The role of sub-optimal preovulatory oestradiol
secretion in the aetiology of premature luteolysis
during the short oestrous cycle in the cow
G.E. Mann
∗, G.E. Lamming
University of Nottingham, School of Biosciences, Division of Animal Physiology, Sutton Bonington, Loughborough LE12 5RD, UK
Received 3 June 1999; received in revised form 23 August 2000; accepted 4 September 2000
Abstract
Premature regression of the corpus luteum, following the first post partum ovulation, is often preceded by sub-optimal preovulatory oestradiol secretion and accompanied by elevated levels of oxytocin receptors early in the luteal phase. We have investigated the role of preovulatory oestradiol in the control of subsequent oxytocin receptor concentration and activity by treating ovariectomised cows, over a simulated 48 h follicular phase, with high (600mg per day) medium (300mg per day) or low (150mg per day) levels of oestradiol. These doses of oestradiol generated mean±S.E.M.
plasma oestradiol concentrations of 12.1±1.0, 4.9±0.5 and 2.9±0.4 pg ml−1, respectively.
In Study 1 (n = 4 per group), we found that by day 4 following oestrus there was a significant (P <0.05) effect of the level of oestradiol on the inhibition of oxytocin binding activity measured in endometrial biopsy samples. This had fallen to mean±S.E.M.concentrations of 25±2 fmol per mg protein in the high group, 47±8 fmol per mg protein in the medium group and 65±12 fmol per mg protein in the low group. In Study 2, cows (n=3 per group) were treated with the same three levels of oestradiol followed by treatment with increasing levels of progesterone from days 3 to 6 following oestrus, generating mean±S.E.M.plasma concentrations of 2.17±0.18 ng ml−1by day 6. On day 6, there was a significant (P < 0.01) effect of the level of oestradiol on PGF2a
release in response to oxytocin challenge. High, medium and low oestradiol groups exhibiting mean±S.E.M., increase plasma PGF2ametabolite concentrations of 10.0±2.2, 21.3±4.3 and
41.3±1.2 pg ml−1, respectively, during the hour after oxytocin administration. From these results,
we postulate that at the first post partum ovulation a low level of preovulatory oestradiol can result in the early generation of a luteolytic mechanism during the subsequent luteal phase due to impaired
∗Corresponding author. Tel.:+44-1159-516326; fax:+44-1159-516326. E-mail address: [email protected] (G.E. Mann).
inhibition of oxytocin receptors allowing increased PGF2arelease. © 2000 Elsevier Science B.V.
All rights reserved.
Keywords: Cattle-endocrinology; Oestradiol; Luteolysis; Corpus luteum
1. Introduction
In cattle, luteolysis results from the release of luteolytic episodes of uterine prostaglandin F2a (PGF2a) in response to the binding of oxytocin to newly developed receptors on the
uterine endometrium (for review see Mann et al., 1999). During the normal luteal phase, progesterone inhibits the development of these oxytocin receptors until the appropriate time (around day 16–17). However, at puberty, and following post partum and seasonal anoestrus, the basic mechanisms regulating cycle length do not function normally. During these periods of transition from a state of acyclicity to one of cyclicity, the first oestrous cycle is often of short duration (for reviews see Lauderdale, 1986; Hunter, 1991; Garverick et al., 1992). Numerous studies have demonstrated that the occurrence of short-lived corpora lutea is a function of a lack of prior exposure to progesterone as pre-treatment with progesterone results in the formation of corpora lutea of normal life span. However, the mechanism by which this lack of prior exposure to progesterone results in premature luteal regression is far from clear.
A number of studies have failed to demonstrate any consistent differences in the charac-teristics of normal and short-lived corpora lutea prior to the initiation of premature luteolysis in the short-lived group (for review see Hunter, 1991; Garverick et al., 1992). This suggests no innate inadequacy of the short-lived corpus luteum. There is, however, strong evidence to suggest that a premature triggering of the luteolytic mechanism, early in the luteal phase, is responsible for the premature demise of short-lived corpora lutea. The premature luteolytic signal in animals with short-lived corpora lutea is associated with an increased level of both endogenous (Peter et al., 1989; Cooper et al., 1991) and oxytocin-induced (Zollers et al., 1989) PGF2a release. Furthermore, treatment with indomethacin to block prostaglandin
synthesis prevents premature luteolysis (Troxel and Kesler, 1984), as does removal of the uterus, the principle source of luteolytic PGF2arelease (Copelin et al., 1987).
