Reprod. Fertil. Dev., 1990, 2, 89-99
Effect of Continuous Infusion of Oxytocin on Prostaglandin FzolSecretion and Luteolysis in the Cyclic Ewe
E. L. Sheldrick and A . P. F.
lint^
AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, UK. A Present address: Institute of Zoology, Regent's Park, London NW1 4RY, UK.
Abstract Circulating concentrations of 13,14-dihydro-15-keto PGF2, (DHKF2,), luteinizing hormone (LH) and prolactin (PRL) have been measured in cyclic ewes treated with continuous infusions of oxytocin, in order to investigate the mechanism by which the treatment delays luteal regression. Continuous infusion of oxytocin reduced prostaglandin F2, (PGF2,) secretion but had no detectable direct effect on LH or PRL. Oxytocin (3 nmol h-' i.v.) given from Day 12 or 13 until Day 18 after oestrus delayed luteolysis, eight out of nine treated ewes not returning to behavioural oestrus until Day 29.1 k 3.2 (mean + s.e.m.; cycle length of control ewes 16.7 f 0 . 3 days). In the ewe in which oxytocin failed to prevent luteolysis, luteal regression had commenced before oxytocin treatment was started. In three ewes undergoing delayed luteolysis (cycle lengths, 21, 24 and 25 days) basal concentrations of PGF2, (measured as DHKF2,) were unchanged, but there was only one episode of PGF2, secretion compared with 20 episodes in three control ewes. Prolactin secretion was pulsatile during oxytocin infusion, and levels were low following infusion in ewes with cycle length >25 days while the corpora lutea were maintained. Circulating PRL concentrations were high in ewes undergoing delayed luteolysis but there was no discrete episode of PRL secretion associated with the pre-ovulatory LH surge in these animals. To investigate the possibility that the pattern of PGF2, secretion was affected by depletion of oxytocin from corpora lutea, ewes previously treated with oxytocin to delay luteolysis were given a luteolytic dose of cloprostenol on Day 21 after oestrus. The amount of oxytocin secreted in response to cloprostenol was < 10% of that seen in ewes similarly treated on Days 11-13 after oestrus. Low levels of luteal oxytocin may therefore reduce PGF2, secretion in ewes undergoing delayed luteolysis. Keywords: luteinizing hormone, prolactin, corpus luteum.
Introduction Oxytocin is synthesized in and secreted by the corpus luteum of the sheep (Flint and Sheldrick 1982; Wathes and Swann 1982). In cyclic ewes, luteal concentrations of oxytocin rise early in the cycle reaching maximal levels between Days 4 and 10, before declining to low levels between Days 12 and 14 (Sheldrick and Flint 1989). Concentrations of mRNA encoding the oxytocin-neurophysin prohormone are highest before Day 5 of the cycle, falling to low levels by Day 9 (Jones and Flint 1988), and the decline in basal circulating and luteal concentrations of oxytocin observed in the latter half of the oestrous cycle can therefore be accounted for by reduced luteal concentrations of mRNA. Evidence is accumulating that luteal oxytocin regulates uterine production of PGF2, in the sheep. Endometrial concentrations of oxytocin receptor rise at the end of the luteal phase of the oestrous cycle, reaching maximal levels at oestrus (Roberts et al. 1976; Sheldrick and Flint 1985). The presence of this receptor allows exogenous oxytocin to cause rapid secretion 1031-3613/9O/OlOO89$O3.OO
E. L. Sheldrick and A. P. F. Flint
of endometrial PGF,,: oxytocin stimulates hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol, thereby providing arachidonic acid as a precursor for PGF2, synthesis (Flint et al. 1986). Exogenous PGF,, and its analogue cloprostenol cause rapid secretion of luteal oxytocin into the ovarian vein (Flint and Sheldrick 1982). Episodic secretion of PGF,, is thought to be required for efficient luteolysis to occur (Schramm et al. 1983), and concurrent episodes of PGF,, and oxytocin or oxytocin-associated neurophysin secretion have been detected at luteolysis in the sheep (Fairclough et al. 1980; Flint and Sheldrick 1983; Moore et al. 1986). Administration of a luteolytic dose of oestradiol induces increased concentrations of uterine oxytocin receptor before the onset of episodic PGF,, secretion and luteolysis (Hixon and Flint 1987). Further evidence that oxytocin and its receptor have a role in endocrine control of luteolysis is provided by the effect of continuous infusion of oxytocin in cyclic sheep. Oxytocin administered to ewes between Days 13 and 21 after oestrus delays return to oestrus by a mean interval of 7 days (Flint and Sheldrick 1985), possibly by down-regulating the oxytocin receptor and thereby blocking oxytocin-stimulated PGF2, secretion. However, the mechanism underlying this effect is not fully understood. The experiments described here were undertaken to determine whether PGF,, secretion is affected by continuous oxytocin administration. 13,14-Dihydro-15-keto-PGF,,(DHKF,,), oxytocin and progesterone have been measured during oxytocin infusion and at luteal regression following withdrawal of oxytocin, in order to determine whether oxytocin blocks or reduces uterine PGF2, secretion and whether delayed luteolysis occurs in a manner similar to luteal regression during the normal oestrous cycle. In addition, the contribution of luteal oxytocin to regulation of PGF,, in delayed.lutea1 regression was assessed by measuring cloprostenol-induced oxytocin secretion in ewes in which luteal function was extended by oxytocin treatment. Circulating plasma concentrations of PRL and of LH during the pre-ovulatory surge were measured in order to investigate possible effects of oxytocin infusion on these parameters. Materials and Methods Animals Eighteen Clun Forest ewes were kept in a paddock or covered yard with a crayon-bearing vasectomized ram. Ewes were checked daily for oestrous behaviour and had exhibited at least two normal oestrous cycles (16.8 + 0.1 days mean + s.e.m.; N = 18, n = 65, where N is number of animals and n is number of observations) prior to experimentation. Experiments were performed in December 1986, January, February and December 1987, and January 1988 at a latitude of 52"N. Animals were assigned at random to four experimental groups, as follows: Group A: three ewes infused with sterile saline (150 mmol L - ' NaC1, 3 mL h-') beginning on Day 13 (oestrus = Day 0); blood samples were collected from these ewes at 90-min intervals from Day 13 until Day 18; Group B: three ewes infused with oxytocin (3 nmol h - ' in sterile saline) from Day 13 to Day 18; blood samples were collected at 90-min intervals from Day 13 to Day 20; Group C: six ewes infused with oxytocin (3 nmol h-') from Day 12 to Day 18, blood samples being collected at 90-min intervals from Day 18 to Day 25 and then daily until return to behavioural oestrus. For the purpose of presenting the results, Groups B and C have been subdivided on the basis of the experimental outcome. A further six ewes were infused with oxytocin (3 nmol h-') from Day 12 to Day 18. Blood samples were collected three times a day for progesterone determination. On Day 21 these ewes were given a luteolytic dose of cloprostenol (Estrumate, Coopers Animal Health Ltd, UK; 125 pg i.m.; N = 4) or saline (0.5 mL ism.; N = 2) at t = 0 and blood samples were collected for oxytocin determination at t = -15, -10, -5, +2, +5, +8, +11.5, +15, +20, +25, +30, +40, +50 and +60min. During experimentation, ewes were kept in metabolism cages under conditions that allowed infusions to be carried out and blood samples to be collected without disturbance (Flint and Sheldrick 1983). They were exposed to ambient temperature and natural daylength. A vasectomized ram was housed with the ewes throughout the experiment. While in metabolism cages, sheep were fed chopped hay and water ad libitum plus 250 g concentrates per day. A polyvinyl chloride catheter 0.d. 2.1 mm was inserted into each external jugular vein, under local anaesthesia by the method of Seldinger (1953). One catheter was used for infusion of oxytocin (Bachem
Oxytocin Infusion in Ewes
Inc. Torrance, CA, USA) which was prepared in sterile physiological saline (150 mmol L-') and infused by means of a peristaltic pump (0.05 mL min-I). The second catheter was used for withdrawal of blood samples during periods of frequent sampling.
