Response of anestrous ewes to the ram effect after follicular wave synchronization with a single dose of estradiol-17beta

Anestrous ewes respond to the introduction of rams with either an ovulation within 2-3 days that may be followed by luteal phases of normal or short length, with delayed ovulations (5-6 days later), or with the luteinization of follicles. The aim of this work was to study the relationship between the growth status of the largest follicle present when rams are introduced and the type of ovarian response in non-treated ewes and in ewes treated with estradiol-17beta before ram introduction. Thirteen anestrous Corriedale ewes were divided into 2 groups: E2 (n = 7) and C (n = 6). The E2 ewes received a single dose of 50 microg estradiol-17beta 5 days before the introduction of the rams to synchronize the onset of their follicle waves, while C ewes remained untreated. When the rams were introduced, all E2 ewes had the largest follicle in a growing stage in contrast with the C ewes (3 out of 6; P < 0.05). Five C and 4 E2 ewes ovulated after the introduction of the rams (Day 3.4 +/- 0.4 for C vs. 4.8 +/- 0.3 for E2 ewes, respectively, P < 0.05). Only one ewe from each group developed a normal luteal phase: 4 C and 3 E2 ewes had short luteal phases. One C ewe and 2 E2 ewes had short luteal phases originating from follicles that did not ovulate. After the first luteal phase, all ewes returned to anesirus without a second ovulation or luteal phase. The remaining E2 ewe did not ovulate or show any changes in progesterone serum concentrations. We conclude that the growth status of the largest follicle alone does not determine the ovarian responding pattern of anestrous ewes to the ram effect.     

Ovarian follicular dynamics after cauterization of the dominant follicle in anestrous ewes

An experiment was conducted to ascertain if follicles could reach ovulatory size after the largest follicle (dominant) has been removed at different times during a progestin treatment in anestrous ewes, and secondly to determine if these new follicles could respond to an hCG-induced ovulation and have similar function as corpora lutea. Mature crossbred sheep (n=44) in anestrous were treated with an intravaginal sponge containing 40 mg of FGA (day 0=sponge insertion) for 9 days. Treatments consisted of cauterization of the largest follicle on the experimental day 3 (T1), day 6 (T2) and day 9 (T3); day 12 to ascertain the size of the largest follicle in control ewes. During laparotomies, the diameters of the largest follicle (DF), and those of the second and third largest follicles (SF1 and SF2, respectively) were determined. On day 12, a second laparotomy was performed for those ewes which had their DF cauterized on days 3, 6 and 9, a fourth group was left intact and only laparotomized on day 12. At this time, the size of the new DF, SF1 and SF2 were determined. Immediately after the laparotomy on day 12, all the ewes were treated with 1000 i.u. of hCG to induce ovulation. Blood samples were collected daily from day 0 to 50 and samples were analyzed for progesterone concentrations. The size of the DF at the time of sponge removal was smaller that those observed on day 3 or 6 of sponge suggesting that follicles in ewes treated with this progestin regress and a new wave of follicular development ensues between day 6 and the time of sponge removal. The size of the DF on day 12 was also smaller in ewes that have the largest follicle removed at the time of sponge removal reflecting that these follicles had a shorter period of growth; however, the rate of growth was greater for these follicles than for follicles arising after cauterization on day 3 or 6 after sponge insertion. There were no differences among treatments, in the number of ewes that formed a corpus luteum (CL) in response to hCG. Life span of the corpora lutea did not differ among ewes having their DF removed on day 6 or 9 or those that served as controls, however, ewes that had their DF removed on day 3 developed longer lived CL in a larger proportion of animals. Average progesterone concentration during the life span of the induced corpora lutea was greater in control ewes than in any other experimental group. These observations allow us to conclude that, (a) the follicular dynamics observed in anestrous ewes treated with a progestin intravaginal sponge resembles that observed during the normal estrous cycle in the ewe; (b) the effects of progesterone on life span of the corpus luteum could not be only related to direct effects at the follicle but also involve changes in other components of the uterine-ovarian-hypothalamic axis; (c) the mechanisms controlling luteal life span seem to be different to those mechanisms controlling the function of the induced corpus luteum.     

