saline to study the effect of E2 on their plasma LH levels and on oestrus and ovulation. In the three groups which received 30 (experiment I), 50 and 30 (experiment II) microgram E2 respectively, 27 out of 28 lambs showed an LH response, the corresponding mean LH peaks being 64.3 +/0 22.5, 153.6 +/-33.4 and 91.7 +/- 16.9 ng/ml at mean intervals of 11.1, 11.2 and 10.5 h, respectively, after injection. None of the 20 lambs in the control groups had an LH level higher than 18 ng/ml 12 h after injection. In the three E2 groups, 41.7, 62.5 and 37.5% of animals showed oestrus within 26 h of injection while in the control groups only one animal showed oestrus. Of 13 animals showing oestrus in the E2 groups, 11 failed to ovulate. The mean pre-injection plasma FSH level in experiment I was 102.7 ng/ml, and in four 5--7-month-old lambs over several weeks uas 155.3 ng/ml. Despite these high pre-injection levels of FSH, it appears that the follicles were unable to respond to the LH peak which followed the E2 injection.     

Active immunization of the cow against oestradiol-17beta

Six beef heifers were immunized over a 4-month period with an oestradiol-17beta-BSA conjugate in Freund's adjuvant. There was an interference with oestrus in the treated heifers; 2 ceased to exhibit oestrus, one exhibited one oestrus and three exhibited oestrus after Day 47 of treatment. The control heifers treated with Freund's adjuvant had normal oestrous cycles. The antiserum titre rose in all treated heifers and attained its highest level in the 2 animals in which oestrus did not recur. The temporal changes in plasma LH, progesterone and oestradiol were normal during the pretreatment period, but became abnormal during the 120 days after immunization. Although plasma oestradiol-17beta rose at the expected time of oestrus after treatment, it was apparently effectively neutralized by the antiserum induced by treatment as evidenced by the absence of an LH surge. Plasma progesterone levels fell to baseline and remained low, indicating lack of formation of corpora lutea.     

Metabolism of oestradiol-17 beta and progesterone in the white rhinoceros (Ceratotherium simum simum)

14C-Labelled oestradiol-17 beta and progesterone (50 mu Ci each) were injected i.v. into an adult female white rhinoceros and all urine and faeces collected separately over the next 4 days. The total recovery of injected label was 61%, 25% being present in the urine and 36% in the faeces. Of the radioactivity recovered, 69% was excreted on Day 2 of the collection period. Repeated extraction of samples obtained on Day 2 showed that, of the radioactivity in faeces, 92.4% was associated with unconjugated steroids whereas in the urine the proportion of conjugated and unconjugated steroids were similar (41.2% and 51.4% respectively). After phenolic separation of urinary steroids, HPLC followed by derivatization and recrystallization techniques identified progesterone as the major component of the unconjugated portion with 4-pregnen-20 alpha-ol-3-one as the principal metabolite in the conjugated fraction. Pregnanediol was not present. Oestrone appeared to be the most abundant oestrogen metabolite with smaller but significant amounts of oestradiol-17 beta and oestradiol-17 alpha in the unconjugated and conjugated fractions respectively. Small amounts of progesterone were found in the faecal extract in which the radioactivity consisted mainly of oestradiol-17 alpha and oestradiol-17 beta. The results have established the major excreted metabolites of oestradiol-17 beta and progesterone in the white rhinoceros and the development of more appropriate assay methods for monitoring ovarian function in African rhinoceroses should now be possible.     

Progesterone, oestradiol-17 beta and LH during the oestrous cycle of muskoxen (Ovibos moschatus)

Progesterone, oestradiol-17 beta and LH were measured in plasma from 6 non-pregnant, captive, female muskoxen during the 1984 and 1985 breeding seasons. Jugular blood samples were taken on an alternating 3/4-day schedule in 1984 and daily or at 4-h intervals over oestrus, via indwelling jugular cannulae, for 6 weeks in 1985. Oestrous cycle length was 19.6 +/- 0.96 (s.d.) days (n = 19) and did not vary between the first and subsequent cycles of the season. Progesterone was lowest at oestrus (less than or equal to 0.1 ng/ml), began to rise on Days 4-5, peaked on Days 10-12 (mean = 2.6 ng/ml) and returned to baseline 2-5 days before the next oestrus. A small rise in progesterone before the first cycle of the breeding season was observed on 7 of 12 occasions. Oestradiol-17 beta was significantly higher (P less than 0.001) 1-4 days before, or coincident with, oestrus. The average duration of the LH peak was 24.6 h (n = 7) and coincided with observations of behavioural oestrus. In one animal behavioural oestrus and an LH peak preceded a small progesterone rise at the beginning of the breeding season. The temporal relationship of these three hormones during the muskox oestrous cycle is very similar to that seen in domestic ruminants.     

Plasma levels of oestradiol-17 beta in the pre-puberal heifer treated to induce superovulation

The plasma concentrations of oestradiol-17β have been measured by radioimmunoassay in pre-puberal calves following treatments used to induce superovulation (PMSG/HCG and PMSG/FGA/HCG). Before treatment, in almost all animals, the concentrations of oestradiol-17β were different from zero (2 to 8 pg/ml). The highest concentrations were measured around 130 h. after the beginning of treatment, before ovulation (150 to 2050 pg/ml). The curves showing the changes in hormonal levels have the same form as those of follicular growth measured using morphological criteria. The two hormonal treatments resulted in similar oestradiol-17β concentrations.     

