Summary. Twenty seasonally anoestrous ewes were pretreated with progesterone for 4 days and divided into four equal groups. Ewes in Group 1 received no GnRH treatment and were slaughtered immediately after progesterone removal. Ewes in Groups 2, 3 and 4 received i.v. injections of 250 ng GnRH every 2 h for 36 h starting at the time of progesterone removal. Ewes in Group 2 were slaughtered immediately after the 36 h GnRH pulsing, while ewes in Groups 3 and 4 were given a bolus injection of 125 μg GnRH at this time and were slaughtered 2 and 10 h after the bolus injection, respectively. Blood samples were collected every 30 min from ewes in Group 4 only, from 4 h before the start of GnRH treatment until 10 h after the bolus injection. Pulsing with GnRH resulted in episodic release of LH, and the bolus injection of GnRH was immediately followed by a preovulatory type LH surge in those ewes in which an endogenous surge had not already begun. The pituitary GnRH receptor numbers were significantly higher for the ewes in Group 2 than for any of the other treatment groups, while there was no significant difference in the receptor numbers between Groups 1, 3 and 4. The results suggest an up-regulation of GnRH receptors resulting from pulsatile GnRH therapy.
M. Khalid, W. Haresign and M. G. Hunter
A. P. Beard and M. G. Hunter
A steroid-treated ovariectomized ewe model was used to investigate the role of progesterone pretreatment in the control of functional oxytocin receptor concentrations during the early luteal phase. Ovariectomized ewes (n = 28) were injected with oestradiol for 2 days (final injection = day 0) with or without progesterone pretreatment (progestagen sponge for 10 days). Ewes were then given high or low concentrations of progesterone combined with high, low or zero concentrations of oestradiol in a pattern known to simulate the early luteal phase profile (n = 4 per group). Ewes were given 1 μg oxytocin (i.v.) on day 4 and plasma was collected to assay 13,14-dihydro-15-keto PGF2α. The concentration of progesterone and oestradiol administered had no effect on the concentration of 13,14-dihydro-15-keto PGF2α following oxytocin administration (P > 0.05). However, the group that was not pretreated exhibited a small but significant 13,14-dihydro-15-keto PGF2α response in comparison with the equivalent pretreated group (P < 0.05). In a subsequent study, ewes were divided into groups pretreated and not pretreated with progesterone; both groups were given oestrous concentrations of oestradiol and high concentrations of progesterone and oestradiol together. On day 0, 2, 3 or 4, ewes from each group (n = 3, 3, 4 and 4, respectively) were given 1 μg of oxytocin i.v., and the endometrium was collected to measure the binding of oxytocin receptors. Oxytocin caused a significant (P < 0.05) increase in the concentration of 13,14-dihydro-15-keto PGF2α in all ewes on day 0 but not on days 2, 3 or 4. Oxytocin receptor concentrations were maximal on day 0 and basal by day 4. The decline in receptor concentrations occurred more rapidly in the progesterone-pretreated than in the ewes that were not pretreated. This study has shown that progesterone pretreatment alters the subsequent steroid hormone control of oxytocin receptor concentrations, and has identified the delayed decline in oxytocin receptor concentrations as the potential cause of premature luteolysis in ewes that are not pretreated.