Radioimmunoassay for Oxytocin Oxytocin was measured in jugular venous plasma as described previously (Sheldrick and Flint 1981). Extraction of 'ZS~-oxytocinfrom sheep plasma was 77 + 1%. The sensitivity of the assay (calculated from 2 x s.d. below the zero point) was 1 . 3 Em01 (equivalent to 1.6 pmol L-' plasma). Intra- and inter-assay coefficients of variation were 9.9 and 6.8970, respectively. Values were corrected for extraction losses. Radioimmunoassay for Progesterone Progesterone was measured in plasma after extraction with diethyl ether as described previously (Sheldrick et al. 1980). Sensitivity of the assay reported here was 41.0 fmol tube (equivalent to 0.452 nmol L - ' plasma). Intra- and inter-assay coefficients of variation were 9.7 and 14.5%. Extraction recovery was 90 + 1% and values were corrected for extraction losses. Radioimmunoassay for I3,14-Dihydro-15-keto-PGF2, Concentrations of DHKF2, were measured in plasma by the method of Mitchell et al. (1976). Assay sensitivity and intra- and inter-assay coefficients of variation were 29.3 pmol L-', 6.0% and 8.5% respectively. Mean extraction recovery was 86 f 1% and values were corrected for extraction losses. Radioimmunoassay for Luteinizing Hormone Plasma concentrations of LH were determined by double-antibody radioimmunoassay using an antiserum raised in a rabbit (Lindsay 1982). Sensitivity of the assay was 0.96 pg L-'. Intra- and inter-assay coefficients of variation were 9 . 0 and 12.1%, respectively. Radioimmunoassay for Prolactin Prolactin was measured in plasma by a radioimmunoassay using an antiserum raised in a rabbit against ovine PRL (NIH-P-Sll; Lindsay 1982). Bovine PRL (LER891, equivalent to NIH-P-B4) was used as radiolabel and standard. Antibody-bound and free PRL were separated by a second precipitating antibody, and Staphylococcus aureus cells (Pansorbin, Calbiochem, Behring Diagnostics, La Jolla, CA, USA; 200 pg tube) were added immediately before centrifugation to increase the size and stability of the pellet. Displacement curves for bovine and ovine PRL were parallel (Bicknell and Chapman 1983); PRL in plasma samples diluted in parallel to the standard curve. Sensitivity of the assay was 1.15 pg L-' plasma, and intra- and inter-assay coefficients of variation were 8 . 8 and 17.9% respectively. Data Analysis and Statistics All values are reported as mean f s.e.m. The number of animals is indicated by N a n d the number of observations by n. Where pooled data are reported mean and s.e.m. are based on N. Hormone pulses are identified as being statistically significant when peak values are greater than 3 standard deviations (s.d.; calculated from the basal values) above the mean basal concentration and are also separated from an adjacent peak by at least one sample lying within 1 s.d. of the basal level and at least 3 s.d. below both adjacent peaks.
Results
Hormone Changes at Spontaneous Luteal Regression In three saline-treated sheep in which luteal regression occurred between Days 13 and 15 (Group A, Fig. 1) changes in circulating concentrations of DHKF,,, oxytocin and progesterone were similar to those described previously (Fairclough et al. 1980; Flint and Sheldrick 1983; Hooper et al. 1986). Episodes of secretion of DHKF,, were noted at mean intervals of between 4 and 12 h; mean basal circulating concentrations of DHKF,, were: (animal a) 176.6 + 7.9, n = 20; (b) 167.3 + 10.7, n = 25; (c) 223.4 + 13.2 pmol L-', n = 23, and the mean peak concentration reached during episodes of secretion was
E. L. Sheldrick and A. P . F. Flint
Days after oesbus
Days after oestrus
Fig. 1. Plasma progesterone (O), DHKF2, (O), LH (hatched areas), prolactin (0)and oxytocin (A) in samples collected at 90-min intervals from animals in Group A, three ewes (a-c) which received vehicle (NaCl, 150 mmol L-', 3 mL h-') between Days 13 and 18.
Days after oestrus
714.4 f 25.4 pmol L-'; N = 3; n = 20). All three ewes showed increased PRL secretion at the time of the pre-ovulatory LH surge, and in one ewe circulating PRL concentrations also increased at luteolysis (Fig. lc). High-amplitude pulses of oxytocin secretion were Table 1. Effect of treatment on luteolysis and return to oestrus in ewes Group
N
Treatment
A
3 2 1 3 3
saline infusion oxytocin infusion oxytocin infusion oxytocin infusion oxytocin infusion
B1 B2 C1 Cz
Treatment Sampling Day on which Day of period period circulating pre-ovulatory (days) (days) progesterone LH surge concentration fell to below 1.5 nmol L-' 13-18 13-18 13-18 12-18 12-18
13-18 13-20 13-20 18-25 18-24
14, 14, 15 >20 14 19, 22, 22 26, 32, 33
16, not 15 21, not
17, 17 observed 23, 25 detected
Day of return to behavioural oestrus
16, 17, 17 22, 48 16 21, 24, 25 26, 32, 35
Oxytocin Infusion in Ewes
detected in all three ewes, 21% of which were synchronous with episodes of DHKF,, secretion.
Hormone Changes in Animals Treated with Oxytocin Oxytocin infusion delayed return to oestrus in eight of nine treated ewes (see Table 1). In three ewes in which peripheral blood samples were collected during oxytocin infusion
Days aftec oestrus Fig. 2. Plasma progesterone (a), DHKF2, (O), and prolactin (0) in two ewes (Group B1),given oxytocin (3 nmol h-I, i.v.) from Day 13 to 18. Duration of the infusion is indicated by the horizontal bar. Peripheral blood samples were collected at 90-min intervals from Day 13 to 20. Ewes a and b returned to oestrus on Days 22 and 48, respectively.
(Group B), circulating oxytocin concentrations were raised from a mean level of 12.9 f 5 . 6 (N = 3; n = 12) to 59.9 k 2 . 3 pmol L-', n = 77 (Ewe a, Fig. 2); 74.8 f 1.9, n = 79 (Ewe b, Fig. 2) and 38.6 f 1 . 0 , n = 80 (Fig. 3) during infusion. In two of these ewes
E. L. Sheldrick and A. P. F. Flint
(Group B1, Fig. 2) progesterone levels remained similar to those observed before oxytocin administration but in the third ewe, in which luteal regression had commenced before oxytocin infusion (Fig. 3), oxytocin failed to prevent completion of luteolysis. In those animals in which luteal regression was blocked (Fig. 2), DHKF2, concentrations remained low during and after infusion; episodes of secretion such as those observed in the control group were absent. Prolactin secretion was pulsatile and LH secretion remained at basal levels. In the ewe (Group B2; Fig. 3) which returned to oestrus on Day 16, oxytocin infusion initially caused a single high-amplitude pulse of DHKF,, secretion; thereafter, circulating concentrations remained at 345.9 + 32.4 pmol L - N = 1; n = 25, and during this period no significant episodes of secretion were detected.