Local versus systemic effects of exogenous estradiol-17 beta on ovarian follicular dynamics in heifers with progestogen implants

Two experiments were designed to determine if the suppressive effect of estradiol treatment on ovarian follicles in progestogen-implanted heifers is mediated directly at the ovary or systemically, at a higher level. The purpose of Experiment 1 was to determine a minimal effective dose of estradiol-17beta (E-17beta) that would induce follicle regression in progestogen-implanted heifers. Beef heifers were implanted with progestogen on Day 2 (Day 0=ovulation) and were assigned randomly to five groups: control (sesame seed oil, n=9); 0. 1 mg of E-17beta (n=8); 0.5 mg of E-17beta (n=8); 1 mg of E-17beta (n=8); or 5 mg of E-17beta (n=8) by intramuscular (im) injection on Day 3. Treatment with 5 and 1 mg of E-17beta resulted in smaller (P<0.05) day-to-day diameter profiles of the dominant follicle compared with controls, whereas 0.1 mg of E-17beta did not have an apparent effect on follicle growth. The effect of a dose of 0.5 mg was intermediate and tended (P<0.06) to result in a smaller diameter profile of the dominant follicle compared with control heifers. Experiment 2 was designed to utilize a subminimal dose of E-17beta (0.1 mg), locally, to determine whether estradiol treatment induces follicle regression through a direct action on the ovary. Beef heifers received a progestogen ear implant on Day 2 and were assigned randomly to five groups on Day 3: control (sesame seed oil, n=8); 5 mg of E-17beta im (n=8); 0.1 mg of E-17beta im (n=8); 0.1 mg of E-17beta given into the wall of the uterus, near the tip of the horn ipsilateral to the dominant follicle (intrauterine (iu), n=8); or 0.1 mg of E-17beta given into the stroma of the ovary, immediately adjacent to the dominant follicle (intraovarian (io), n=6). Local (iu and io) treatments were given via a transvaginal ultrasound-guided needle injection. Treatment with 5 mg of E-17beta im resulted in suppression of the dominant follicle of the first follicular wave and early emergence of the second follicular wave (P<0.05). Diameter profiles of the dominant follicle in heifers treated with 0.1 mg im or 0.1 mg iu differed from those of control heifers on Day 5, whereas diameter profiles of the dominant follicle in heifers treated with 0.1 mg io did not differ from the controls. Daily changes in diameter of the dominant follicle did not differ among the three groups treated with 0.1 mg of E-17beta (im, iu and io). Hourly changes in circulating concentrations of FSH and LH were not detected following estradiol treatment either before or after the results were combined for all estradiol-treated groups. Results are supportive of the hypothesis that the suppressive effect of estradiol in cattle is exerted indirectly through a systemic route rather than directly at the ovary. Although low plasma concentrations of FSH and LH were not detected, systemic treatments with high E-17beta dosages resulted in follicular suppression whereas local treatments with subminimal dosages, within the ovary bearing the dominant follicle, were without effect.     

Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glucosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9)) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFP and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.     

Markers of atresia in ovarian follicular components from rhesus monkeys treated with estradiol-17 beta

A new model for the investigation of atresia in rhesus monkeys is presented. This model is based upon the reliable induction of an atresia-like process in the dominant preovulatory follicle (DF) by estradiol-17 beta administered subcutaneously via Silastic capsules for 24 h. Data obtained from follicular contents aspirated from treated animals demonstrated alterations in the putative markers of atresia similar to those described in other models of atresia. Although follicle size and appearance and volume of follicular fluid (FF) were unaltered in treated animals, FF was much more viscous than that aspirated from follicles in untreated animals; this was apparently due to a greater quantity of intercellular matrix that was sensitive to digestion by hyaluronidase. In treated animals, FF concentrations of estrogen (E) and progesterone (P) were depressed 3- and 6.6-fold, respectively. Viability of granulosa cells (GC) from these animals was reduced by 40%, as was their ability to release basal amounts of E and P in vitro. Accumulation of P by GC from treated animals approximated unstimulated control levels when human follicle-stimulating hormone (hFSH) was included in the culture. Therefore, FSH may have a limited capability to "rescue" GC from atresia induced by estradiol. The percentage of cells that bound 125I-hFSH maximally, as measured by autoradiography following 72 h in culture, was not altered by treatment. Oocytes from animals treated with estradiol showed signs of degeneration at aspiration, and deteriorated further in culture. This model is unique in that atresia can be induced in the single DF of a primate species, and thus avoids the disadvantages inherent to studying atresia of heterogeneous follicles in polytocous species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos

Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.     

Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 × 0.47 or 5 × 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.     