A. P. Beard and M. G. Hunter
The role of oestradiol in the control of premature luteolysis (previously shown to occur by the normal luteolytic mechanism involving PGF2α and oxytocin) was investigated in anoestrous ewes induced to ovulate using GnRH (250 ng every 2 h for 24 h followed by 125 μg on day 0) without progesterone pretreatment. Seven ewes were administered charcoal stripped bovine follicular fluid (bFF) on days 1–5 (2 ml by s.c. injection every 8 h) together with oestradiol on days 2–4 (4 μg in 1 ml of corn oil by i.m. injection every 8 h). Ten ewes were treated with bFF and corn oil (as above), and ten ewes received saline and corn oil (control group). All ewes were treated with 1 μg oxytocin (i.v.) on day 4 and plasma was collected for measurement of 13,14-dihydro-15-keto PGF2α (PGFM). Blood samples were collected for measurement of progesterone and oestradiol (day−2 to 15). The ewes in the control group that responded to GnRH formed either normal (50% of ewes) or short-lived (50% of ewes) corpora lutea identified by progesterone profiles. The proportion of ewes that displayed premature luteolysis was reduced (P < 0.05) by bFF treatment alone (to 11% of ewes), and increased (P < 0.001) by bFF plus oestradiol treatment (to 100%). bFF treatment suppressed oestradiol concentrations (P < 0.01), whereas bFF plus oestradiol treatment increased oestradiol concentrations (P < 0.001) on days 1–5. The high oestradiol concentrations appeared to stimulate the premature luteolytic mechanism as the mean PGFM response to oxytocin was higher in the ewes treated with bFF plus oestradiol than in the other two groups (P < 0.001). In addition, the control ewes that formed short-lived corpora lutea had higher oestradiol concentrations (days 1–5) than did ewes with normal corpora lutea (P = 0.05). This study suggests that short-lifespan corpora lutea are the result of increased oestrogenic stimulation of the luteolytic mechanism during the early luteal phase (following a lack of prior exposure to progesterone) which can be overcome by suppressing oestradiol secretion. This finding demonstrates that oestradiol plays a key role in the initiation of premature luteolysis, probably through stimulation of the prostaglandin response to oxytocin.
L. S. Faillace and M. G. Hunter
A study was carried out to compare preovulatory ovarian events in prolific Meishan gilts (which have high levels of embryo survival) with contemporary white hybrid control gilts. Gilts of similar reproductive ages (second or third oestrous cycle) were observed three times a day for oestrous behaviour and ovaries recovered at a time estimated as within 7 h of ovulation (white hybrid, n = 13; Meishan, n = 16). Preovulatory follicles were recovered (n = 195, white hybrid; n = 252, Meishan), and oocytes were aspirated and fixed for later identification of meiotic stage, and follicular fluid was frozen for subsequent determination of progesterone. The number of presumed ovulatory follicles recovered per animal was similar to the expected ovulation rate (15.0, white hybrid; 15.75, Meishan; P > 0.1); however, follicles from Meishan gilts were smaller (8.1 versus 9.0 mm, P < 0.001) and contained less follicular fluid (139.9 versus 187.6 μl, P < 0.02) than did white hybrid gilts. Total follicular progesterone (88.7 ng per follicle, white hybrid; 77.4 ng per follicle, Meishan) and concentration of progesterone in follicular fluid (526 ng ml−1, white hybrid; 640 ng ml−1, Meishan) did not differ between the two breeds (P > 0.1). There were no breed differences in the extent of heterogeneity in follicular diameter, volume of follicular fluid, and total follicular progesterone and progesterone concentration (P > 0.1). However, classification of recovered oocytes into seven discrete meiotic stages revealed that more oocytes recovered from Meishan preovulatory follicles were in the more advanced stages of meiosis than were those recovered from white hybrid gilts (P < 0.001). In conclusion, it is suggested that the advanced oocyte maturation in follicles of Meishan pigs before ovulation may be important for ensuring the prolificacy of this breed.
A. P. Beard and M. G. Hunter
Two experiments investigate the effects of oxytocin and progesterone on premature luteolysis in ewes. In Expt 1, 20 anoestrous ewes were induced to ovulate by multiple injections of GnRH (250 ng i.v. every 2 h for 24 h) followed by a bolus injection of GnRH (125 μg, i.v.). Ten ewes received a continuous infusion of oxytocin from the day after the GnRH bolus injection and the other ten ewes were infused with saline. Oxytocin infusion had no significant effect on the proportion of ewes with short luteal phases (P > 0.05). All ewes that had luteal phases of normal duration from either group (n = 9) exhibited a transient increase in plasma concentrations of progesterone 2 h after insertion of the pump. In Expt 2, 25 anoestrous ewes were treated with GnRH as in Expt 1. Five ewes were pretreated with progestagen for 11 days and ten ewes received progesterone (12 mg, i.m.) 24 h after the bolus injection of GnRH. All animals received an oxytocin injection (1 μg, i.v.) on day 4 after the GnRH bolus. All five ewes that were pretreated with progestagen had normal luteal function and none exhibited a 13,14-dihydro-15-keto PGF2α (PGFM) response to oxytocin. None of the ten ewes injected with progesterone had a normal luteal phase and six ewes exhibited a PGFM response to oxytocin. Four ewes in the control group had normal luteal function and three had short luteal phases. It is concluded that (1) administration of oxytocin from about the time of ovulation does not prevent premature luteal regression; (2) a transient increase in progesterone at about the time of ovulation is associated with luteal phases of normal duration; (3) a more extended exposure to progesterone at about the time of ovulation prevents normal luteal function and may inhibit luteinization and (4) pretreatment with progesterone prevents luteolysis by reducing the uterine response to oxytocin early in the luteal phase.