',
Days after oestrus DHKF2, (a),LH (hatched area), prolactin (0)and oxytocin (A) concenFig. 3. Progesterone (O), trations in one ewe (Group B2) given oxytocin infusion from Day 13 to Day 18 (as indicated by horizontal bar). The ewe returned to oestrus on Day 16. As is apparent from falling progesterone concentrations and increased DHKF2, secretion at the start of the oxytocin infusion, luteolysis had commenced in this ewe before treatment was started.
In a further six ewes, blood samples were obtained after the withdrawal of oxytocin infusion; in three of these animals blood samples were collected during luteal regression between Days 19 and 22 (Group C1; Fig. 4), and in three ewes the sampling period preceded luteolysis (Group CZ, Fig. 5). In Group C1, luteal regression occurred without frequent high-amplitude episodes of DHKF,, secretion comparable to those observed in the control group (Group A); in one of these ewes (Fig. 4b) one significant episode of DHKF,, secretion was detected at luteolysis, while in the other two animals (Fig. 4a, c) no significant episodes were detected. During luteolysis, ewes in Group C1 (Fig. 4a-c) had the following basal circulating concentrations of DHKF,,: (animal a) 289.3 + 27 3 pmol mLn = 37; (b) 221.4 k 12.1, n = 42; (c) 194.9 k 17.5, n = 20, and oxytocin concentrations remained low throughout luteolysis (a) 3.2 + 0 . 2 pmol L-', n = 37; (b) 3.1 f 0.5, n = 42; (c) 5.1 k 0.6, n = 20. In all three ewes in Group C1, luteal regression was accompanied by circulating PRL levels that were high compared with those in ewes not
',
Oxytocin Infusion in Ewes
Days aftw
OBS~~US
Fig. 4. Progesterone (O), DHKF2, (a),LH (hatched areas), prolactin (0)and oxytocin (A) in three ewes (a-c) sampled at 90-min intervals from withdrawal of oxytocin until day of oestrus (Group C,).
Days after oesbus
Days aftec oesbus
E. L. Sheldrick and A. P. F. Flint
Days after oestrus
Days after oestrus
Days after oestrus
DHKF2, (e), prolactin (0)and oxytocin (A) concentrations in jugular Fig. 5. Progesterone (O), venous plasma from three ewes (a-c) in which oxytocin delayed luteolysis for more than 10 days (Group C2).
Oxytocin Infusion in Ewes
undergoing luteolysis at a comparable stage after oestrus (Group C2, Fig. 5), and there was no discrete PRL release associated with the pre-ovulatory LH surge. Concentrations of LH were consistently low ( ~ 7 . 4pg L-') in the animals sampled after oxytocin infusion but before luteolysis (Group C2); circulating PRL levels were low ( ~ 5 1 . 0pg L-') with the exception of one sample where the animal was handled during sampling on Day 21 (Fig. 5a). DHKF,, concentrations were low (mean, 140-3 pmol L-'; N = 2; n = 207) in two of these ewes but raised basal secretion was observed in the animal that returned to oestrus earliest in this group (253.2 k 9.9 pmol L-'; n = 101; Fig. 5a). There were no surges of oxytocin associated with DHKF,, secretion in these animals.
Fig. 6 . Concentrations of oxytocin in jugular venous plasma of oxytocin-treated ewes (N = 4) given a luteolytic dose of cloprostenol Day 21 after oestrus. Mean maximal jugular venous concentrations in control ewes treated with cloprostenol on Days 11-13 after oestrus were 113 k 38 pmol L-'; this level was reached 10-20 min after treatment (Flint and Sheldrick 1983).
i7me (minutes)
Oxytocin Secretion in Response to Cloprostenol in Animals in which Luteal Function was Prolonged Following Oxytocin Infusion The mean concentration of oxytocin in jugular venous plasma before treatment was 2.4 f 0.5 pmol L-' (N = 6; n = 20). In four ewes treated with cloprostenol on Day 21 after oestrus the mean circulating oxytocin concentration rose to maximal levels (13.0 f 6.6 pmol L-'; N = 4; n = 4) 11.5 min after treatment (Fig. 6), and declined to 3.8 f 1.2 pmol L-' (N = 4; n = 4) after 60 min. In two control ewes, mean circulating oxytocin concentrations were 1.51 pmol L-' (N = 2; n = 6) before and 1.55 pmol L-' (N = 2; n = 14) during the 60 min after saline administration. Mean circulating concentration of progesterone was 12.7 3.2 nmol L-' ( N = 6; n = 20) before treatment, indicating that the ewes had functional corpora lutea. Progesterone levels fell to < 1.5 nmol L-' within 24 h of cloprostenol treatment and behavioural oestrus was detected within 48 h. Discussion The experiments described here show that continuous oxytocin infusion blocks the episodic secretion of PGF2, normally associated with luteal regression. This observation is consistent with the hypothesis that oxytocin infusion down-regulates the uterine oxytocin receptor (Flint and Sheldrick 1985). Oxytocin receptor concentrations are low on Days 12 or 13 of the oestrous cycle (Sheldrick and Flint 1985), when the infusions were started in these experiments, and therefore it must be assumed that the low level of receptor expressed in the uterus at this time is sufficient to lead to down-regulation.
