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TEMPORAL RELATIONSHIPS BETWEEN PERIPHERAL PLASMA CONCENTRATIONS OF OXYTOCIN, PROGESTERONE AND 13,14- DIHYDRO-15-KETO-PROSTAGLANDIN F DURING THE OESTROUS

CYCLE AND EARLY PREGNAN& IN THE EWE

R. Webb '1 $2

M. D. Mitchell , J. Falconer *+3

and J. S. Roblnsonff3.

*Nuffield Department of Obstetrics and Gynaecoloqy and the +Nuffield Institute for Medical Research, University of Oxford, Headington, Oxford. OX3 9DU, U.K.

Present addresses:

1. ARC Animal Breeding Research Organisation, West Mains Road, Edinburgh, EH9 3JQ, U.K.

2. Cecil H and Ida Green Center for Reproductive Biology Sciences and the Departments of Biochemistry and Obstetrics and Gynecology, The University of Texas, Dallas, U.S.A.

3. Faculty of Medicine, The University of Newcastle, New South IrJales, Australia.

ABSTRACT

Peripheral plasma concentrations of oxytocin, 13,14-dihydro-15-keto-prostaqlandin F2a (PGFM), proges- terone and LH were determined at 3 hourly intervals during the oestrous cycle (n = 3) and in early preg- nancy (n = 4) in sheep. The progesterone and LH concen- trations showed that the cyclinq ewes were sampled during the periods of luteal regression (decreasing progesterone concentrations), the preovulator;r gonadotrophin surge +nd the beginning of the next luteal phase (increasing progesterone concentrations). The pregnant ewes had basa LH concentrations and luteal phase concentrations of progesterone (>lng/ml after day 5 following mating) throuqhout the whole of the sampling period. Oxytocin concentrations in the non-pregnant ewes decreased around the time of luteal reqression to reach low concentrations (mean concentrations of approximately lBpg/ml) during the preovulatory period and then increased after the pre- ovulatory surge. PGFM concentrations exhibited a pul- satile pattern with increasing concentrations as progesterone levels fell. In the pregnant ewes oxytocin concentrations gradually fell until approximately 16 days post-mating (approximately 7-8pg/ml). The magnitude of the pulses in PGFM concentrations were also lower than in

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the cycling ewes. These results demonstrate that the increased concentrations of PGFM which are found during the period of luteal regression are not caused by increased peripheral concentrations of oxytocin.

INTRODUCTION

Recent experimental evidence has suggested that towards the end of the oestrous cycle in the ewe, uterine prostaglandin F (PGF 1 causes regression of the corpus luteum CZP;) (l-i?. Furthermore, increased concentrations of PGF2, have been found in utero-ovarian blood when progesterone concentrations are decreasing (5-7). However, the mechanism responsible for the release and synthesis of PGFza is not known.

Two hormones that have been closely implicated in the process of luteal regression are oestradiol-17P and oxytocin. Oestradiol-17B has been shown to cause luteal regression in the ewe (8) and stimulate the release of PGF2, from the uterus (6, g-11), but only after the uterus has been previously exposed to progesterone (9, 12, 13). Oxytocin administration has also been found to increase utero-ovarian venous PGF2, concen- trations (14). Moreover, it has been demonstrated that oxytocin receptors can be found in both the myometrium and endometrium of the uterus, with the number of binding sites reaching a peak at oestrus (15). It was postulated that the increase in the number of oxytocin binding sites around oestrus was probably influenced by increasing oestrogen concentrations. This conclusion agreed with the previous finding of Sharma and Fitzpatrick (10) that oxytocin alone has negligible effects on prostaglandin concentrations in blood collected from the posterior vena cava of anoestrous ewes, whereas when oxytocin was administered to oestrogen primed anoestrous ewes the prostaglandin concentrations rose dramatically. Further evidence indicating that oxytocin may be involved in ovine luteolysis was provided by the finding that active immunisation of sheep against oxytocin prolonged the luteal phase of the oestrous cycle (16). From temporal hormone data it has recently been postulated that oxytocin, which is considered to be released in association with neurophysins, may play an important role in ovine luteolysis by stimulating the secretion of prostaglandin F2a from the uterus during days 13-15 of the oestrous cycle (17).

The aim of this study was to investigate the temporal relationships between peripheral plasma

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concentrations of oxytocin, PGFM, the major circulating metabolite of PGF2, (181, progesterone and LH in order to determine whether increased PGFM concentrations around the time of luteolysis are correlated with increased concen- trations of oxytocin.