Ovulation and lambing rates in Karaguniki ewes after treatment with follicular fluid

The aim of the present study was to examine the effect of steroid-free bovine follicular fluid (bFF) on both ovulation and lambing rates. For this purpose, 30 adult ewes of the Karaguniki breed were randomly allocated to three treatment groups (A,B and C; n=10 ewes each) during the breeding season of 1988. The ewes in Group A received bFF (6 ml iv) twice daily during their luteal phase, starting on Day 5 and lasting until Day 9. The ewes in Group B received a mixture of bFF/arachid oil (3 ml sc, 2:1) on Days 3, 4, 5, 10 and 12 of the estrous cycle. The ewes in Group C (Controls) were treated subcutanecusly with a mixture of steroid-free bovine plasma and arachid oil (2:1) on the same days as the ewes in Group B. Plasma concentrations of progesterone showed that the luteal function during the treatment cycle was normal in all treated and control ewes. The ovulation and lambing rates, however, were greater in Group A (2.5 +/- 0.2 and 1.9 +/- 0.3, respectively) and in Group B (2.1 +/- 0.2 and 1.6 +/- 0.1, respectively) than in Group C (1.5 +/- 0.2 and 1.2 +/- 0.3, respectively). Precipitating antibodies were detected in the plasma of Group B ewes only.     

Serum estradiol-17beta concentrations during spontaneous silent estrus and after prostaglandin treatment in the mare

Serum estradiol-17beta concentrations were determined during silent estrus in the mare. Relationships between serum estradiol-17beta concentration, corpus luteum regression, follicular development, ovulation, prostaglandin treatment and behavioral estrus were investigated. The expression of behavioral estrus was found to be related to the patterns of progesterone and estradiol-17beta secretion during the periovulatory period. When compared to normal estrous cycles, silent estrus was accompanied by a significantly lower maximum serum estradiol-17beta concentration (47.8 vs 34.6 pg/ml), a significantly longer interval from maximum estradiol-17beta concentration to ovulation (1.7 vs 4.0 days), and a significantly shorter interval from corpus luteum regression to ovulation (5.3 vs 2.8 days). Silent estrus following prostaglandin treatment was related to a significantly shorter interval from prostaglandin treatment to ovulation (3.6 +/- 0.4 days) than from normal corpus luteum regression to ovulation (5.3 +/- 0.3 days). Silent estrus appeared to be related to changes in follicular estradiol-17beta secretion and to the pattern of its secretion as related to regression of the corpus luteum. There appeared to be not only less estradiol-17beta present, but also less time available after luteal regression for it to interact with the central nervous system to elicit the changes necessary to cause behavioral estrus. There fore, unusual relationships between luteal function and folliculogenesis can result in one type of silent estrus. Significant correlations (P<0.05) were found between follicle size and serum estradiol-17beta concentration whenever behavioral estrus occurred [follicle diameter in mm = 0.96 (serum estradiol-17beta in pg/ml) + 6.08 and 0.73 (serum estradiol-17beta + 13.32 for control and normal estrus following prostaglandin treatment groups, respectively]. During silent estrus, however, no significant correlations between follicle size and serum estradiol-17beta concentration were observed.     

Rat iso-alpha1-fetoproteins. Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha 1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adsorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glu-cosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha 1-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFT and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.     

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.     

Persistent infertility in ewes after prolonged exposure to oestradiol-17 beta

Merino ewes were treated with implants which released 300 micrograms oestradiol-17 beta per day or 5 mg progesterone per day, or both, for 9 months (Months 1-9), and after an 11-month intermission were treated again for 6 months (Months 20-26). Ewes were run with rams at Months 16, 28 and 40. Fertility was not affected by the first exposure period, but the second exposure to oestradiol reduced the fertility of ewes at both subsequent mating periods. Affected ewes returned to service more frequently (P less than 0.01) and were less likely to conceive (P less than 0.05). After mating, a normal population of spermatozoa was established in the caudal cervix, but transport through the cervix was impaired in affected ewes and there were fewer spermatozoa (P less than 0.01) in the cranial cervix. In affected ewes, the spinnbarkeit of cervical mucus was reduced (P less than 0.05), and the histological appearance of the cervix changed, looking like that of the uterus. Treatment with progesterone did not affect fertility, cervical mucus or sperm transport, but diminished the histological abnormalities produced by oestradiol (P less than 0.05). These results show that oestradiol-17 beta given after puberty can cause the same kind of permanent sexual transdifferentiation that is produced by the oestrogenic isoflavones in ewes with clover disease. The results suggest that this change may require more than a single exposure to oestrogen.     

Radioimmunoassay of estradiol-17beta in bovine peripheral plasma with and without chromatography.

