ELSEVIEd Animal Reproduction Science 46 (1997) 213-222

Use of a GnRH antagonist in conjunction amplitude, high frequency LH pulses to

follicular growth without an LH surge and in ewes

with low induce ovulation

H. Dobson *, B.K. Campbell I, R.J. Scaramuzzi * CSIRO Dihion of Animal Production, Private Bag I, Blacktown, NS’. W. 2148, Australia

Accepted 7 October 1996

Abstract

The present study was undertaken to develop an experimental sheep model which could be used to investigate the abnormal follicle growth that is associated with the absence of the LH surge. On Day 10 of the oestrous cycle, 16 ewes were treated with an analogue of prostaglandin (cloprostenol; PG) and blood sampled every 4 h thereafter to determine the normal timing of the preovulatory LH surge. Three oestrous cycles later, all ewes were simultaneously treated with PG and a gonadotrophin-releasing hormone (G&I-I) antagonist ([AC-DNal’, D4C1Phe2, DTrp’, DArg6, DAla”] GnRH.HOAc; 50 pg kg-’ subcutaneously). Group 1 ewes (n = 6) received no further treatment. Group 2 ewes (n = 5) were additionally treated for a total of 7 days, starting at the time of PG injection, with purified ovine luteinising hormone (LH; preparation P 3 R3-5; equivalent to 1.25 pg NIH-oLH-S26) administered i.v. over a 2 min period. For the first 24 h, LH was given at 3 h intervals for 12 h, then every 2 h for 12 h, and thereafter hourly for 6 days. Group 3 ewes (n = 5) were treated as Group 2 but at 72 h received an additional antagonist injection (50 pg kg- ’ subcutaneously).

* Correspondence address: Department of Veterinary Clinical Science and Animal Husbandry, University of Liverpool, Leahurst, Neston, L64 7TE, UK. Tel.: f(44) 151-794-6080; fax: +(44) 151-794-6005; e-mail: hdobson@liverpool.ac.uk.

’ Present address: Department of Obstetrics and Gynaecology, University of Edinburgh, 37 Chalmers Street, Edinburgh EH3 9EW, UK.

* Present address: Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 OTU, UK.

037X-4320/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved, PII SO378-4320(96)01624-7

214 H. Dobson et al./Animal Reproduction Science 46 (1997) 213-222

Mean values of LH from 24 to 96 h were significantly lower in untreated controls and Group 1 than in the other two groups (0.58 f 0.2 ng ml-’ and 0.55 + 0.2 ng ml-’ vs. 1.63 k 0.5 and 1.68 f 0.6 ng ml-‘, respectively; P < 0.01). After treatment with PG alone in the untreated control group, the preovulatory LH surge began in all ewes at 59.9 f 2.8 h after PG. All Group 1 ewes also had an LH surge but the period from PG injection to the onset of the surge was 124 + 17.3 h (range 96-152 h). Only two of the Group 2 ewes had an LH surge (at 160 and 168 h, respectively) and no surge was detected in Group 3 ewes. In Group 1, mean values of follicle-stimulating hormone (FSH; 0.78 k 0.07 ng ml-‘) were not affected by treatment with antagonist alone; however, in the two groups receiving exogenous LH pulses there was a marked decrease in FSH concentrations during the period 24-96 h. Progesterone concentrations increased 9 days after PG treatment in five out of six ewes in Group 1. In Group 2, there was evidence of a variable luteinisation response, but in Group 3 progesterone remained less than 0.08 ng ml-’ throughout. Endoscopy 112-l 15 h after PG confirmed that none of the 16 antagonist-treated ewes had ovulated; an event normally expected approximately 80 h after PG in sheep.

The experimental protocol of Group 3 provides the basis for a model which will enable examination of the long-term functional capacity of ovarian follicles which have not been exposed to an LH surge. 0 1997 Elsevier Science B.V.