E. L. Sheldrick and A. P. F. Flint
Blood samples were taken too infrequently to provide any information on whether oxytocin decreases LH pulse frequency or amplitude in those animals in which ovulation was blocked. However, as shown previously (Flint and Sheldrick 1985), oxytocin infusion failed to prevent completion of luteolysis in one ewe in which luteal regression had started before oxytocin infusion had begun and in this ewe there was a normal pre-ovulatory LH surge followed by luteinization; therefore it appears unlikely that oxytocin infusion acts primarily by inhibiting LH secretion. This ewe responded to oxytocin with a single highamplitude pulse of prostaglandin secretion which is indicative of the presence of functional uterine oxytocin receptors. Similar episodes of PGF,, secretion were seen in cattle given oxytocin infusion starting on Day 15 after oestrus (Kotwica et al. 1988), a treatment which failed to prolong luteal function. It seems possible therefore that oxytocin infusion fails to down-regulate the receptor once levels of receptor reach a certain point; under these conditions oxytocin administration causes PGF,, secretion and luteal regression. There was no immediate rise in PGF,, production following withdrawal of oxytocin infusion (Fig. 2), and when luteolysis did occur following withdrawal of oxytocin it was associated with reduced PGF,, release, compared with that in untreated ewes at luteolysis. This suggests that the trigger to episodic PGF,, release was blocked or ameliorated for a long period. One explanation for the absence of episodes of PGF,, secretion is that when luteal function is extended beyond the normal time of luteolysis, the corpus luteum loses its ability to synthesize oxytocin; this occurs in pregnancy and after hysterectomy (Sheldrick and Flint 1983a, 19833) as well as after immunization against PGF,, or oxytocin (Sheldrick and Flint 1984), and may reflect loss from the tissue of oxytocin-neurophysin messenger RNA (Jones and Flint 1988). This mechanism was confirmed in the present study in animals treated with a luteolytic dose of cloprostenol on Day 21, following oxytocin infusion between Days 12 and 18; peripheral circulating oxytocin concentrations in these ewes were raised in response to cloprostenol (Fig. 6 ) , but the level reached was less than 10% that observed in otherwise untreated mid-cycle ewes (Flint and Sheldrick 1983). Cloprostenol fails to stimulate oxytocin secretion in ovariectomized ewes (Flint and Sheldrick 1982), therefore it seems unlikely that a pituitary component is involved in this response. The occurrence of luteal regression in the absence of episodes of PGF,, secretion noted here following oxytocin withdrawal is difficult to explain as PGF,, is the only currently recognised luteolysin. It is unlikely to reflect increasing sensitivity of the corpus luteum to low levels of PGF,, since no such hypersensitivity has been reported during pregnancy. An alternative possibility is that a different compound causes luteolysis at this time. Prolactin causes structural luteolysis in aged corpora lutea in the mouse (Grandison and Meites 1972) and in rats in which luteal function has been extended by hypophysectomy (Malven and Sawyer 1966; Malven 1969), and has recently been shown to be luteolytic in the tammar wallaby (Tyndale-Biscoe et al. 1988). Furthermore, the circulating concentrations of prolactin were raised at luteal regression following oxytocin infusion. Therefore the possibility exists that PRL may be luteolytic in sheep when luteal function is extended beyond the normal time of luteolysis, although other mechanisms, such as withdrawal of an unidentified luteotrophin or secretion of an unidentified luteolysin, cannot be excluded. References Bicknell, R. J., and Chapman, C. (1983). Bombesin stimulates growth hormone secretion from cultured bovine pituitary cells. Neuroendocrinology 36, 33-8. Fairclough, R. J., Moore, L. G., McGowan, L. T., Peterson, A. J., Smith, J. F., Tervit, H. R., and Watkins, W. B. (1980). Temporal relationship between plasma concentrations of 13,14-dihydro-15keto-prostaglandin F and neurophysin 1/11 around luteolysis in sheep. Prostaglandins 20, 199-208. Flint, A. P. F., and Sheldrick, E. L. (1982). Ovarian secretion of oxytocin is stimulated by prostaglandin. Nature (Lond.) 297, 587-8. Flint, A. P. F., and Sheldrick, E. L. (1983). Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep. J. Reprod. Fertil. 67, 215-25.
Oxytocin Infusion in Ewes
Flint, A. P. F., and Sheldrick, E. L. (1985). Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes. J. Reprod. Fertil. 75, 623-31. Flint, A. P. F., Leat, W. M. F., Sheldrick, E. L., and Stewart, H. J . (1986). Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium. Biochem. J. 237, 797-805. Grandison, L., and Meites, J . (1972). Luteolytic action of prolactin during estrous cycle of the mouse. Proc. Soc. Exp. Biol. 140, 323-5. Hixon, J. E., and Flint, A. P. F. (1987). Effects of a luteolytic dose of oestradiol benzoate on uterine oxytocin receptor concentrations, phosphoinositide turnover and prostaglandin F-2a secretion in sheep. J. Reprod. Fertil. 79, 457-67. Hooper, S. B., Watkins, W. B., and Thorburn, G. D. (1986). Oxytocin, oxytocin associated neurophysin, and prostaglandin F2a concentrations in the utero-ovarian vein of pregnant and non-pregnant sheep. Endocrinology 119, 2590-7. Jones, D. S. C., and Flint, A. P. F. (1988). Concentrations of oxytocin-neurophysin prohormone mRNA in corpora lutea of sheep during the oestrous cycle and in early pregnancy. J. Endocrinol. 117, 409-14. Kotwica, J., Schams, D., Meyer, H. H. D., and Mittermeier, Th. (1988). Effect of continuous infusion of oxytocin on length of the oestrous cycle and luteolysis in cattle. J. Reprod. Fertil. 83, 284-94. Lindsay, K. S. (1982). Pituitary gonadotrophin responsiveness in sheep: effects of reproductive stage and nutrition. Ph.D. Thesis, University of Cambridge. Malven, P . V. (1969). Luteotrophic and luteolytic responses to prolactin in hypophysectomised rats. Endocrinology 84, 1224-9. Malven, P. V., and Sawyer, C. H. (1966). A luteolytic action of prolactin in hypophysectomised rats. Endocrinology 79, 268-74. Mitchell, M. D., Flint, A. P. F., and Turnbull, A. C. (1976). Plasma concentrations of 13,14-dihydro15-keto-prostaglandin F during pregnancy in sheep. Prostaglandins 11, 319-29. Moore, L. G., Choy, V. J., Elliot, R. L., and Watkins, W. B. (1986). Evidence for the pulsatile release of PGF2, inducing the release of ovarian oxytocin during luteolysis in the ewe. J. Reprod. Fertil. 76, 159-66. Roberts, J. S., McCracken, J . A., Gavagan, J. E., and Soloff, M. S. (1976). Oxytocin-stimulated release of prostaglandin F from ovine endometrium in vitro: correlation with estrous cycle and oxytocin-receptor binding. Endocrinology 99, 1107-14. Schramm, W., Bovaird, L., Glew, M. E., Schramm, G., and McCracken, J. A. (1983). Corpus luteum regression induced by ultra-low pulses of prostaglandin F2a. Prostaglandins 26, 347-64. Seldinger, S. I. (1953). Catheter replacement of the needle in percutaneous arteriography. Acta Radiol. 39, 368-76. Sheldrick, E. L., and Flint, A . P. F. (1981). Circulating concentrations of oxytocin during the estrous cycle and early pregnancy in sheep. Prostaglandins 22, 631-6. Sheldrick, E. L., and Flint, A P . F. (1983a). Luteal concentrations of oxytocin decline during early pregnancy in the ewe. J. Reprod. Fertil. 68, 477-80. Sheldrick, E. L., and Flint, A. P . F. (1983b). Regression of the corpora lutea in sheep in response to cloprostenol is not affected by loss of luteal oxytocin after hysterectomy. J. Reprod. Fertil. 68, 155-60. Sheldrick, E. L., and Flint, A. P. F. (1984). Ovarian oxytocin. In 'Gonadal Proteins and Peptides and their Biological Significance'. (Eds M. R. Sairam and L. E. Atkinson.) pp. 257-72. (World Scientific Publishing: Singapore.) Sheldrick, E. L., and Flint, A P . F. (1985). Endocrine control of uterine oxytocin receptors in the ewe. J. Endocrinol. 106, 249-58. Sheldrick, E. L., and Flint, A. P. F. (1989). Post-translational processing of oxytocin-neurophysin prohormone in the ovine corpus luteum: activity of peptidyl glycine a-amidating mono-oxygenase and concentrations of its cofactor, ascorbic acid. J. Endocrinol. 122, 313-22. Sheldrick, E. L., Mitchell, M. D., and Flint, A. P. F. (1980). Delayed luteal regression in ewes immunised against oxytocin. J. Reprod. Fertil. 59, 37-42. Tyndale-Biscoe, C. H., Hinds, L. A,, and Horn, C. A. (1988). Fetal role in the control of parturition in the tammar, Macropus eugenii. J. Reprod. Fertil. 82, 419-28. Wathes, D. C., and Swann, R. W. (1982). Is oxytocin an ovarian hormone? Nature (Lond.) 297, 225-7. Manuscript received 25 August 1989, accepted 3 October 1989
Effect of Continuous Infusion of Oxytocin on Prostaglandin FzolSecretion and Luteolysis in the Cyclic Ewe
E. L. Sheldrick and A . P. F.
lint^
AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, UK. A Present address: Institute of Zoology, Regent's Park, London NW1 4RY, UK.
