The use of an oxytocin antagonist to study the function of ovarian oxytocin during luteolysis in cattle

ELSEVIER

THE USE

OF AN OXYTOCIN ANTAGONIST OXYTOCIN DURING

J. Kotwica,lva

D. Skatzynski,’

TO STUDY LUTEOLYSIS

M. Bogacki,’

P. Melin

‘Institute

Division

THE FUNCTION IN CATTLE

of Animal Reproduction and Food The Polish Academy of Sciences of Reproductive Endocrinology and Pathophysiology, 2Fening Research Institute AB, Malmo, Received

for publication: Accepted:

October -July

2,

and Research

OF OVARIAN

B. Starostkal of

Olsztyn-Kortowo, Sweden

Poland

22, 1996 1997

ABSTRACT The importance of ovarian oxytocin (OT) in cattle during luteolysis and the mid-luteal phase using a highly specific OT antagonist (CAP-527) was studied. To establish the effective dose of CAP, heifers (n=4) were infused with saline for 30 min, followed by 50 IU OT into the abdominal aorta on Days 17 and 18 of the estrous cycle. After 5.5 hours later, either 4, 6, 8 or 10 mg of CAP was infused for 30 min, followed by 50 IU OT. Plasma concentrations of 15 keto-13,14-dihydro-prostagiandin F2a. (PGFM) increased after 4 and 6 mg CAP. Therefore, in Experiment 2,8 mg of CAP was infused and 50 IU OT was given after 3,4,6 and 9 h to define how long CAP saturates OT receptors. Concentrations of PGFM increased after 6 and 9 h of OT treatment only. We concluded that 8 mg CAP effectively blocked uterine OT receptors for 4 h in our model; hence in further experiments this dosage of CAP was used. In Experiment 3, CAP was given to 4 heifers every 4 h on Days 15 to 22 of the cycle, and 4 additional heifers received saline and served as the control. The CAP treatment changed neither the duration of the cycle, progesterone, PGFM nor OT plasma concentrations compared with that of the controls. Experiment 4 was designed to study the involvement of OT in noradrenaline (NA)stimulated progesterone secretion. It was found earlier that ovarian OT stimulates progesterone secretion, and that NA was able to evoke concomitant release of both progesterone and OT. Therefore, in Experiment 4, NA was infused on Days 11 and 12 of the cycle in heifers (n=4) but it was preceded with 8 mg CAP or with a saline (control) infusion. Concentrations of plasma progesterone concentrations increased after NA treatment in both the experimental and control groups. Thus, we conclude that if ovarian OT and uterine/ovarian OT receptors are involved in luteolysis and steroidogenesis in cattle, they play a more facilitating than mandatory role. 0 1997 by Elsev~er Scmce

Inc

Key words:

antagonist,

oxytocin

and oxytocin,

prostaglandins,

luteolysis.

cattle,

noradrenaline

Acknowledgments We thank Drs. S. Okrasa, G. Kotwica and B. Szafranska, University of Agriculture and Technology, Olsztyn, Poland, and W.J. Silvia, University of Kentucky, Lexington, USA, for progesterone, oxytocin, estradiol-176 and PGFM antiserum. This study was supported by the National Research Council (5PO6D 051 09). Part of these results were presented during 1995 Hanseatic Endocrine Conference in Stade, Germany and the XXX Simposio lntemationale di Zootecnia, Milano, Italy, 1995. a Correspondence and reprint requests: Dr. Jan Kotwica, Fax (+48) 89 5237347; e-mail: [email protected]. Thenogenology 48:1287-1299, 0 1997 by Elsewer Science

1997 Inc.

0093.691W97/$17.00 PII SOO93-691X(97)00371-3

Theriogenology

1288

INTRODUCTION Ovarian oxytocin (OT) is suggested to be a trigger for luteolysis in ruminants (7,35,47) that affects uterine PGF2a pulsatile release. Noradrenaline (NA) has been found to stimulate the release of OT from the corpus luteum (CL; 26,27) with an efficiency comparable to that shown by PGFzc, (i.e., approximately 98%; 7). It should be underscored that OT, once depleted from the CL, can not be restored (20). However, depleting the CL of OT by means of NA on Days 12 or 16 by up to 80% (27) affected neither the time of luteolysis nor the duration of the estrous cycle, though it was possible that the residue of OT in the CL (i.e., 20%) could still induce luteolysis. Thus the blocking of OT receptors by the continuous treatment of mature heifers with a specific OT antagonist on Days 15 to 22 of the cycle would be useful for assessing the role of OT in luteolysis. Ovarian OT is also assumed to affect progesterone release from early- and mid-cycle CL in cattle (34,38), whereas noradrenaline (NA) stimulates concomitant secretion of progesterone and ovarian OT (26). Thus, it is possible that NA stimulates OT secretion which, in turn, influences progesterone release. This supposition was also tested in our study using the OT antagonist.