Keywords: Sheep-endocrinology; FSH; Oestradiol; Progesterone

1. Introduction

Ovarian follicles grow continuously and when fully developed either ovulate or degenerate rapidly through atresia (Scaramuzzi et al., 1993). Disruption of this process leads to the formation of persistent follicles (cysts) resulting in subfertility in women (Hamilton et al., 1985), cattle (Borsberry and Dobson, 1989) and sheep (Winter and Dobson, 1992). It has been suggested that cystic follicles are formed in ruminants during the post-partum period due to the absence of a preovulatory luteinising hormone (LH) surge (Kesler et al., 1979) and 50% of clinical cases of cystic ovaries in cattle fail to release an LH surge in response to exogenous oestradiol (Dobson and Nanda, 1992). Detailed investigation using clinical material has serious limitations because when a case is presented the duration of cyst existence is not known. The present study was undertaken to develop an experimental sheep model which could be used to investigate abnormal follicle growth induced by inhibition of the LH surge.

Administration of a gonadotrophin releasing hormone (GnRH) antagonist acutely inhibited the tonic pulsatile secretion of LH in ewes for at least 68 h during the early follicular phase of the oestrous cycle (Campbell et al., 1990a) but it is not known whether the LH surge in response to oestradiol positive feedback could be inhibited by similar doses of antagonist. Furthermore, low amplitude, high frequency LH pulses administered to ewes in anoestrus stimulated follicular development similar to that of the normal follicular phase in the breeding season (Baird and McNeilly, 1981). The present study sought to develop a regime of pulsatile gonadotrophin replacement in GnRH antagonist-treated ewes to promote normal follicle growth. In this way, follicles could be induced to grow in a controlled manner but continued antagonist treatment would suppress an oestradiol-induced LH surge. Thus, oestrogenic follicles could be produced and their long-term functional capacity observed in the absence of the LH surge.

H. Dobson et al/Animal Reproduction Science 46 (1997) 213-222 215

2. Materials and methods

2.1. Experimental animals

This study was conducted with the approval of the AEEC ethical committee, CSIRO Prospect, in accordance with the CSIRO/NH and MRC guidelines.

Border Leicester X Merino ewes (n = 16; mean weight 66 f 3 kg) in the mid-breed- ing season were housed in conditions of natural lighting to which they had been habituated prior to the start of the experiment. They were fed a daily maintenance diet of 800 g pellets containing 60% luceme and 40% oats, with water ad libitum. Oestrous cycles were synchronised by treatment with a combination of intravaginal progestagen pessaries (medroxy-progesterone-acetate, Repromap, Upjohn Pty. Ltd., Rydalmere, N.S.W., Australia) and injections of cloprostenol, an analogue of prostaglandin-FZj, (PG: 125 pg i.m., Estrumate, ICI Australia). Oestrus was detected by a ram fitted with a mating harness and crayon.

On the day before the start of each frequent blood collection period, all animals had a silastic catheter inserted into a jugular vein under local anaesthesia (Xylocaine; Astra Pharmaceuticals Pty Ltd, North Ryde, N.S.W., Australia). For the administration of exogenous LH, a catheter was also inserted in the opposite jugular vein; one catheter was used exclusively for blood sampling and the other for LH administration. The catheters were filled with heparinised saline (200 or 1000 IU ml-‘) between blood samples. All animals received daily prophylactic antibiotic treatment (4 ml i.m.; Hydropen, Bomac Laboratories, Castle Hill, N.S.W., Australia) starting on the day of catheterisation. The catheters were removed after taking the last sample in each frequent collection period.