Abstract Circulating concentrations of 13,14-dihydro-15-keto PGF2, (DHKF2,), luteinizing hormone (LH) and prolactin (PRL) have been measured in cyclic ewes treated with continuous infusions of oxytocin, in order to investigate the mechanism by which the treatment delays luteal regression. Continuous infusion of oxytocin reduced prostaglandin F2, (PGF2,) secretion but had no detectable direct effect on LH or PRL. Oxytocin (3 nmol h-' i.v.) given from Day 12 or 13 until Day 18 after oestrus delayed luteolysis, eight out of nine treated ewes not returning to behavioural oestrus until Day 29.1 k 3.2 (mean + s.e.m.; cycle length of control ewes 16.7 f 0 . 3 days). In the ewe in which oxytocin failed to prevent luteolysis, luteal regression had commenced before oxytocin treatment was started. In three ewes undergoing delayed luteolysis (cycle lengths, 21, 24 and 25 days) basal concentrations of PGF2, (measured as DHKF2,) were unchanged, but there was only one episode of PGF2, secretion compared with 20 episodes in three control ewes. Prolactin secretion was pulsatile during oxytocin infusion, and levels were low following infusion in ewes with cycle length >25 days while the corpora lutea were maintained. Circulating PRL concentrations were high in ewes undergoing delayed luteolysis but there was no discrete episode of PRL secretion associated with the pre-ovulatory LH surge in these animals. To investigate the possibility that the pattern of PGF2, secretion was affected by depletion of oxytocin from corpora lutea, ewes previously treated with oxytocin to delay luteolysis were given a luteolytic dose of cloprostenol on Day 21 after oestrus. The amount of oxytocin secreted in response to cloprostenol was < 10% of that seen in ewes similarly treated on Days 11-13 after oestrus. Low levels of luteal oxytocin may therefore reduce PGF2, secretion in ewes undergoing delayed luteolysis. Keywords: luteinizing hormone, prolactin, corpus luteum.
Introduction Oxytocin is synthesized in and secreted by the corpus luteum of the sheep (Flint and Sheldrick 1982; Wathes and Swann 1982). In cyclic ewes, luteal concentrations of oxytocin rise early in the cycle reaching maximal levels between Days 4 and 10, before declining to low levels between Days 12 and 14 (Sheldrick and Flint 1989). Concentrations of mRNA encoding the oxytocin-neurophysin prohormone are highest before Day 5 of the cycle, falling to low levels by Day 9 (Jones and Flint 1988), and the decline in basal circulating and luteal concentrations of oxytocin observed in the latter half of the oestrous cycle can therefore be accounted for by reduced luteal concentrations of mRNA. Evidence is accumulating that luteal oxytocin regulates uterine production of PGF2, in the sheep. Endometrial concentrations of oxytocin receptor rise at the end of the luteal phase of the oestrous cycle, reaching maximal levels at oestrus (Roberts et al. 1976; Sheldrick and Flint 1985). The presence of this receptor allows exogenous oxytocin to cause rapid secretion 1031-3613/9O/OlOO89$O3.OO
E. L. Sheldrick and A. P. F. Flint
of endometrial PGF,,: oxytocin stimulates hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol, thereby providing arachidonic acid as a precursor for PGF2, synthesis (Flint et al. 1986). Exogenous PGF,, and its analogue cloprostenol cause rapid secretion of luteal oxytocin into the ovarian vein (Flint and Sheldrick 1982). Episodic secretion of PGF,, is thought to be required for efficient luteolysis to occur (Schramm et al. 1983), and concurrent episodes of PGF,, and oxytocin or oxytocin-associated neurophysin secretion have been detected at luteolysis in the sheep (Fairclough et al. 1980; Flint and Sheldrick 1983; Moore et al. 1986). Administration of a luteolytic dose of oestradiol induces increased concentrations of uterine oxytocin receptor before the onset of episodic PGF,, secretion and luteolysis (Hixon and Flint 1987). Further evidence that oxytocin and its receptor have a role in endocrine control of luteolysis is provided by the effect of continuous infusion of oxytocin in cyclic sheep. Oxytocin administered to ewes between Days 13 and 21 after oestrus delays return to oestrus by a mean interval of 7 days (Flint and Sheldrick 1985), possibly by down-regulating the oxytocin receptor and thereby blocking oxytocin-stimulated PGF2, secretion. However, the mechanism underlying this effect is not fully understood. The experiments described here were undertaken to determine whether PGF,, secretion is affected by continuous oxytocin administration. 13,14-Dihydro-15-keto-PGF,,(DHKF,,), oxytocin and progesterone have been measured during oxytocin infusion and at luteal regression following withdrawal of oxytocin, in order to determine whether oxytocin blocks or reduces uterine PGF2, secretion and whether delayed luteolysis occurs in a manner similar to luteal regression during the normal oestrous cycle. In addition, the contribution of luteal oxytocin to regulation of PGF,, in delayed.lutea1 regression was assessed by measuring cloprostenol-induced oxytocin secretion in ewes in which luteal function was extended by oxytocin treatment. Circulating plasma concentrations of PRL and of LH during the pre-ovulatory surge were measured in order to investigate possible effects of oxytocin infusion on these parameters. Materials and Methods Animals Eighteen Clun Forest ewes were kept in a paddock or covered yard with a crayon-bearing vasectomized ram. Ewes were checked daily for oestrous behaviour and had exhibited at least two normal oestrous cycles (16.8 + 0.1 days mean + s.e.m.; N = 18, n = 65, where N is number of animals and n is number of observations) prior to experimentation. Experiments were performed in December 1986, January, February and December 1987, and January 1988 at a latitude of 52"N. Animals were assigned at random to four experimental groups, as follows: Group A: three ewes infused with sterile saline (150 mmol L - ' NaC1, 3 mL h-') beginning on Day 13 (oestrus = Day 0); blood samples were collected from these ewes at 90-min intervals from Day 13 until Day 18; Group B: three ewes infused with oxytocin (3 nmol h - ' in sterile saline) from Day 13 to Day 18; blood samples were collected at 90-min intervals from Day 13 to Day 20; Group C: six ewes infused with oxytocin (3 nmol h-') from Day 12 to Day 18, blood samples being collected at 90-min intervals from Day 18 to Day 25 and then daily until return to behavioural oestrus. For the purpose of presenting the results, Groups B and C have been subdivided on the basis of the experimental outcome. A further six ewes were infused with oxytocin (3 nmol h-') from Day 12 to Day 18. Blood samples were collected three times a day for progesterone determination. On Day 21 these ewes were given a luteolytic dose of cloprostenol (Estrumate, Coopers Animal Health Ltd, UK; 125 pg i.m.; N = 4) or saline (0.5 mL ism.; N = 2) at t = 0 and blood samples were collected for oxytocin determination at t = -15, -10, -5, +2, +5, +8, +11.5, +15, +20, +25, +30, +40, +50 and +60min. During experimentation, ewes were kept in metabolism cages under conditions that allowed infusions to be carried out and blood samples to be collected without disturbance (Flint and Sheldrick 1983). They were exposed to ambient temperature and natural daylength. A vasectomized ram was housed with the ewes throughout the experiment. While in metabolism cages, sheep were fed chopped hay and water ad libitum plus 250 g concentrates per day. A polyvinyl chloride catheter 0.d. 2.1 mm was inserted into each external jugular vein, under local anaesthesia by the method of Seldinger (1953). One catheter was used for infusion of oxytocin (Bachem
Oxytocin Infusion in Ewes
Inc. Torrance, CA, USA) which was prepared in sterile physiological saline (150 mmol L-') and infused by means of a peristaltic pump (0.05 mL min-I). The second catheter was used for withdrawal of blood samples during periods of frequent sampling.
