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Corpora lutea with a short life-span induced by rams in seasonally .... (1983a). The limit of detection of the standard curve was 01 ng/ml plasma and the mean.
Corpora lutea with a short life-span induced by rams in seasonally anovulatory ewes are prevented by progesterone delaying the preovulatory surge of LH D. T. Pearce, G. B. Martin and C. M. Oldham School ofAgriculture (Animal Science), University of Western Australia, Nedlands, Western Australia 6009, Australia

Summary. Seasonally anovulatory Merino ewes isolated from

rams were allocated to three treatments before the re-introduction of rams. Ten ewes received a single injection of progesterone (20 mg), 18 ewes received the injection of progesterone but had the ram-induced preovulatory surge of LH replaced by a series of injections of GnRH 24 h after the introduction of the rams, and 20 control ewes had no hormone treatment. Of the 48 ewes, 44 ovulated within 5 days of the introduction of rams and the treatments had no significant effect on the incidence of ovulation. The frequency of corpora lutea with a short life span (the interval between successive preovulatory surges of LH being 5\m=.\1 \m=+-\0\m=.\9days) was 72% for control ewes and 58% for ewes treated with progesterone and GnRH, but such CL were prevented completely after the injection of progesterone alone (P < 0\m=.\001).The injection of progesterone also delayed the preovulatory surge of LH (P < 0\m=.\001).These results suggest that progesterone assures normality of corpora lutea by lengthening the period of gonadotrophin priming of follicles before ovulation.

Introduction

Stimulation of the pulsatile secretion of luteinizing hormone (LH) in the seasonally anovulatory by use of the 'ram effect' (Martin, Oldham & Lindsay, 1980b), infusion of LH (McNeilly, O'Connell & Baird, 1982), or gonadotrophin-releasing hormone (GnRH) (McLeod, Haresign & Lamming, 1982a; McNatty, Ball, Gibb, Hudson & Thurley, 1982) can induce ovulation. These ovulations frequently result in corpora lutea (CL) which have a short life span (Oldham & Martin, 1978) and which produce very little progesterone (Knight, Tervitt & Fairclough, 1981; McLeod et

ewe

al., 1982a).

Progesterone pretreatment, in conjunction with the 'ram effect' or treatment with GnRH, that all CL persist for the normal period, irrespective of whether progesterone pretreatment is for 10-14 days (Oldham, Cognie, Poindron & Gayerie, 1980; Martin, Scaramuzzi & Lindsay, 1981; McLeod et al., 1982a) or as a single intramuscular injection (Cognie et al., 1982; Oldham, Pearce & Gray, 1985). Progesterone pretreatment also delays the timing of the preovulatory surge of LH (Martin et al., 1980a; McLeod et al., 1982a). It has also been shown that the frequency and amplitude of pulses of LH are reduced in ewes treated with progesterone and exposed to rams (Martin, Scaramuzzi, Oldham & Lindsay, 1983b) and that progesterone blocks positive feedback by oestrogen in the ovariectomized ewe (Scaramuzzi, Tillson, Thorneycroft & Caldwell, 1971). Together, all of these observations suggest that progesterone pretreatment may ensure a normal life ensures

*

Present address: MRC

Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EN3 9EW, U.K.

by delaying the preovulatory surge of LH and thereby exposing the developing longer period of stimulation by gonadotrophins. This hypothesis was tested in progesterone-primed ewes by giving GnRH to induce a premature preovulatory surge of LH after span of induced CL

follicle

to a

exposure to

rams.