M. G. Hunter, J. A. Southee and G. E. Lamming
Summary. Normal and abnormal corpora lutea were recovered from anoestrous Romney Marsh ewes on Days 3, 4, 5 and 6 after treatment with small-dose (250 ng) multiple injections of GnRH followed by a bolus injection (125 μg) with (+P) and without (−P) progesterone pretreatment and a study made of their characteristics in vitro. Plasma progesterone concentrations initially rose concurrently in all animals but abnormal luteal function occurred in 70% of the −P ewes and was defined on Day 5 when plasma progesterone concentrations declined relative to those in the +P ewes.
All corpora lutea recovered on Days 3 and 4 appeared macroscopically similar and there were no significant differences between the +P and −P groups in terms of luteal weight, progesterone content and binding of 125I-labelled hCG on these days. However, corpora lutea from the −P animals only exhibited a decline in progesterone production in vitro on Day 4 (P < 0·01), and morphological differences became apparent on Days 5 and 6 when the abnormal corpora lutea from the −P animals also decreased in weight (P < 0·01) and progesterone content (P < 0·001). Binding of 125I-labelled hCG increased on Day 5 in the normal corpora lutea only.
These results show that, although abnormal luteal function induced by GnRH treatment of anoestrous ewes could not be distinguished from normal corpora lutea before Day 5 by measurement of progesterone in peripheral plasma, a significant decline in progesterone production in vitro occurred on Day 4 in the abnormal corpora lutea. This was followed by significant decreases in weight and progesterone content and a failure to increase 125I-labelled hCG binding. Abnormal corpora lutea are therefore capable of some initial growth and progesterone production, before undergoing a rapid and premature regression from Day 4, which has similar characteristics to natural luteolysis.
Keywords: abnormal luteal function; in vitro; sheep; anoestrus
A. P. Beard, M. G. Hunter and G. E. Lamming
The effect of oestradiol and progesterone concentrations on the uterine PGF2α response to oxytocin was investigated by measuring 13,14-dihydro-15-keto PGF2α (PGFM) secretion. One week after ovariectomy, 27 ewes were administered progestagen for 10 days followed by oestradiol for 2 days. Day 0 was designated as the time of the last 'oestrous' oestradiol injection. Six groups of ewes (n = 4) were then treated for 12 days with a high or low dose of progesterone (36 or 12 mg day−1) either alone or with a high or low dose of oestradiol (36 or 12 μg day−1) administered (in 1 ml of corn oil by i.m. injection, at intervals of 8 h) in a pattern designed to simulate a natural oestrous cycle profile. A control group (n = 3) was given corn oil alone. Ewes were treated with 1 μg oxytocin (i.v.) on days 4, 8 and 12 of the simulated cycle and plasma was collected for assay of PGFM. An oxytocin-induced PGFM response occurred only on day 12, when the response was suppressed by high doses of progesterone and stimulated by high oestradiol doses. There was a significant effect of progesterone (P < 0.05) and a highly significant effect of oestradiol (P < 0.01) on the pattern of PGFM release in response to oxytocin. Low progesterone/high oestradiol stimulated the largest and most sustained increase in PGFM following oxytocin. There was a significant relationship between the oestradiol:progesterone ratio and the mean PGFM response on day 12 (P < 0.05). This is the first demonstration of a quantitative effect of steroid hormone concentrations on the PGFM response to oxytocin in ewes, and indicates that in early pregnancy, ewes with a high oestradiol:progesterone ratio may generate larger PGF2α episodes thus increasing the risk of a failure of the maternal recognition of pregnancy.