Radioimmunoassay for Oxytocin Oxytocin was measured in jugular venous plasma as described previously (Sheldrick and Flint 1981). Extraction of 'ZS~-oxytocinfrom sheep plasma was 77 + 1%. The sensitivity of the assay (calculated from 2 x s.d. below the zero point) was 1 . 3 Em01 (equivalent to 1.6 pmol L-' plasma). Intra- and inter-assay coefficients of variation were 9.9 and 6.8970, respectively. Values were corrected for extraction losses. Radioimmunoassay for Progesterone Progesterone was measured in plasma after extraction with diethyl ether as described previously (Sheldrick et al. 1980). Sensitivity of the assay reported here was 41.0 fmol tube (equivalent to 0.452 nmol L - ' plasma). Intra- and inter-assay coefficients of variation were 9.7 and 14.5%. Extraction recovery was 90 + 1% and values were corrected for extraction losses. Radioimmunoassay for I3,14-Dihydro-15-keto-PGF2, Concentrations of DHKF2, were measured in plasma by the method of Mitchell et al. (1976). Assay sensitivity and intra- and inter-assay coefficients of variation were 29.3 pmol L-', 6.0% and 8.5% respectively. Mean extraction recovery was 86 f 1% and values were corrected for extraction losses. Radioimmunoassay for Luteinizing Hormone Plasma concentrations of LH were determined by double-antibody radioimmunoassay using an antiserum raised in a rabbit (Lindsay 1982). Sensitivity of the assay was 0.96 pg L-'. Intra- and inter-assay coefficients of variation were 9 . 0 and 12.1%, respectively. Radioimmunoassay for Prolactin Prolactin was measured in plasma by a radioimmunoassay using an antiserum raised in a rabbit against ovine PRL (NIH-P-Sll; Lindsay 1982). Bovine PRL (LER891, equivalent to NIH-P-B4) was used as radiolabel and standard. Antibody-bound and free PRL were separated by a second precipitating antibody, and Staphylococcus aureus cells (Pansorbin, Calbiochem, Behring Diagnostics, La Jolla, CA, USA; 200 pg tube) were added immediately before centrifugation to increase the size and stability of the pellet. Displacement curves for bovine and ovine PRL were parallel (Bicknell and Chapman 1983); PRL in plasma samples diluted in parallel to the standard curve. Sensitivity of the assay was 1.15 pg L-' plasma, and intra- and inter-assay coefficients of variation were 8 . 8 and 17.9% respectively. Data Analysis and Statistics All values are reported as mean f s.e.m. The number of animals is indicated by N a n d the number of observations by n. Where pooled data are reported mean and s.e.m. are based on N. Hormone pulses are identified as being statistically significant when peak values are greater than 3 standard deviations (s.d.; calculated from the basal values) above the mean basal concentration and are also separated from an adjacent peak by at least one sample lying within 1 s.d. of the basal level and at least 3 s.d. below both adjacent peaks.
Results
Hormone Changes at Spontaneous Luteal Regression In three saline-treated sheep in which luteal regression occurred between Days 13 and 15 (Group A, Fig. 1) changes in circulating concentrations of DHKF,,, oxytocin and progesterone were similar to those described previously (Fairclough et al. 1980; Flint and Sheldrick 1983; Hooper et al. 1986). Episodes of secretion of DHKF,, were noted at mean intervals of between 4 and 12 h; mean basal circulating concentrations of DHKF,, were: (animal a) 176.6 + 7.9, n = 20; (b) 167.3 + 10.7, n = 25; (c) 223.4 + 13.2 pmol L-', n = 23, and the mean peak concentration reached during episodes of secretion was
E. L. Sheldrick and A. P . F. Flint
Days after oesbus
Days after oestrus
Fig. 1. Plasma progesterone (O), DHKF2, (O), LH (hatched areas), prolactin (0)and oxytocin (A) in samples collected at 90-min intervals from animals in Group A, three ewes (a-c) which received vehicle (NaCl, 150 mmol L-', 3 mL h-') between Days 13 and 18.
Days after oestrus
714.4 f 25.4 pmol L-'; N = 3; n = 20). All three ewes showed increased PRL secretion at the time of the pre-ovulatory LH surge, and in one ewe circulating PRL concentrations also increased at luteolysis (Fig. lc). High-amplitude pulses of oxytocin secretion were Table 1. Effect of treatment on luteolysis and return to oestrus in ewes Group
N
Treatment
A
3 2 1 3 3
saline infusion oxytocin infusion oxytocin infusion oxytocin infusion oxytocin infusion
B1 B2 C1 Cz
Treatment Sampling Day on which Day of period period circulating pre-ovulatory (days) (days) progesterone LH surge concentration fell to below 1.5 nmol L-' 13-18 13-18 13-18 12-18 12-18
13-18 13-20 13-20 18-25 18-24
14, 14, 15 >20 14 19, 22, 22 26, 32, 33
16, not 15 21, not
17, 17 observed 23, 25 detected
Day of return to behavioural oestrus
16, 17, 17 22, 48 16 21, 24, 25 26, 32, 35
Oxytocin Infusion in Ewes
detected in all three ewes, 21% of which were synchronous with episodes of DHKF,, secretion.
Hormone Changes in Animals Treated with Oxytocin Oxytocin infusion delayed return to oestrus in eight of nine treated ewes (see Table 1). In three ewes in which peripheral blood samples were collected during oxytocin infusion
Days aftec oestrus Fig. 2. Plasma progesterone (a), DHKF2, (O), and prolactin (0) in two ewes (Group B1),given oxytocin (3 nmol h-I, i.v.) from Day 13 to 18. Duration of the infusion is indicated by the horizontal bar. Peripheral blood samples were collected at 90-min intervals from Day 13 to 20. Ewes a and b returned to oestrus on Days 22 and 48, respectively.
(Group B), circulating oxytocin concentrations were raised from a mean level of 12.9 f 5 . 6 (N = 3; n = 12) to 59.9 k 2 . 3 pmol L-', n = 77 (Ewe a, Fig. 2); 74.8 f 1.9, n = 79 (Ewe b, Fig. 2) and 38.6 f 1 . 0 , n = 80 (Fig. 3) during infusion. In two of these ewes
E. L. Sheldrick and A. P. F. Flint
(Group B1, Fig. 2) progesterone levels remained similar to those observed before oxytocin administration but in the third ewe, in which luteal regression had commenced before oxytocin infusion (Fig. 3), oxytocin failed to prevent completion of luteolysis. In those animals in which luteal regression was blocked (Fig. 2), DHKF2, concentrations remained low during and after infusion; episodes of secretion such as those observed in the control group were absent. Prolactin secretion was pulsatile and LH secretion remained at basal levels. In the ewe (Group B2; Fig. 3) which returned to oestrus on Day 16, oxytocin infusion initially caused a single high-amplitude pulse of DHKF,, secretion; thereafter, circulating concentrations remained at 345.9 + 32.4 pmol L - N = 1; n = 25, and during this period no significant episodes of secretion were detected.