MATERIALS Animals

and Surgical

AND

METHODS

Procedure

Twenty-four heifers (380 to 450 kg) with regular estrous cycles (18 to 22 days) were used in this study (day of onset of estrus was Day 0). One day before the study, a polyvinyl chloride catheter was inserted into the abdominal aorta through the coccygeal artery (25) for the infusion of either saline, noradrenaline (NA; 0.3 pg/kg/min), oxytocin or an oxytocin antagonist (CAP-527). The NA dosage was predetermined (26). The tip of each cannula was positioned cranial to the origin of the ovarian artery, as established earlier (25) to allow for the direct application of drugs to the reproductive tract, which permitted a lower dosage of drug to be used and thus avoided unwanted side-effects. A second catheter was inserted into the jugular vein to collect the blood samples. Experiment

1

Four heifers were used to establish the effective dose of oxytocin antagonist (CAP, Ferring AB, Sweden). This OT antagonist (I-deamino-2-D-Tyr(OEt)+Thr-8-Om-oxytocin) binds directly to OT receptors (36) and blocks the action of OT. On Days 17 and 18 of the estrous cycle, the heifers were infused with saline into the abdominal aorta for 30 min followed by 50 IU OT. Three hundred and 30 min later either 4, 6, 8 or 10 mg of CAP were infused for 30 min followed by 50 IU CAP in bolus infusion. Blood samples (8 ml) were taken every 5 to 10 min during the experiment, and 13,14-dihydro-15keto-prostagiandin Fza (PGFM) concentrations were measured as uterine response to OT treatment (6,15).

Theriogenology Experiment

1289

2

To study how long CAP occupies OT receptors, 8 mg of CAP were infused for 30 min to heifers (n=4) once on Days 17 and 18 followed by i.a. injection of OT (50 IU) 3, 4, 6 or 9 h later. Concentrations of PGFM were measured in response to OT treatment in jugular blood samples taken every 5 or 10 min. Experiment

3

To verify the hypothesis that ovarian OT is important for luteolysis in cattle. 8 mg CAP (n=4) in 20 ml of saline or saline alone (n=4) were infused for 30 min every 4 h from Day 15 until the first signs of estrus were observed or until Day 22 of the estrous cycle. Blood samples were collected every hour during the experiment. Experiment

4

To determine if NA-stimulated progesterone secretion can be released independently of OT on Days 11 and 12 of the cycle, heifers (n=4) were infused with 4 mg NA (26,27), and in accordance with a Latin square design they were preinfused (30 min) with saline or with 8 mg CAP as described in Experiment 1. Hormone

Analysis

Concentrations of progesterone (25) and OT (43) were determined by RIA. The rabbit progesterone antiserum (IFP4) used in the progesterone RIA (29) and the anti-rabbit oxytocin antiserum (R-1;27) were described. The sensitivity of the assay averaged 0.3 to 0.6 nglml for progesterone and 3 pg/ml for OT; the coefficient of correlation between added and recovered amounts of progesterone and OT in the plasma for 4 different concentrations were 93 and 95%, respectively. The intra- and inter-assay CVs were 8.1 and 15.9% for progesterone and 5.6 and 10.5% for OT, respectively. Plasma concentrations of PGFM were determined in blood samples, as described by Homanics and Silvia (15). The antiserum (WS4468 BD 6/237/21) and free and antibody-bound tracers were separated by charcoal precipitation, The sensitivity of the assay was 65 pg/ml, and the intra- and inter-assay CVs were 6.8 and 11.3%, respectively. Estradiol-178 in plasma was determined according to Hotchkiss et al. (17). Specificity of antibodies for estradiol-178 was given earlier (48). Extraction efficiency was above 80%, and concentrations were corrected for the procedural losses. The sensitivity of the method was 2 to 3 pglml. The intra- and inter-assay CVs were 5.5 and 11.2%, respectively. Statistical