2.2. LH purijkation

The ovine LH was prepared from frozen sheep pituitaries by ammonium sulphate precipitation (Papkoff et al., 1965) and anion exchange chromatography following the method of Curlewis et al. (1992). Briefly, the modified procedure included mincing 1 kg ovine pituitaries followed by the addition of 2 1 of 0.15 M (NH,)SO,; the pH was then adjusted to 4.0 and stirred for 2-3 h. The supematant obtained by centrifugation was adjusted to pH 3.0 with 0.2 M metaphosphoric acid and the precipitate discarded. The pH of the supematant was adjusted to 6.5-7.0 with NaOH and a crude LH preparation obtained by addition of 311 g (NH,)SO, 1-l. Following dialysis and lyophilisation, 1 g of this precipitate was dissolved in 15 ml 10 mM TRIS pH 7.0 and passed through a column (25 mm X 205 mm) of DEAE-Sepharose CLBB (Pharmacia, Uppsala, Sweden) previously equilibrated with 10 mM TRIS pH 7.0. Under these conditions, LH passed through the column unabsorbed whereas follicle stimulating hormone (FSH) contamina- tion was eluted using a linear gradient of 0.5 M NaCl. The LH-rich fractions from duplicate runs were pooled, dialysed and lyophilised.

The resultant material had an immunoactivity equivalent to 1.2 times NIH-oLH-Sl and contained 0.8% FSH contamination; similar purification methods resulted in prepa- rations with less than 1% contamination with growth hormone (GH) or thyroid-stimulat-

216 H. Dobson et aL/Animal Reproduction Science 46 (1997) 213-222

Fig. 1. Diagram

Hours 0 24 48 72

of the experimental protocol for ewes treated with GnRH antagonist (open arrow) and replacement LH (vertical lines). Treatment began on Day 10 of a synchronised oestrous cycle and a PG injection was also given at time zero; see text for further details. Blood samples were taken every 4 h (before an LH injection).

Antagonist

Group 1 1 LH InjectIons

Group 2

Group 3

ing hormone (TSH) (Papkoff et al., 1965). The bioactivity of the material was confirmed by showing that a 2.5 kg dose was equipotent with 1.25 pg NIH-oLH-S26 (which has a bioactivity of 2.3 NIH-oLH-Sl) in stimulating the pulsatile secretion of oestradiol by the ovary of ewes with an ovarian autotransplant (B.K. Campbell and M. Friend, unpub- lished observations, 1992).

2.3. Experimental design

On the tenth day of one synchronised oestrous cycle, luteolysis was induced with an injection of PG and six ewes were bled every 4 h for 72 h to determine the normal timing of the preovulatory LH surge (untreated control period). Three oestrous cycles later, again on the tenth day of the cycle (time zero), luteolysis was induced by an injection of PG, and all ewes were simultaneously treated with a GnRH antagonist ([AC-DNal’, D4C1Phe2, DTrp3, DArg6, DAla”] GnRI-LHOAc; 50 p,g kg-’ subcuta- neously). As shown in Fig. 1, Group 1 ewes (n = 6) received no further treatment except saline vehicle for injections subsequently described. Group 2 ewes (n = 5) were additionally treated for 7 days, starting at the time of PG injection, with exogenous purified ovine LH dissolved in 1 .O% normal sheep plasma in saline. The LH preparation (P 3 R3-5; 2.5 l.~ g per 2 ml injection; equivalent to 1.25 kg NIH-oLH-S26) was infused i.v. each time over a 2 min period. For the first 24 h, LH was administered three-hourly for 12 h, then every 2 h for 12 h, and thereafter hourly for 6 days. Group 3 ewes (n = 5) were treated as Group 2 but at 72 h received an additional injection of antagonist (50 pg kg-’ subcutaneously).

2.4. Blood sampling

In all antagonist-treated ewes, jugular venous blood samples (3 ml) were taken at 6-h intervals for 36 h from the injection of PG and then at 4-h intervals for a further 6.5 days. During the administration of exogenous LH, blood samples for hormone analysis were taken from the sampling catheter immediately before one of the hourly LH pulses.

H. Dobson et al/Animal Reproduction Science 46 (1997) 213-222 217

To confirm the appropriateness of the dose of exogenous LH, six ewes were chosen at random from Groups 2 and 3 and blood sampled (3 ml) every 10 min for 2 h around an LH injection on Day 1 (20 h after PG treatment); for comparison, six ewes during an untreated control period were sampled every 10 min from 20 to 28 h after PG. To assess subsequent luteal development by progesterone analysis, 3-ml blood samples were taken from all treated ewes daily for 4 days after the cessation of the LH injections.