',
Days after oestrus DHKF2, (a),LH (hatched area), prolactin (0)and oxytocin (A) concenFig. 3. Progesterone (O), trations in one ewe (Group B2) given oxytocin infusion from Day 13 to Day 18 (as indicated by horizontal bar). The ewe returned to oestrus on Day 16. As is apparent from falling progesterone concentrations and increased DHKF2, secretion at the start of the oxytocin infusion, luteolysis had commenced in this ewe before treatment was started.
In a further six ewes, blood samples were obtained after the withdrawal of oxytocin infusion; in three of these animals blood samples were collected during luteal regression between Days 19 and 22 (Group C1; Fig. 4), and in three ewes the sampling period preceded luteolysis (Group CZ, Fig. 5). In Group C1, luteal regression occurred without frequent high-amplitude episodes of DHKF,, secretion comparable to those observed in the control group (Group A); in one of these ewes (Fig. 4b) one significant episode of DHKF,, secretion was detected at luteolysis, while in the other two animals (Fig. 4a, c) no significant episodes were detected. During luteolysis, ewes in Group C1 (Fig. 4a-c) had the following basal circulating concentrations of DHKF,,: (animal a) 289.3 + 27 3 pmol mLn = 37; (b) 221.4 k 12.1, n = 42; (c) 194.9 k 17.5, n = 20, and oxytocin concentrations remained low throughout luteolysis (a) 3.2 + 0 . 2 pmol L-', n = 37; (b) 3.1 f 0.5, n = 42; (c) 5.1 k 0.6, n = 20. In all three ewes in Group C1, luteal regression was accompanied by circulating PRL levels that were high compared with those in ewes not
',
Oxytocin Infusion in Ewes
Days aftw
OBS~~US
Fig. 4. Progesterone (O), DHKF2, (a),LH (hatched areas), prolactin (0)and oxytocin (A) in three ewes (a-c) sampled at 90-min intervals from withdrawal of oxytocin until day of oestrus (Group C,).
Days after oesbus
Days aftec oesbus
E. L. Sheldrick and A. P. F. Flint
Days after oestrus
Days after oestrus
Days after oestrus
DHKF2, (e), prolactin (0)and oxytocin (A) concentrations in jugular Fig. 5. Progesterone (O), venous plasma from three ewes (a-c) in which oxytocin delayed luteolysis for more than 10 days (Group C2).
Oxytocin Infusion in Ewes
undergoing luteolysis at a comparable stage after oestrus (Group C2, Fig. 5), and there was no discrete PRL release associated with the pre-ovulatory LH surge. Concentrations of LH were consistently low ( ~ 7 . 4pg L-') in the animals sampled after oxytocin infusion but before luteolysis (Group C2); circulating PRL levels were low ( ~ 5 1 . 0pg L-') with the exception of one sample where the animal was handled during sampling on Day 21 (Fig. 5a). DHKF,, concentrations were low (mean, 140-3 pmol L-'; N = 2; n = 207) in two of these ewes but raised basal secretion was observed in the animal that returned to oestrus earliest in this group (253.2 k 9.9 pmol L-'; n = 101; Fig. 5a). There were no surges of oxytocin associated with DHKF,, secretion in these animals.
Fig. 6 . Concentrations of oxytocin in jugular venous plasma of oxytocin-treated ewes (N = 4) given a luteolytic dose of cloprostenol Day 21 after oestrus. Mean maximal jugular venous concentrations in control ewes treated with cloprostenol on Days 11-13 after oestrus were 113 k 38 pmol L-'; this level was reached 10-20 min after treatment (Flint and Sheldrick 1983).
i7me (minutes)
Oxytocin Secretion in Response to Cloprostenol in Animals in which Luteal Function was Prolonged Following Oxytocin Infusion The mean concentration of oxytocin in jugular venous plasma before treatment was 2.4 f 0.5 pmol L-' (N = 6; n = 20). In four ewes treated with cloprostenol on Day 21 after oestrus the mean circulating oxytocin concentration rose to maximal levels (13.0 f 6.6 pmol L-'; N = 4; n = 4) 11.5 min after treatment (Fig. 6), and declined to 3.8 f 1.2 pmol L-' (N = 4; n = 4) after 60 min. In two control ewes, mean circulating oxytocin concentrations were 1.51 pmol L-' (N = 2; n = 6) before and 1.55 pmol L-' (N = 2; n = 14) during the 60 min after saline administration. Mean circulating concentration of progesterone was 12.7 3.2 nmol L-' ( N = 6; n = 20) before treatment, indicating that the ewes had functional corpora lutea. Progesterone levels fell to < 1.5 nmol L-' within 24 h of cloprostenol treatment and behavioural oestrus was detected within 48 h. Discussion The experiments described here show that continuous oxytocin infusion blocks the episodic secretion of PGF2, normally associated with luteal regression. This observation is consistent with the hypothesis that oxytocin infusion down-regulates the uterine oxytocin receptor (Flint and Sheldrick 1985). Oxytocin receptor concentrations are low on Days 12 or 13 of the oestrous cycle (Sheldrick and Flint 1985), when the infusions were started in these experiments, and therefore it must be assumed that the low level of receptor expressed in the uterus at this time is sufficient to lead to down-regulation.