M. G. Hunter, S. A. Grant and G. R. Foxcroft
Summary. Ovaries were collected from naturally cycling gilts during the preovulatory period and the stage relative to the LH surge estimated by measurement of oestradiol and progesterone concentrations in follicular fluid. Many of the follicles recovered had become flaccid with an associated increase in follicular fluid viscosity. Marked infolding of both the granulosa and theca tissue in some follicles suggested early luteinization. However, these morphological changes did not necessarily occur simultaneously in the same follicle, or in all follicles within an ovary. Moreover, they were not consistently related to characteristic differences in the concentration of follicular fluid steroids, suggesting either that the morphological and biochemical aspects of the luteinization of follicles may be independently controlled, or may respond at different rates to the same signal.
Keywords: pig; follicle; histology; heterogeneity
S. A. Grant, M. G. Hunter and G. R. Foxcroft
Summary. Ovaries were recovered from groups of naturally cyclic pigs (N = 5) on each of Days 16, 18, 20 and 21 of the oestrous cycle. Follicular diameter, follicular fluid volume and concentrations of oestradiol, testosterone and progesterone, and granulosa cell number were determined in all follicles ⩾2 mm in diameter (n = 511). In alternate follicles either granulosa cell aromatase activity and theca testosterone content or 125I-labelled hCG binding to granulosa and theca were determined. The mean total number of follicles recovered per animal decreased as the follicular phase progressed and a strong positive relationship (P < 0·001) existed between follicular diameter and volume on all days. The number of granulosa cells recovered per follicle was variable, and not related to oestrogenic activity of the follicles. Mean follicular fluid oestradiol, testosterone and 125I-labelled hCG binding all increased until Day 20 and decreased on Day 21, whereas mean theca testosterone content, 125I-labelled hCG binding to theca tissue and aromatase were all maximal on Day 21. On Days 20 and 21 a subset of 14–16 large follicles was readily distinguishable from the remaining smaller, less oestrogenically active population in each animal. Yet, consistently within these subsets there was a difference in follicular diameter of ∼ 2·0 mm and also a considerable range of biochemical development even among follicles of equal size. These results indicate asynchrony at the time of recruitment and selection among follicles destined to ovulate and suggest that heterogeneity continues into the immediate preovulatory period.
Keywords: follicle; development; pig; heterogeneity
T. Wiesak, M. G. Hunter and G. R. Foxcroft
Summary. Luteal cells were obtained by digestion of luteal tissue of cyclic (day 12) and early pregnant (days 12, 20 and 30) pigs. Suspensions of the dispersed luteal cells (5 × 104 cells ml−1) were incubated for 2 h in minimum essential medium (MEM) alone (control) and MEM with different concentrations of prostaglandin F2α (PGF2α) and PGE2 (0·01, 0·1, 1, 10, 100 and 1000 ng ml−1) and luteinizing hormone (LH) 100 and 1000 ng ml−1, or with combinations of LH + PGF2αand LH + PGE2. Net progesterone production was measured in the incubation media by direct radioimmunoassay.
The overall response pattern of the luteal cells to exogenous hormones on day 12 of the oestrous cycle and pregnancy differed (P < 0·5) from treatment on day 20 and 30 of pregnancy. In general progesterone production was higher (P < 0·5) and the response to PGF2α and PGE2 treatment was most obvious on day 12 of the oestrous cycle and pregnancy. Overall, PGF2α stimulated progesterone production in a dosedependent manner (P < 0·05). The response to PGE2 was of a quadratic nature (P < 0·05) in which the lowest and the highest doses of PGE2 were associated with a greater production of progesterone than were the intermediate doses. Treatment of luteal cells with PGF2α + LH or PGE2 + LH caused overall inhibition (P < 0·05) of progesterone production compared with treatment with each hormone alone. This interaction was not affected by the dose of LH used.
These findings indicate that PGF2α and PGE2 are involved in the autocrine control of corpus luteum function.
Keywords: pig; luteal cells; prostaglandins; pregnancy