Blood was centrifuged at 1000 X s for 15 min at 4°C and plasma stored at - 20°C.

2.5. Hormone analyses

Plasma concentrations of LH, FSH and progesterone were determined using previ- ously described radioimmunoassays (Campbell et al., 1990b, 1994). The sensitivity of the assays for LH, FSH and progesterone were 0.2 ng (NIH-LH-S20) ml-‘, 0.1 ng (NIAMDD-FSH-RPl) ml-’ and 0.05 ng ml- * respectively. The intra- and inter-coeffi- cients of variation for LH, FSH and progesterone were 4.3% and 5.4%, 1.7% and 2.8% and 7.9% and lO.O%, respectively.

2.6. Endoscopy

The ovaries of all treated ewes were examined by endoscopy under xylocaine-in- duced local anaesthesia between 112 and 115 h after PG. At 113 and 114 h none of the ewes received LH pulses.

2.7. Statistical analysis

Statistical analysis of hormonal profile data was conducted by repeated measures ANOVA with individual comparisons between times and between treatments being made using a pooled error term. The preovulatory LH surge was defined to have occurred when at least two consecutive samples with a concentration of LH greater than 10 ng ml-’ were detected.

3. Results

3. I. LH pulses

During the frequent sampling period in the untreated control ewes (20-28 h after PG), the baseline value was 0.58 + 0.19 ng ml-’ and endogenous LH pulses had an amplitude of 1.19 _+ 0.11 ng ml-’ with an inter-pulse frequency of 62.6 & 3.4 min.

In GnRH antagonist-treated ewes on Day 1 (20 h after PG), the baseline concentra- tion of LH was 0.76 k 0.11 ng ml-’ in six ewes sampled frequently around an LH injection. Values increased by 1.29 & 0.23 ng ml-’ to a maximum of 2.05 f 0.43 ng ml-’ by 5 min after administration of exogenous LH. Concentrations then declined to 1.26 f 0.20 and 0.89 + 0.15 ng ml-’ at 25 and 55 min, respectively, after injection (Fig. 2). The amplitude and pattern of the LH pulse after LH injection was similar to the endogenous pulses in control ewes (P > 0.05).

218 H. Dobson et al./Animal Reproduction Science 46 (1997) 213-222

(L,H9 ml-‘) 2.5 1 1 T

0.03 -40 -20 0 20 40 60 60 100 120

Time (mln)

Fig. 2. Plasma concentration of LH (expressed as NM-LH-S20 equivalents; ng ml-’ ) for 2 h around an i.v. injection of purified ovine LH (equivalent to 1.25 pg NIH-oLH-S26) given, as indicated by the arrow, 20 h after injection of GnRH antagonist.

3.2. LH basal values

Mean values of LH from 24 h to 96 h were significantly lower in the controls and Group 1 than in the other two groups (0.58 f 0.2 ng ml-’ and 0.55 f 0.2 ng ml- ’ vs. 1.63 + 0.5 and 1.68 f 0.6 ng ml-‘, respectively; P < 0.01).

In Group 1, basal values of LH declined from 24 h after the injection of G&I-I antagonist and remained at 0.55 k 0.2 ng ml-’ until 96 h after antagonist treatment (Fig. 3(a)). The concentration of LH from 24 to 96 h in Groups 2 and 3 (i.e. from the start of hourly LH injections to the onset of the LH surge in Group 1 ewes) was significantly higher than in Group 1 (Fig. 3(b and c)); 1.63 f 0.5 and 1.68 f 0.6 ng ml-‘, respectively; P < 0.01).