MATERIALS AND METHODS

Animals

Non-pregnant sheep: Four mule ewes were maintained under natural Oxford environmental conditions and observed at regular intervals during at least one normal oestrous cycle prior to treatment. Raddled vasectomized rams were used to check for oestrus. A jugular vein of each ewe was cannulated on approximately day 6 of the oestrous cycle (oestrus = day 0). Two days later lOm1 blood samples were collected at 3h intervals for the next 14 days.

Pregnant sheep: Four mule ewes were maintained under natural Oxford environmental conditions and observed at regular intervals during at least one normal oestrous cycle. Raddled vasectomized rams were used to check for oestrus. The ewes were then mated with a fertile ram. Two days after mating a jugular vein of each ewe was cannulated. Blood samples (10ml) were collected at 3h intervals from day 4 until day 17 after mating. All 4 ewes were laparotomised at least 50 days post-mating to confirm that conception and implantation had occurred.

All blood samples were collected into chilled hepariniseg tubes, placed on ice and immediately centri- fuged at 4 C and 15009 for 15 min. The plasma was divided into 2 aliquots and stored at -2OOC.

Hormone Assays

Concentrations of LH were measured using a previously described radioimmunoassay (19). LH concentrations, expressed in terms of NIH-LH-S22, were determined in duplicate in 5, 10, 100 and 200~1 aliquots of each plasma sample. The limit of detection defined as two standard deviations from the buffer controls was 67pg/ml when assaying 200 cl1 of plasma. The inter-assay coefficient of variation (CV) averaged 8.8% and the intra-assay CV averaged 5.6%. Oxytocin concentrations were measured as described previously (20). When varying concen- trations of plasma were extracted (0.5, 1, 1.5 and 2.0 ml)

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the values were parallel to the standard curve, wit9 a mean concentration (corrected for volume) of 118.5 - 2.7 (SEMI pg/ml. The limit of detection averaged 0.6 pg/tube. The inter-assay CV averaged 10.3% and the intra-assay CV averaged 9.4%. PGFM and progesterone concentrations were determined by previously described radioirnmunoassays (16,18,21). Inter-assay CV averaged 18.1% (PGFM) and 3.8% (progesterone) and the intra-assay CV averaqed 3.4% (PGFM) and 4.6% (proqesterone).

RESULTS

Non-pregnant animals: Peripheral plasma progesterone and LH concentrations indicated that 3 of the 4 ewes were exhibiting normal oestrous cycles; as demonstrated by the occurrence of a preovulatory LH surge which was preceded by a fall and then a subsequent rise in progesterone concentrations. The ewe that failed to exhibit this pattern was removed from the experiment. Plasma concentrations of oxytocin, PGFM, progesterone and LH in the rcmaininq 3 ewes are shown in Figure la, b and c. Peripheral plasma PGFM concentrations showed, a pulsatile pattern. During the period of luteal regression (decreasing progesterone concentrations) PGFM concentrations increased to reach peak values after the progesterone concentrations reached basal levels. However, the frequency and the height of the PGFM pulses subsequently decreased and remained low for the remainder of the period of investigation. Oxytocin concentrations seemed to follow a Qmilar pattern to that shown by progesterone4 with mean ( - SEM) concentrations decreasing from 24.1 - 2.7 pq/ml on day -5 (see Figure 1) to 18.3 + 3.4 pq/ml on day -1 (p>O.l, Mann Whitney Rank Sum 'Test). After the preovulatory LH surge mean oxytocin concentrations then increased significantly (p<O.OOl) to 48.7 2 3.3 pq/ml on day +4. The rise in the oxytocin concentrations seemed to be positively correlated with the rise in the progesterone concentrations that are associated with the formation of a new CL.

Pregnant ewes: Plasma concentrations of oxytocin, PGFM, progesterone and LH are shown in Figure 2a, b, c and d. Plasma LH concentrations remained low throughout the whole of the sampling period, whereas progesterone concentrations remained at luteal phase concentrations with no decreases around days 12 to 13 post-matinq. PGFM concentrations remained low throughout the study period. No large pulses in PGFM concentrations were found in the preqnant ewes similar to those found in

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Figure la, b and c. Peripheral plasma concentrations of oxytocin (o---O 1, PGFM (W ), LH ( _) and progesterone ( -1 in 3 cyclinq ewes.