Materials and Methods

Forty-eight ewes were selected as being seasonally anovulatory by laparoscopy from a flock of Merino ewes which had been kept in isolation (sight, sound and smell) from rams for at least 1 month. These anovulatory ewes were allocated to three treatments: 10 (Group P) were injected intramuscularly with 20 mg progesterone (Sigma, St Louis, MO, U.S.A.) in 2 ml olive oil, 18 (Group + GnRH) were injected with progesterone followed 24 h later by GnRH (Gonadorelin: Hoechst Australia, Melbourne, Australia) in saline (0-9% (w/v) NaCl), and 20 ewes (Group C) received no hormone treatment. Immediately after the injection of progesterone, Day 0 of the experiment, 5 vasectomized rams were introduced. GnRH was given intravenously as a series of injections of 10 µg doses every 30 min for 6h. Such a treatment has been shown to stimulate a preovulatory type surge of LH (Crighton, Foster, Haresign & Scott, 1975). All ewes were kept together in a single flock. Ewes were fitted with indwelling jugular catheters. Sampling of blood, 2 ml per time, began 2 h before the introduction of rams and continued every 4 h for 72 h for 10 of the ewes in Group C and all of the ewes in Group + GnRH, and for 120 h for all of the ewes in Group P. The remaining 10 ewes in Group C were bled every 4 h for 7 days. At the end of this intensive sampling all ewes were bled daily at 06:00 and 18:00 h until Day 17. The plasma was stored at 10°C until assayed for LH or progesterone. Ovulation and premature regression of CL were determined by laparoscopy at Days 3 and 10 (Groups C and + GnRH) or Day 5 and Day 10 (Group P) as described previously (Oldham & Martin, 1978; Oldham & Lindsay, 1980). CL were defined as having a normal life span if they were present at both times of laparoscopy. The concentration of progesterone was measured in duplicate 0-1 ml samples of plasma with a double-antibody radioimmunoassay, described in detail by Martin, Scaramuzzi & Henstridge (1983a). The limit of detection of the standard curve was 01 ng/ml plasma and the mean (± s.e.m.) extraction efficiency was 85 + 1%. A pooled sample of plasma containing 306 + 0-16ng progesterone/ml was used to measure the coefficients of variation within assays (13-3 + 10%) and between assays (13%). The principal cross-reactions exhibited by the antiserum were with 5ß-pregnane-3,20-dione (25%), 11 ß-hydroxyprogesterone (11-5%), 21-deoxycortisol (4%) and corticosterone (1%). Concentrations of LH were measured in duplicate 005ml samples of plasma by doubleantibody radioimmunoassay as described previously (Martin et ., 1980b). The activity of the LH standard (M3-CNRS) was 1-8 i.u./mg NIH-LH-S1 (M. Jutisz: College de France, Paris), and the limit of detection for the standard curve was 0-6 ng/ml plasma. Six replicates of three pooled samples, containing 21 + 0-3,8-9 + 1-0 and 12-9 + 1-2 ng/ml, were included in each assay to allow estimation of variation within and between the 5 assays used in this study. The coefficients of variation within assays were 10· 1 + 20%, 11-0 + 3-2% and 81 + 2-8% for the respective pools. The coefficients of variation between assays were 12-2%, 161% and 16-9% respectively. The antiserum (UWA.3B) exhibited minor cross-reactions with NIH-FSH-S12 (0-9%), NIH-P-S12 (0-2%), NIH-TSH-S8 (4-6%) and NIH-GH-S11 (1-3%). The time of the preovulatory surge of LH was defined as the first time the concentration of LH was sustained above 10 ng/ml. The effects of treatments on the proportion of ewes ovulating and frequency of CL with a short life span were tested by 2 analysis. The effects of treatments on concentrations of LH were examined by t tests. -

Results In the ewes in Groups and + GnRH the plasma concentration of progesterone increased from 0-5 to 3-3 ng/ml within 6 h of injection, and then declined to 0-5 ng/ml by 38 h (Text-fig. 1).

10

50 40 30 Hours after injection

20

60

70

Text-fig. 1. The effect of a single injection of progesterone (20 mg) on the concentration of pro¬ gesterone in the jugular plasma of ewes in Group and Group + GnRH (·) and Group C (O). Values are mean + s.e.m. Table 1. Effect of treatments on incidence of ovulation, time of the start of the surge of LH and frequency of ewes with CL with a short life span after the introduction of rams time of the surge of LH

Ewes with a CL of short life span

(h)

(%)

11-5 ± 2 6 260 71-8 + 6-2

13/18(72) 7/12(58) 0/9 (0)

Mean +

Ewes

ovulating (%) Group C Group + GnRH Group

18/20(90) 12/14(86) 9/10(90)

s.e.m.

The mean time of the start of the preovulatory surge of LH was 11-5 h after rams were intro¬ duced in Group C (Table 1), which was earlier than in ewes from Groups ( < 0001) and + GnRH ( < 001). Seven ewes in Group C had a preovulatory surge within 6 h after the intro¬ duction of rams. The mean time of the preovulatory surge for the ewes in Group + GnRH was 260 h and was less than in ewes from Group ( < 0001). Four ewes in Group + GnRH were excluded because they had spontaneous surges before the injection of GnRH. By contrast, in Group P, the time of the surge of LH was delayed to a mean of 71-8 h after the introduction of rams.

None of the three treatments had any effect on the proportion of ewes ovulating but 72% and 58% of ewes had CL with a short life span in Groups C and + GnRH compared with no ewes with CL with a short life span in Group ( < 0001, Table 1). Within the ewes in Group C the mean time of preovulatory surge of LH for those that had CL with a short life span was 100 ± 2-7 h ( = 10) after the introduction of rams compared to 16-7 + 7-1 h (N 3) for ewes with normal life span CL, but this difference was not statistically significant. =

(a) Group C LH 20 r surge 1-5

1-0-

0-5

1

0

4

2

(b) Group

6

8

10

12

14

16

P + GnRH

20 r

4

Days

6

8

10

12

14

after introduction of rams

Text-fig. 2. The concentration of progesterone in the jugular plasma of ewes that had normal corpora lutea (·) or corpora lutea with a short life span followed by normal corpora lutea (O) induced by rams. Ewes were in (a) Group C (controls), (b) Group + GnRH, or (c) Group P. Each figure is aligned to the time of the preovulatory surge of LH. Values are mean ± s.e.m. and the numbers in parentheses indicate the number of ewes per group. The concentration of progesterone in the plasma of ewes with CL with a normal life span increased to 1-5-2 ng/ml but no rise was detected in ewes with CL with a short life span (Text-fig. 2). Among the ewes in Group C and Group + GnRH the concentration of progesterone in ewes with normal life span CL began to increase 3 or 5 days respectively after the introduction of rams.