3.3. LH surge

During the untreated control period, an LH surge of 18.6 + 2.7 ng ml-’ was detected in all ewes 59.9 + 2.8 h after PG. In all six ewes treated with antagonist alone (Group l), the period from PG to the onset of the LH surge was 124 + 17.3 h (range 96-152 h; P > 0.05 compared with controls; peak values ranged from 11 to 40 ng ml- ’ ). Only two of the five ewes in Group 2 had an LH surge (19 and 31 ng ml- ’ at 160 and 168 h, respectively) having been treated with one injection of antagonist plus pulsatile LH. None of the five ewes in Group 3 (antagonist at 0 and 72 h) had an LH surge.

3.4. FSH

In ewes during the untreated control period, mean FSH values declined from 1.10 + 0.10 ng ml- ’ at the time of PG injection to 0.60 f 0.05 ng ml-’ between 24 and 48 h later. In the treated ewes, mean values of FSH (0.78 f 0.07 ng ml-‘) were not affected by administration of antagonist alone (Group 1; Fig. 3(a)). However, in the two groups receiving exogenous LH pulses there was a marked decrease in FSH concentra-

H. Dobson et al./Animal Reproduction Science 46 (1997) 213-222 219

a) Group 1

LH (ng ml-l ) 10

T FSH (rig ml -I )

6- 1.0

4-

0.5 2-

o- 0.0 0 24 46 72 96 120 144 166

b) Group 2 10 - 2.0

0 - 1.6

6 - 1.0

4 - 0.5

2

0 - 0.0 0 24 46 72 66 120 144 166

c) Group 3 10.

8.

6,

4

2

0

j-2.0

J, ILO. 0 24 40 72 S6 120 144 168

Hours from PG + first antagonist Injection

Fig. 3. Plasma concentrations (mean& SE) of LH (e) and FSH (0) in ewes given prostaglandin on day 10 of the oestrous cycle (time zero). The six ewes in (a) were given one injection of GnRH antagonist (50 t.~g kg- ’ ) at time zero as shown by the closed arrow; the five ewes in (b) were similarly treated with GnRH antagonist as well as hourly injections of purified LH (equivalent to 1.25 p,g NM-oLH-S26) beginning at time zero; whereas the five ewes in (cl were given a second GnRH antagonist injection 72 h after the first (open arrow), in addition to hourly LH injections.

tions with a nadir equivalent to 46% of initial values in both Groups 2 and 3. This was followed by an increase beginning approximately 112 h after PG treatment (Fig. 3(b and c)).

220 H. Dobson et al. /Animal Reproduction Science 46 (1997) 213-222

3.5. Progesterone

Progesterone concentrations remained basal (less than 0.08 ng ml- ’ > throughout the follicular phase in all ewes. Values increased 9 days after PG treatment in five out of six ewes in Group 1 to reach over 1.5 ng ml - ’ by the end of the experiment 2 days later. The remaining ewe in this group was the last to have an LH surge at 152 h but had a progesterone value of 0.56 ng ml-’ by 11 days after PG treatment. In Group 2, two ewes maintained low progesterone concentrations until the end of the experiment; one ewe had values of 0.2 and 0.4 ng ml- ’ 10 and 11 days after PG, respectively; and the remaining two ewes had values of 1.0 ng ml-’ on the 11th day. In Group 3, progesterone concentrations remained less than 0.08 ng ml-’ throughout.

3.6. Follicular growth

Observations during endoscopy 112- 115 h after PG revealed the presence of one to five medium to large sized follicles in four of the five ewes treated with antagonist alone (the sixth ewe had abdominal adhesions and the ovaries were not seen), and in four of the five ewes in both the LH injected groups. No ewe in any experimental group had ovulated by this time.