-6-7-6-5-4-3-2-l 0 12 3 4 5 6

TIME (daya)

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Figure 2a, b, c and d. of oxytocin CC?--+ ),

Peripheral plasma concentrations PGFM (M 1, LH ( C--Q) and

progesterone CM 1 in 4 pregnant ewes.

3 6 8 7 8 2 to ii 12 ia 14 15 16 17

DAY6 AFTER LuTllJa

4= Lo 50

ii 100 30

10

4 4

35 5 e 7 a 2 lo 11 12 ia 14 15 16 17

DAYS AFTER MATWQ

DAY8 AFTER MAM

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3

%

2 I

3 ’ f

DAY8 AFTER UAW

the cycling animals during the period of luteal regression. However, one of the animals did have higher PGFM concentrations compared to the other 3 ewes. Mean oxytogin concentrations 15.1

declined $ignificantly from - 2.0 pg/ml on day 5 to 8.5 - 1.0 pg/ml (p<O.Oll

on day 16 post-mating.

DISCUSSION

The data presented here demonstrate that in cycling ewes peripheral plasma oxytocin concentrations exhibit a similar pattern to that shown by progesterone. This study does not support the hypothesis that increased plasma concentrations of oxytocin stimulate increased uterine production of PGF around the time of luteal regression (Figure 1). I%?eed oxytocin concentrations actually decrease around the time of luteolysis. Oxytocin associated neurophysin concentrations have been found to increase at the time of luteal regression (17). From the data described here it would seem that at certain stages of the sheep oestrous cycle neurophysin concentrations in the peripheral plasma may not always be positively correlated with oxytocin concentrations.

The fall in oxytocin concentrations around the time of luteolysis raises the question of whether oxytocin is involved in the luteolytic mechanism. The temporal relationships demonstrated here do not provide support for such an involvement. Although oxytocin binding to the uterine endometrium increases to a peak around oestrus (111, the increase was thought to be associated with increased oestrogen concentrations. However, the oestrogen rise does not occur until progesterone concentrations have started to decline (71, thus inferring that the increase in the number of oxytocin

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binding sites does not occur until after the initiation of luteal reqrcssion.

As reported previously (17,22), we found a pulsatile pattern of the peripheral plasma PGFM concentrations with peak levels occurring when progesterone concen- trations had reached basal levels. Furthermore the pul- satile pattern of plasma PGFM secretion is consistent with the finding of earlier studies that PGF

* Z” in the

ewe is released from the uterus in a pulsate e fashion with increased concentrations around the time of luteal regression (5-7). Previous studies have further demonstrated that after progesterone concentrations have started to decrease oestradiol concentrations increase (7,25). Therefore the results presented here support previous conclusions that PGF2, release is enhanced when physiological amounts of oestrogen are imposed on a proqesterone primed uterus (7,131. After the pre- ovulatory surge, however, the frequency and height of the PGFM pulses seemed to decrease and exhibited a pattern of release similar to that in the pregnant ewes (Figure 2). The absence of large peaks of PGFM in pregnant ewes on days 13-14 after mating agrees with an earlier study (22) and also with measurements of PGF concentrations in the utero-ovarian vein of ewes at Zfe same staqe following mating (6).

It is interesting to note that the pregnant ewes had lower concentrations of oxytocin than the non-pregnant ewes, except around the time of the preovulatory LH surge. One possible explanation could be related to the time of year when the experiments were carried out as the pregnant ewes were sampled during October whereas the non-pregnant ewes were sampled in January. However in a more recent study (23) oxytocin concentrations in peripheral blood samples collected from cycling ewes during October showed similar concentrations to those found in the non-pregnant ewes in this study. It there- fore appears that the time of the year is not the reason for the difference. One further explanation could pertain to the different physiological states of the ewes and this might imply that the embryo, even prior to implantation, at approximately 16 days post-mating (24) can exert an influence resulting in decreased plasma concentrations of oxytocin. f-low this decrease could be effected is not known. There was, however, a decrease in the concentrations of oxytocin throughout the period of investigation in the pregnant ewes suggesting that the influence may be enhanced as pregnancy progresses.

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ACKNOWLEDGEMENTS

The technical assistance of Jane Brunt, Linda Clover, Linda Gaden, Lynne Harrison and Lesley Mountford, and the secretarial assistance of Ray Anson are gratefully appreciated. The antiserum to oxytocin and authentic standards were the kind gift of Drs. J. Dogterom and D. F. Swaab (Institute for Brain Research, Amsterdam). MDM is an MRC Senior Fellow.

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