E. L. Sheldrick and A. P. F. Flint
Blood samples were taken too infrequently to provide any information on whether oxytocin decreases LH pulse frequency or amplitude in those animals in which ovulation was blocked. However, as shown previously (Flint and Sheldrick 1985), oxytocin infusion failed to prevent completion of luteolysis in one ewe in which luteal regression had started before oxytocin infusion had begun and in this ewe there was a normal pre-ovulatory LH surge followed by luteinization; therefore it appears unlikely that oxytocin infusion acts primarily by inhibiting LH secretion. This ewe responded to oxytocin with a single highamplitude pulse of prostaglandin secretion which is indicative of the presence of functional uterine oxytocin receptors. Similar episodes of PGF,, secretion were seen in cattle given oxytocin infusion starting on Day 15 after oestrus (Kotwica et al. 1988), a treatment which failed to prolong luteal function. It seems possible therefore that oxytocin infusion fails to down-regulate the receptor once levels of receptor reach a certain point; under these conditions oxytocin administration causes PGF,, secretion and luteal regression. There was no immediate rise in PGF,, production following withdrawal of oxytocin infusion (Fig. 2), and when luteolysis did occur following withdrawal of oxytocin it was associated with reduced PGF,, release, compared with that in untreated ewes at luteolysis. This suggests that the trigger to episodic PGF,, release was blocked or ameliorated for a long period. One explanation for the absence of episodes of PGF,, secretion is that when luteal function is extended beyond the normal time of luteolysis, the corpus luteum loses its ability to synthesize oxytocin; this occurs in pregnancy and after hysterectomy (Sheldrick and Flint 1983a, 19833) as well as after immunization against PGF,, or oxytocin (Sheldrick and Flint 1984), and may reflect loss from the tissue of oxytocin-neurophysin messenger RNA (Jones and Flint 1988). This mechanism was confirmed in the present study in animals treated with a luteolytic dose of cloprostenol on Day 21, following oxytocin infusion between Days 12 and 18; peripheral circulating oxytocin concentrations in these ewes were raised in response to cloprostenol (Fig. 6 ) , but the level reached was less than 10% that observed in otherwise untreated mid-cycle ewes (Flint and Sheldrick 1983). Cloprostenol fails to stimulate oxytocin secretion in ovariectomized ewes (Flint and Sheldrick 1982), therefore it seems unlikely that a pituitary component is involved in this response. The occurrence of luteal regression in the absence of episodes of PGF,, secretion noted here following oxytocin withdrawal is difficult to explain as PGF,, is the only currently recognised luteolysin. It is unlikely to reflect increasing sensitivity of the corpus luteum to low levels of PGF,, since no such hypersensitivity has been reported during pregnancy. An alternative possibility is that a different compound causes luteolysis at this time. Prolactin causes structural luteolysis in aged corpora lutea in the mouse (Grandison and Meites 1972) and in rats in which luteal function has been extended by hypophysectomy (Malven and Sawyer 1966; Malven 1969), and has recently been shown to be luteolytic in the tammar wallaby (Tyndale-Biscoe et al. 1988). Furthermore, the circulating concentrations of prolactin were raised at luteal regression following oxytocin infusion. Therefore the possibility exists that PRL may be luteolytic in sheep when luteal function is extended beyond the normal time of luteolysis, although other mechanisms, such as withdrawal of an unidentified luteotrophin or secretion of an unidentified luteolysin, cannot be excluded. References Bicknell, R. J., and Chapman, C. (1983). Bombesin stimulates growth hormone secretion from cultured bovine pituitary cells. Neuroendocrinology 36, 33-8. Fairclough, R. J., Moore, L. G., McGowan, L. T., Peterson, A. J., Smith, J. F., Tervit, H. R., and Watkins, W. B. (1980). Temporal relationship between plasma concentrations of 13,14-dihydro-15keto-prostaglandin F and neurophysin 1/11 around luteolysis in sheep. Prostaglandins 20, 199-208. Flint, A. P. F., and Sheldrick, E. L. (1982). Ovarian secretion of oxytocin is stimulated by prostaglandin. Nature (Lond.) 297, 587-8. Flint, A. P. F., and Sheldrick, E. L. (1983). Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep. J. Reprod. Fertil. 67, 215-25.
Oxytocin Infusion in Ewes
Flint, A. P. F., and Sheldrick, E. L. (1985). Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes. J. Reprod. Fertil. 75, 623-31. Flint, A. P. F., Leat, W. M. F., Sheldrick, E. L., and Stewart, H. J . (1986). Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium. Biochem. J. 237, 797-805. Grandison, L., and Meites, J . (1972). Luteolytic action of prolactin during estrous cycle of the mouse. Proc. Soc. Exp. Biol. 140, 323-5. Hixon, J. E., and Flint, A. P. F. (1987). Effects of a luteolytic dose of oestradiol benzoate on uterine oxytocin receptor concentrations, phosphoinositide turnover and prostaglandin F-2a secretion in sheep. J. Reprod. Fertil. 79, 457-67. Hooper, S. B., Watkins, W. B., and Thorburn, G. D. (1986). Oxytocin, oxytocin associated neurophysin, and prostaglandin F2a concentrations in the utero-ovarian vein of pregnant and non-pregnant sheep. Endocrinology 119, 2590-7. Jones, D. S. C., and Flint, A. P. F. (1988). Concentrations of oxytocin-neurophysin prohormone mRNA in corpora lutea of sheep during the oestrous cycle and in early pregnancy. J. Endocrinol. 117, 409-14. Kotwica, J., Schams, D., Meyer, H. H. D., and Mittermeier, Th. (1988). Effect of continuous infusion of oxytocin on length of the oestrous cycle and luteolysis in cattle. J. Reprod. Fertil. 83, 284-94. Lindsay, K. S. (1982). Pituitary gonadotrophin responsiveness in sheep: effects of reproductive stage and nutrition. Ph.D. Thesis, University of Cambridge. Malven, P . V. (1969). Luteotrophic and luteolytic responses to prolactin in hypophysectomised rats. Endocrinology 84, 1224-9. Malven, P. V., and Sawyer, C. H. (1966). A luteolytic action of prolactin in hypophysectomised rats. Endocrinology 79, 268-74. Mitchell, M. D., Flint, A. P. F., and Turnbull, A. C. (1976). Plasma concentrations of 13,14-dihydro15-keto-prostaglandin F during pregnancy in sheep. Prostaglandins 11, 319-29. Moore, L. G., Choy, V. J., Elliot, R. L., and Watkins, W. B. (1986). Evidence for the pulsatile release of PGF2, inducing the release of ovarian oxytocin during luteolysis in the ewe. J. Reprod. Fertil. 76, 159-66. Roberts, J. S., McCracken, J . A., Gavagan, J. E., and Soloff, M. S. (1976). Oxytocin-stimulated release of prostaglandin F from ovine endometrium in vitro: correlation with estrous cycle and oxytocin-receptor binding. Endocrinology 99, 1107-14. Schramm, W., Bovaird, L., Glew, M. E., Schramm, G., and McCracken, J. A. (1983). Corpus luteum regression induced by ultra-low pulses of prostaglandin F2a. Prostaglandins 26, 347-64. Seldinger, S. I. (1953). Catheter replacement of the needle in percutaneous arteriography. Acta Radiol. 39, 368-76. Sheldrick, E. L., and Flint, A . P. F. (1981). Circulating concentrations of oxytocin during the estrous cycle and early pregnancy in sheep. Prostaglandins 22, 631-6. Sheldrick, E. L., and Flint, A P . F. (1983a). Luteal concentrations of oxytocin decline during early pregnancy in the ewe. J. Reprod. Fertil. 68, 477-80. Sheldrick, E. L., and Flint, A. P . F. (1983b). Regression of the corpora lutea in sheep in response to cloprostenol is not affected by loss of luteal oxytocin after hysterectomy. J. Reprod. Fertil. 68, 155-60. Sheldrick, E. L., and Flint, A. P. F. (1984). Ovarian oxytocin. In 'Gonadal Proteins and Peptides and their Biological Significance'. (Eds M. R. Sairam and L. E. Atkinson.) pp. 257-72. (World Scientific Publishing: Singapore.) Sheldrick, E. L., and Flint, A P . F. (1985). Endocrine control of uterine oxytocin receptors in the ewe. J. Endocrinol. 106, 249-58. Sheldrick, E. L., and Flint, A. P. F. (1989). Post-translational processing of oxytocin-neurophysin prohormone in the ovine corpus luteum: activity of peptidyl glycine a-amidating mono-oxygenase and concentrations of its cofactor, ascorbic acid. J. Endocrinol. 122, 313-22. Sheldrick, E. L., Mitchell, M. D., and Flint, A. P. F. (1980). Delayed luteal regression in ewes immunised against oxytocin. J. Reprod. Fertil. 59, 37-42. Tyndale-Biscoe, C. H., Hinds, L. A,, and Horn, C. A. (1988). Fetal role in the control of parturition in the tammar, Macropus eugenii. J. Reprod. Fertil. 82, 419-28. Wathes, D. C., and Swann, R. W. (1982). Is oxytocin an ovarian hormone? Nature (Lond.) 297, 225-7. Manuscript received 25 August 1989, accepted 3 October 1989