4. Discussion

The results of this experiment confirm and extend the results of previous studies using a similar dose of this GnRH antagonist in sheep; Campbell et al. (199Oa) inhibited pulsatile LH secretion in the early follicular phase for at least 68 h after one injection, and the LH response to a low dose of exogenous G&I-I (150 ng) was suppressed for at least 7 days in anoestrous ewes (Campbell et al., 1992). The present study has shown for the first time that a single 50 p,g kg-’ dose of antagonist given at the time of PG injection delayed LH surge release by an average of 64 h in ewes in the breeding season. As in anoestrus, FSH concentrations were unaltered by treatment with GnRH antagonist in the breeding season, concurring with the commonly held view that basal FSH release is not acutely dependent on GnRH secretion (Narayana and Dobson, 1979; Campbell et al., 1990a). Group 1 ewes, treated with one dose of antagonist alone, did have an LH surge, albeit delayed, so there must have been an earlier release of tonic LH from the suppressive effects of the antagonist for new follicular development to have taken place. This was evidenced, not only by the subsequent positive feed-back to produce the LH surge, but also by the presence of medium to large sized follicles and subsequent elevated progesterone concentrations. From the 4-h profiles of LH it was not possible to establish exactly when the influence of the antagonist ceased; however, a second antagonist injection given 72 h after PG in Group 3 delayed the LH surge beyond the end of the 4-hourly sampling period (168 h after PG).

The surges of LH were inhibited or delayed (to at least 168 h after PG treatment) in antagonist-treated ewes that were given exogenous pulses of LH. The prolonged suppression in FSH concentrations in Groups 2 and 3 suggest that these follicles were

H. Dobson et al. /Animal Reproduction Science 46 (1997) 213-222 221

secreting more oestradiol than the follicles in Group 1 ewes. Exogenous LH pulses stimulate the theta to secrete androstenedione which is aromatised to oestradiol by the granulosa leading to increased oestradiol secretion from the ovary and a suppression in FSH concentrations (Campbell et al., 1990a). In a subsequent experiment with different sheep, direct measurement of oestradiol in ovarian venous blood has now confirmed that the decrease in FSH concentrations occurring over the first 36 h of antagonist plus exogenous LH treatment was the result of enhanced oestradiol negative feed-back (H. Dobson et al., 1997). In Group 1 of the present experiment it is not known if the follicle(s) which began to grow at the beginning of the sampling period remained oestrogenic, or if a new crop of follicles had grown.

The LH plasma profile produced by the exogenous LH injections in antagonist-treated ewes was similar to spontaneous release in these same animals during the early follicular phase 20-28 h after PG treatment. The absence of LH pulses at 113 and 114 h (due to endoscopy procedures) was unlikely to be deleterious (Campbell et al., 1990a).

The timing of the endoscopy (112- 115 h after PG) was chosen to establish whether ovulation had occurred at the normal time (75-85 h after PG). In all experimental groups there was no evidence of ovulation, but in most ewes there were one to five medium to large sized follicles present. Very precise measurement of follicular size and number was not attempted as such results are unreliable using endoscopy. Raised progesterone values in the early luteal phase suggest immediate luteinisation in all Group 1 ewes. In Group 2 ewes there was some luteinisation but it was totally absent in Group 3. In Group 2 ewes, surge release of LH may have occurred after blood sampling stopped, but luteinisation is not thought to have occurred as a result of raised basal LH concentrations as there was no evidence of luteinisation in Group 3. The absence of luteinisation after periods of persistent follicular growth is reminiscent of that in cows with spontaneously occurring ovarian cysts (Dobson and Nanda, 1992; Ribadu et al., 1994).

The objectives of the present study (to block the LH surge and ovulation, and thus inhibit luteinisationl were achieved in Group 3 using a dose of antagonist previously given only to anoestrous ewes or during the very early follicular phase.

Acknowledgements

This work was supported by a grant from the Australian Wool Research and Development Corporation (CPBSl); H.D. was a Development Fellow of the Association of Commonwealth Universities and B.K.C. was a National Research Fellow of the Australian Research Council. We gratefully acknowledge the assistance of Dr J.A. Downing, B.M. Gordon and D.L. Paliskis, the gift of the GnRH antagonist from Dr M. Karten, Contraceptive Development Branch, NICHD, Bethesda, USA and gonadotrophin assay reagents from Dr S. Raiti of NIAMDD, Bethesda, USA.

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