A number of authors have reported sub-optimal levels of preovulatory oestradiol secretion preceding the formation of short-lived corpora lutea (Garcia-Winder et al., 1986; Garverick et al., 1988; Braden et al., 1989a,b). During the normal cycle, high levels of preovulatory oestradiol secretion result in the eventual disappearance of endometrial oxytocin receptors (Lamming and Mann, 1995a,b), producing a situation early in the luteal phase of low oxy-tocin receptor and no oxyoxy-tocin stimulated luteolytic PGF2aepisodes. However, premature
luteolysis in animals exhibiting short cycles is accompanied, early in the luteal phase, by elevated levels of oxytocin receptor (Zollers et al., 1991) and increased oxytocin-induced release of PGF2a(Zollers et al., 1989).
effects of the level of preovulatory oestradiol on subsequent oxytocin receptor concentra-tions and activity using a hormone treated ovariectomised cow model.
2. Materials and methods
2.1. Experimental animals
Two studies were undertaken in mature, ovariectomised Blue-Grey cows. All cows had been ovariectomised between 6 and 7 years previously. Since then cows had been used in between four and five progesterone/oestradiol replacement experiments. Prior to the current study, cows had not been used for at least 6 months, and were then randomly allocated to treatment groups. In the current study, all cows were administered a 2 day 17b-oestradiol treatment regimen to replace preovulatory oestradiol secretion during a simulated follicular phase. All treatments are described relative to the second day of oestradiol treatment, defined as day 0, on which all cows exhibited behavioural oestrus (mounting activity; standing to be mounted; vaginal “sliming”).
2.2. Experimental design
2.2.1. Study 1
In this study, three groups of four cows were treated with different levels of 17b-oestradiol (Sigma, Fancy Road, Poole, UK), administered by i.m. injection in corn oil at 8 h intervals over a 48 h period according to Table 1 to simulate three levels of preovulatory oestradiol production. The effect of the level of oestradiol on subsequent oxytocin receptor decrease was assessed by measuring oxytocin binding activity in endometrial biopsy samples col-lected on days 2, 4 and 6 following oestrus (day 0). Throughout the study, plasma samples were collected to monitor plasma oestradiol concentrations.
2.2.2. Study 2
In this study, the same three levels of oestradiol were administered to produce three levels of simulated preovulatory oestradiol secretion. However, in this study cows were then treated with progesterone by i.m. injection in corn oil twice daily, from days 3 to 6 following oestrus
Table 1
Quantities of oestradiol (mg per i.m. injection) administered to ovariectomised cows during simulated follicular phases
Time (h) High (n=4) Medium (n=4) Low (n=4)
0 50 25 12.5
8 100 50 25
16 150 75 37.5
24 200 100 50
32 200 100 50
(day 3: 15 mg×1; day 4: 30 mg×2; day 5: 60 mg×2; day 6: 90 mg×1) to simulate a post-ovulatory progesterone rise. The potential for a premature luteolytic signal was then determined on day 6 by measuring plasma concentrations of 13,14-dihydro-15-keto-PGF2a
(PGFM), the principle metabolite of PGF2a, in samples collected before and after a 50 i.u.
oxytocin challenge.
2.3. Blood sampling, biopsy collection and oxytocin challenge
Prior to the start of oestradiol treatment, a jugular vein of each animal was cannu-lated under local anaesthesia (2 ml lignocaine s.c. as Lignovet 2%, C-Vet, Bury St Ed-munds, UK) with a 30 cm indwelling jugular catheter (Secalon universal tubing, BOC Health Care, Swindon, UK) using a 12 gauge needle and guide wire. Cannulae were then maintained for the duration of the experiment, and used for the collection of all blood samples.