Morphogenic change in the cervix of the ewe after prolonged exposure to oestradiol-17 beta

This study examined the effects of prolonged exposure to oestradiol-17 beta on the morphology of the cervix of the ewe. Merino ewe lambs were implanted subcutaneously with 3 Silastic capsules which released a total of approximately 300 micrograms oestradiol-17 beta per day. After exposure for 200 days the uterus was more markedly bicornuate, and the cervix was broader and softer, than in controls. The cervical folds were shorter and contained many stromal cells. The amount of lamina propria under the folds was increased and altered so that it contained tubular glands and more stromal cells. The endocervix thus came to resemble endometrium. This appearance developed within 80 days of exposure, and remained for at least 170 days after implant removal. In a second experiment, mature multiparous Merino ewes were ovariectomized and implanted with 1, 2 or 4 similar oestradiol capsules for 140 days. Similar features developed in these ewes, and the degree of change was almost as great with 1 implant as with 4. Changes of a similar nature can be produced in other species by oestrogen given during organogenesis but not during adult life. The changes indicate that the ewe has an ability to display a degree of morphogenic response to oestradiol during adult life.     

Nature and characteristics of the binding of oestradiol-17beta to a uterine macromolecular fraction

The binding of oestradiol-17β to two proteins, namely serum albumin and a uterus fraction, was studied in vitro. The former protein has a physiological function in the transport of the hormone and the latter is involved in the selective uptake of the steroid by the target organ. The uterus fraction shows a high degree of stereospecificity for the binding of the steroid. Cortisone, oestradiol-17α and testosterone are bound negligibly and progesterone to a much smaller extent than is oestradiol-17β. This property is in contrast with the wide variety of ligands bound by the serum albumin. The temperature and the presence of the steroid influence markedly the binding properties. Oestradiol binding to the uterus fraction is optimum at 37° and at pH7–8·5. It is markedly decreased at pH values above or below this range, suggesting stringent conformational requirements. The tissue `receptor' protein is a macromolecule with a minimum molecular weight of 100000. The protein moiety is essential for the binding function. The probable concentration of the total binding sites for oestradiol in the ovariectomized-rat uterus cytoplasmic fraction as determined in vitro is about 1mμm at a steroid concentration of 50mμm.     

Progesterone and oestradiol-17 beta concentration profiles throughout the reproductive cycle in harbour seals (Phoca vitulina)

Serum samples of harbour seals kept in captivity were analysed for progesterone and oestradiol-17 beta. The hormone profiles obtained were used to describe a complete reproductive cycle. A clear peak in oestradiol values, indicative of ovulation, was followed by elevated concentrations of progesterone. Implantation probably occurred 3-3.5 months thereafter. Progesterone concentrations rose significantly in the last 3-4 months of gestation, whereas oestradiol concentrations gradually increased after implantation. Lactational oestrus was marked by a peak of oestradiol on average 25 days after parturition and lactation lasted 4-5 weeks. Previous pregnancy had a marked influence on the timing of oestrus; females with offspring started a new reproductive cycle about 14 days later than previously non-pregnant seals. No differences in timing of parturition between the 2 groups were observed. This was probably the result of a flexible period of delayed implantation.     

Protein synthesis in the uterus of the immature rat: the effects of short exposures to oestradiol-17 beta

Three methods were used to study the effect of oestrogen on the incorporation of radioactive precursor into uterine protein. Intact tissue was incubated in vitro. Isolated uterine epithelial, stromal and myometrial cells were labelled in vitro. Isolated polysomes were translated in cell free protein synthesising systems. In all of these systems, minor qualitative changes in protein synthesis were detected where the uteri were derived from oestrogen-treated rats. These changes were most dramatic in isolated stromal cells and were accompanied by a marked overall increase in protein synthesis. The translation of mRNA on isolated uterine polysomes revealed a sequence of minor, but reproducible, oestradiol-induced changes. It was difficult, however, to relate these changes to those detected in incubated tissue or cells, possibly because the cell free translation products were not subject to normal post-translational modification and processing.     

Response of ovariectomized ewes to injection of oestradiol-17 beta at different times of the year

Long-term ovariectomized Clun Forest ewes were challenged with a range of doses (12.5-50.0 micrograms/injection) of oestradiol benzoate every 2 months from March to November. All treatments induced a biphasic pattern of change in LH concentrations, consisting of an initial depression in concentrations followed by an LH peak, similar to a preovulatory LH surge. The positive feedback response to 12.5 micrograms oestradiol was significantly lower than that after the two higher dose levels, but the magnitude of the response showed no significant seasonal variation. It is concluded that a seasonal change in responsiveness to positive feedback is unlikely to contribute to the absence of ovulation during seasonal anoestrus.     

Influence of progesterone and oestradiol-17 beta on blastocysts of the tammar wallaby (Macropus eugenii) during seasonal diapause

Forty tammar wallabies, presumed to be carrying quiescent blastocysts, were injected with progesterone and oestradiol alone, or in combination, during seasonal quiescence when the corpus luteum is inactive. Plasma progesterone concentrations were increased to values equivalent to those of late pregnancy for the duration of the treatment in progesterone-treated groups but otherwise remained at values equivalent to seasonal quiescence. Tammars treated with low doses of oestradiol showed no measurable increase in plasma oestradiol concentrations but in those treated with high doses plasma concentrations were increased to oestrous levels. At autopsy on Day 18 after the start of treatment the embryos and reproductive tracts were assessed. While progesterone alone caused reactivation of about 50% of the embryos, blastocysts in tammars treated with oestradiol alone remained in diapause (low dose) or disappeared from the uterus (high dose): 2 blastocysts collapsed after some slight expansion. No synergistic effect on pregnancy was noted in tammars receiving both oestradiol and progesterone. We conclude that oestrogen alone is not capable of stimulating normal growth of blastocysts, and its role during early pregnancy in tammars remains unclear.     

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.