An assay of estradiol-17beta (E217beta) in bovine peripheral plasma is described. The plasma is incubated with an antiserum to E217beta-BSA and the gamma-globulin fraction precipitated with ammonium sulphate. After extraction with diethyl ether E217beta in the precipitate is estimated by radioimmunoassay using a specific antiserum against E217beta-6-BSA. Plasma concentrations of E217beta during the normal estrous cycle determined by this method and by a method involving Sephadex LH-20 chromatography range from 4 to 23 pg/ml.     

Follicle and oocyte morphology in ewes after treatment with insulin in the late follicular phase

Stress reduces fertility in ruminants. Various experimental models, such as insulin-induced hypoglycaemia, have been used to investigate the mechanisms involved, and have revealed abnormal LH profiles (both pulse and surge secretion). This disruption affects follicular function and it is proposed there may be negative consequences on subsequent oocyte morphology. Insulin (5 iu/kg), administered to ewes in the late follicular phase, induced hypoglycemia for 10 h, decreased estradiol concentrations for 8-12 h and delayed the LH surge by 15 h. Although the diameters of dominant follicles just before ovulation were not affected, granulosa cells had fewer pycnotic nuclei, less apoptosis and increased proliferation 16-17 h after the LH surge. Nevertheless, we did not observe gross ultra-structural differences in nuclear, cytoplasmic or cumulus maturity between oocytes from insulin-treated and control animals. This suggests that reduced LH pulsatility and a delay in the LH surge may only produce very subtle changes in gross oocyte morphology, imperceptible by electron microscopy.     

The preparation of estradiol-17 beta sulfates with triethylamine-sulfur trioxide

Reaction of estradiol-17 beta with triethylamine-sulfur trioxide in pyridine gives exclusively monosulfation at the C17-hydroxyl group with the preparation of 17 beta-sulfooxyestra-1,3,5(10)-trien-3-ol triethylammonium salt (V). The structural assignment suggested by spectroscopic measurements was confirmed by synthetic studies. (Formula: see text) A synthesis of 3-sulfooxyestra-1,3,5(10)-trien-17 beta-ol triethylammonium salt (II) has been accomplished based on the preparation of 17 beta-formyloxyestra-1,3,5(10)-trien-3-ol (XIII). Fusion of the 3-sulfate triethylammonium salt II gives rise to the 17-sulfate triethylamine salt V. The preparation of estradiol-17 beta disulfate has also been achieved.     

Ovarian response to PMSG treatment in ewes immunized against oestradiol-17 beta

The effects of active immunization against oestradiol-17 beta on the ovarian response to pregnant mare serum gonadotrophin (PMSG) was investigated in Merino ewes. Immunized (79) and control (41) ewes were synchronized with intravaginal sponges, given either 750 or 1500 i.u. PMSG and then mated to rams or inseminated laparoscopically with fresh diluted semen. All control ewes mated naturally exhibited oestrus and 40 out of 41 control ewes ovulated. The ovulation rate was higher in the controls receiving 1500 i.u. PMSG than in those ewes which received 750 i.u. PMSG (10.2 v. 3.3). Immunization against oestradiol-17 beta resulted in antibody titres varying from 100 to more than 100 000 in plasma taken 1-4 days after mating. The ovarian response increased significantly in the lowest titre group (100-1000) in conjunction with stimulation with 1500 i.u. PMSG. In these ewes the ovulation rate increased over controls (16.7 v. 10.2) as did the total ovarian response, which includes follicles greater than 10 mm diameter (22.3 v. 11.1). The total ovarian response was also increased in those ewes given 750 i.u. PMSG which had titres in the 1000-10 000 and 10 000-100 000 range, but this was not accompanied by significant increases in the ovulation rate. In general, the higher titre levels (greater than 1000) were correlated with decreases in the proportion of ewes showing oestrus and ovulating and in the embryo recovery rate. The 1500 i.u. PMSG treatment group with the highest titres (greater than 10 000) also showed a significant drop in the ovulation rate as compared to the 1500 i.u. PMSG controls.     

Plasma estradiol-17beta level in the domestic fowl

Using healthy white Leghorn chickens the estradiol-17β level in plasma were determined by radioimmunoassay and the dependency on age and sex examined. In one and two year old laying hens average values between 48.0 and 54.4 pg/ml were found. The estradiol level in cocks of the same age were between 6.0 and 7.3 pg/ml. Significant difference with respect to the estradiol level were already noticeable in sexually immature 4 month old chickens ♀: 8.1 pg/ml, ♂: 1.0 pg/ml). The significance of the estradiol-17β level with respect to plasma lipids and electrophoretic mobility of serum lipoprotein is shown.