Endometrial biopsies were collected via a trans-cervical technique (Mann and Lamming, 1994). During biopsy collection, animals were first sedated by an i.m. injection of 20 mg xylazine (Rompun; Bayer U.K. Ltd, Science Park, Milton Road, Cambridge) and the biopsy forceps were guided through the cervix by trans-rectal manipulation. Once in the uterus, a single sample of 300–600 mg endometrial tissue was collected. Animals were then ad-ministered prophylactic antibiotic (Duplocillin LA; Mycofarm UK, Science Park, Milton Road, Cambridge). Immediately following collection, samples were snap frozen in liquid nitrogen and then stored in liquid nitrogen until processed for determination of endometrial oxytocin binding.
To monitor endometrial responsiveness to oxytocin, cows were injected with a single iv. bolus of 50 i.u. oxytocin (Hoechst UK Ltd, Walton Manor, Milton Keynes) in 5 ml saline, flushed in with a further 5 ml saline. Plasma concentrations of PGFM were measured in blood samples collected at 20 min intervals for 1 h before the injection of oxytocin, and then at 10 min intervals for 1 h after the challenge. All samples were collected into heparinised tubes, centrifuged at 1500×gfor 10 min and the plasma stored at−20◦C.
2.4. Radioimmunoassays
2.5. Statistical analysis
Plasma concentrations of oestradiol were analysed by repeated sample analysis of vari-ance with group and day as main factors. Changes in plasma concentration of PGFM follow-ing oxytocin challenge were analysed by repeated sample analysis of variance with group and time as main factors. Endometrial oxytocin binding results were analysed by analysis of variance with group and day as the main factors. The effects of level of oestradiol on oxytocin binding were analysed by regression analysis with mean oestradiol concentration on day 0 and oxytocin receptor concentration on day 6 as factors. The effects of level of oestradiol on oxytocin challenge response on day 6 were analysed by regression analysis with mean oestradiol concentration on day 0 and mean PGFM increase on day 6 (expressed as percentage increase above pre-treatment baseline) as factors.
3. Results
3.1. Study 1
Plasma concentrations of oestradiol rose markedly in all groups (P <0.01) following the initiation of oestradiol injections to significantly different (P < 0.01) concentrations between the three groups (Fig. 1). Mean±S.E.M.concentrations on the second day of oestradiol treatment, on which all animals exhibited behavioural oestrus, were 12.1±1.0, 4.9±0.5 and 2.9±0.4 pg ml−1in the high, medium and low groups, respectively. Following the last injection, plasma concentration of oestradiol fell markedly (P <0.01) in all groups with values reaching similar levels in the three groups within 72 h.
On day 2 following oestrus, oxytocin binding in endometrial membrane preparations was similar in the three groups (Fig. 2). However, by day 4, oxytocin binding had fallen
Fig. 2. Mean±S.E.M.oxytocin binding activity (fmol mg protein−1) in uterine endometrial biopsies collected on days 2, 4 and 6 following oestrus in ovariectomised cows treated with high (j;n=4), medium ( ;n=4) or low (h;n=4) levels of oestradiol.
significantly (P <0.05) in the high oestradiol group, and by day 6 oxytocin binding in the high group was significantly (P <0.05) lower than in the other two groups. Furthermore, there was a significant correlation between mean oestradiol concentration on the second day of treatment (day 0) and oxytocin binding on day 6 (r2 =0.71;P < 0.001), higher oestradiol on day 0 being associated with lower oxytocin binding on day 6.
3.2. Study 2
Plasma concentrations of oestradiol generated by the three treatment regimens were similar to those achieved in Study 1 with concentrations showing a significant rise
Fig. 3. Mean±S.E.M.plasma concentrations of oestradiol (open symbols) and progesterone (closed symbols) generated in ovariectomised cows treated with high (s;n=4), medium (h;n=4) or low (△;n=4) levels of oestradiol administered by i.m. injection in corn oil at 8 h intervals (indicated by▽) and a standard level of progesterone (d;n=12 cows combined) administered by i.m. injection in corn oil at 12 intervals (indicated by
Fig. 4. Mean±S.E.M.plasma concentrations of PGFM in ovariectomised cows treated with high (s;n=4), medium (h;n=4) or low (△;n=4) levels of oestradiol and administered a 50 i.u. OT challenge at time 0 (indicated by arrow).