Among ewes with CL with a short life span, the concentrations of progesterone began to rise 9 or 10 days after the introduction of rams, i.e. after they had reovulated, and indicate that CL with a short life span persisted for 5-6 days. For 3 ewes in Group C with CL with a short life span and bled intensively the interval between successive preovulatory surges of LH was 5-1 ± 0-9 days.

Discussion Our data support the hypothesis that an injection of progesterone immediately before the introduc¬ tion of rams to anovulatory ewes guarantees normal luteal function by delaying the preovulatory surge of LH. Progesterone does not block the increased secretion of LH caused by the introduction of rams (Martin et al., 1983b) but delays the LH surge and so lengthens the duration of gonadotrophin priming of follicles. Perhaps this ensures that all follicles were 'mature' when they ovulated. Such a proposal is supported by the work of McNeilly et al. (1982) who induced CL with a normal life span from a preovulatory surge 55 h after the first injection of LH in anovulatory ewes. McLeod et al. (1982a) induced CL with normal life span in only 5/19 ewes from a pre¬ ovulatory surge of LH about 20 h after the first injection of GnRH, but all CL had a normal life span when the preovulatory surge was delayed after progesterone priming. Cognie et al. (1982) reported that injections of progesterone with doses less than 20 mg were associated with increasing proportions of CL with a short life span. This suggests that only doses of progesterone of 20 mg or more can delay the preovulatory surge of LH sufficiently to ensure a normal CL. In addition, progesterone may affect the ovary directly (McLeod & Haresign, 1984). In this case progesterone may act by exceeding a critical concentration or by priming the ewe for a critical period. In the.experiment described here the injection of progesterone raised jugular concentrations for 30-38 h and it was only by the alteration of the time of the preovulatory surge of LH that the frequency of CL with a short life span was changed. The preovulatory surge of LH began at 11 -5 h in the control ewes which is earlier than has been previously reported (27 h: Oldham, Martin & Knight, 1978; 35 h: Knight, Peterson & Payne, 1978; 21 h: Cognie, Poindron & Orgeur, 1978). In some ewes the preovulatory surge of LH began within a few hours of the introduction of rams. Early surges in individual ewes have been reported pre¬ viously (Oldham et al., 1978; Knight et al., 1978) but because exogenous oestrogen does not induce a preovulatory surge within 12 h of injection (Scaramuzzi et al., 1971), it seems unlikely that these surges are mediated by oestrogen feedback. It is more probable that the ewes that had early surges of LH ovulated directly in response to a dramatic increase in the frequency of pulses of LH evoked by the 'ram effect' (Martin et al., 1980b). CL with a short life span did not produce measurable concentrations of progesterone despite a normal appearance at the first laparoscopy on Day 3 but Knight et al. (1981), using a different breed of sheep, detected a transient increase in the concentrations of progesterone associated with CL with a short life span. McLeod et al. (1982a, b) who induced ovulation in anovulatory ewes with GnRH, found that in some ewes progesterone concentrations did not rise for 8-10 days and reported this as delayed luteal function. However, they did not use laparoscopy to monitor ovarian activity and so their results could well be explained by CL with a short life span preceding CL with a normal life span. This argument is supported by the similarity of the interval between successive preovulatory surges of LH in this study and that of McLeod et al. (1982b) and both support the suggestion of Oldham & Martin (1978) that CL with a short life span regress after 5-6 days.

We thank the Australian Meat Research Committee for financial support; Peter Moore, David

Suckling and other colleagues for technical assistance; and D. R. Lindsay for help with preparation of the manuscript. D.T.P. was holder of the E.H.B. Lefroy Fellowship.

References

Cognie, Y., Poindron, P. & Orgeur, P. (1978) Rupture de la période de repos sexuel saissonier chez la brebis par l'effet bélier et le blocage de la secretion de prolactine. 4e Journées de la Recherche Ovine et Caprine, p. 339 (Paris). Cognie, Y., Gray, S.J., Lindsay, D.R., Oldham, CM., Pearce, D.T. & Signoret, J.P. (1982) A new approach to controlled breeding in sheep using the 'ram effect'. Proc. Aust. Soc. Anim. Prod.

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Crighton, D.B., Foster, J.P., Haresign, W. & Scott, S.A. (1975) Plasma LH and progesterone levels after single or multiple injections of synthetic LH-RH in anoestrous ewes and comparison with levels during the oestrous cycle. J. Reprod. Fert. 44, 121-124. Knight, T.W., Peterson, A.J. & Payne, E. (1978) The

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ewe

ment. /.

Received 15 November 1984