(P < 0.01) rising in all groups to significantly different concentrations (P < 0.001) (Fig. 3). Treatment with progesterone resulted in a similar steady increase (P < 0.001) in the plasma concentration of the hormone in the three groups to similar concentrations (Fig. 3). Treatment with oxytocin on day 6 resulted in significantly (P < 0.01) different increases in the mean plasma PGFM concentration during the hour following oxytocin between groups with the high, medium and low oestradiol groups exhibiting 10.0±2.2, 21.3±4.3 and 41.3±1.2 pg ml−1increases, respectively (Fig. 4). Furthermore, when the three groups were combined there was a significant correlation between oestradiol con-centration on day 0 and the magnitude of the oxytocin challenge response expressed as a percentage increase over basal (r2 = 0.63;P < 0.01;n = 9), higher oestradiol being associated with lower PGF2asecretion.
4. Discussion
In the present study, we have confirmed the ability of high levels of oestradiol to cause the decrease in endometrial oxytocin receptor concentrations in the absence of progesterone, and have shown that the degree of oxytocin receptor is related to the level of follicular phase oestradiol. Furthermore, we have demonstrated that after only 3 days exposure to proges-terone, the amount of PGF2areleased in response to oxytocin is related to the concentration
of oxytocin receptors remaining on the endometrium.
preovulatory oestradiol seen prior to short cycles may result in a loss of progesterone domi-nance earlier following ovulation allowing the early synthesis of oxytocin receptors (Zollers et al., 1993). These theories revolve around the potential inhibitory action of progesterone on the luteolytic mechanism.
A number of studies have demonstrated a fall in endometrial oxytocin receptors in ovariectomised sheep and cattle following treatment with high levels of oestradiol (Val-let et al., 1990; Lamming and Mann, 1995a,b). In the present study, we have confirmed the ability of high levels of oestradiol to inhibit endometrial oxytocin receptor concentra-tions in the absence of progesterone, and have shown that the degree to which oxytocin receptor concentrations are decreased is related to the level of oestradiol administered to simulate circulating concentrations during oestrus. These results are in full agreement with the hypothesis that at the first ovulation the reduced secretion of oestradiol by the preovulatory follicle could lead to impaired inhibition of endometrial oxytocin receptor concentrations.
The presence of oxytocin receptors on the endometrium does not guarantee the ability of oxytocin to induce PGF2asecretion, some progesterone priming of the uterus being required
to allow the functioning of the prostaglandin production pathways (Mann and Lamming, 1994). In Study 2, the generation of physiological concentrations of progesterone resulted in the release of PGF2ain response to oxytocin in the medium and low oestradiol groups, but
not in the high oestradiol group in which oxytocin receptor concentrations were effectively inhibited.
It can be hypothesised, therefore, that at the first post partum ovulation a sub-optimal level of preovulatory oestradiol may result in impaired inhibition of oxytocin receptor concentrations. Binding of oxytocin to these receptors may then result in the premature generation of luteolytic episodes of PGF2aby the uterus resulting in early luteolysis. This
is supported by the observation that treatment of anoestrous ewes with additional oestradiol at the time of ovulation induction led to a significant reduction in endogenous PGF2a
secretion at the anticipated time of premature luteolysis (Mann et al., 1997).
These results support the hypothesis that short cycles result from an inadequate level of preovulatory oestradiol secretion at the first ovulation following a period of ovarian quiescence. A reduced level of preovulatory oestradiol secretion does not provide a suffi-ciently strong signal to induce the full inhibitory oxytocin receptors on the endometrium. Oxytocin then binds to these remaining oxytocin receptors causing premature luteolytic PGF2a release, a process controlled by the steroid hormone environment to which the
animal has been exposed. Thus, premature luteolysis does not result from a direct effect of the lack of previous progesterone action on the uterus, but rather from an in-direct effect mediated through an inadequate level of preovulatory oestradiol production.
Acknowledgements
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