Analysis

The total amount of PGFM after OT infusion was measured by calculating area under the curve and then compared by one-way ANOVA with that of saline pretreated (Experiment 1) or with control group (Experiment 2). Baseline for OT and PGFM in Experiment 3 was defined on the basis of the first 24 determinations of each hormone. A pulse was defined when any 3 consecutive concentrations of hormone exceeded the baseline by 50%. Relationship between OT and PGFM pulses was determined by calculating the number of pulses that appeared either concomitantly or within 1 hour of the start of a pulse of another hormone. Mean duration of the estrous cycle in control and experimental group (Experiment 3) was compared. The

1290

Theriogenology

amount of OT and progesterone released only during NA infusion measured by calculating the area under the curve. Baselines for these on the basis of 13 sample concentrations that preceded NA infusion. mean values were estimated by split-plot ANOVA of repeated measures.

in Experiment 4 was hormones was defined Differences between

RESULTS Plasma PGFM in response to a bolus injection of 50 IU OT did not change after either a 30 min infusion of 8 and 10 mg CAP (Figure 1). Moreover, when a bolus injection OT was administered to heifers at 3, 4, 6 or 9 h before a 30 min infusion of 8 mg CAP in Experiment 2, a change in PGFM was not observed after 3 and 4 h (Figure 2). Thus we concluded that 8 mg CAP blocked OT receptors for at least 4 h in our model.

02GOO

350

1500

250

Time Figure

(min)

1. Plasma 13,1Cdihydro-ldketo prostaglandin F (PGFM) and oxytocin concentrations in individual heifers in response to bolus injection of 50 IU of oxytocin (arrow) into the abdominal aorta preceded with infusion (30 min) of saline (SAL) or 4 mg, 6 mg, 8 mg and 10 mg of oxytocin antagonist (CAP). Data for heifers 1, 2, 3 and 4 used on Day 17 are presented.

Theriogenology

1291

1250-

.250

200 B

.= $ -5 0

,150 4 500

g ,100 z =

250

50

-60

-30

0

30

60

90

120

0

Time (minutes) Figure

2.

Concentrations of PGFM and response to 50 IU of OT. Heifers 6, or 9 h before OT.

oxytocin received

(OT) in two heifers (mean + SEM) in a 30-min infusion of 8 mg of CAP at 3, 4,

When CAP was administered every 4 h from Day 15 of the cycle until evident signs of estrus in Experiment 3, no significant effect (P>O.O5) was observed on the profile of studied hormones (Figure 3) compared with those of saline group (Figure 4). The number of OT and PGFM peaks and the relationship between them are given in Table 1. Similar number of OT (18 and 19) and PGFM (19 and 18) surges was measured in the control and in CAP-treated heifers, respectively. Most of PGFM pulses were observed without concomitant OT surges. It should be noted that only in 1 control heifer (Heifer 15) was the first pulse of PGFM accompanied by an OT pulse, whereas in 3 other heifers first surge of PGFM observed on Day 17 or 18 of the estrous cycle was not followed by OT. In CAP-treated heifers the first pulse of PGFM was concomitant with the OT surge in 3 heifers, and in 1 heifer the PGFM pulse not accompanied by OT. Heifers receiving CAP had estrous cycle durations (21.3 f 0.5 d) similar of those of the control group (21 .O f 0.6 d). To determine whether NA-stimulated progesterone secretion can be released independently of OT, heifers (n=4) were infused with NA (4 mg/30 min) on the Day 11 and 12 of the estrous cycle; but in Experiment 4 in Latin square design, they were pm-infused (30 min) with saline or with 8 mg of CAP. NA treatment increased OT and progesterone concentrations in both groups, as shown by area under the curves calculation (Table 2) and given on Figure 5 despite pm-treatment with CAP. Values of the same hormone between groups were not different (P>O.O5; Table 2).

Theriogenology Table

1.

Number and average frequency of oxytocin (OT) and PGFM peaks and the associations among these peaks to each other in control (n=4) and OT antagonist (CAP) -treated (n=4) heifers on Days 15 to 21 of the estrous cycle Total

numbers peaks

of

Agreement peaks

between (%)

Average peak frequency per 74 hours

Groups OT

PGFM

0T:PGFM

CAP

19

18

41.2

Control

18

19

55.5

PGFM:OT

OT

PGFM

41.0

1.3+0.8

1 .7tio.4

51.7

1.4*0.5

2.2 ti.2

DISCUSSION It has been shown that CAP decreases OT-induced contractile uterine activity in vitro in rats (36) and it bound competitively to the OT receptor of the myometrium and inhibited the hydrolysis of phosphatidylinositol (31). Results of Experiments 1 and 2 confirmed the effectiveness the selected dose (8 mg) of CAP to suppress OT-induced PGF2, release. Hence, we assumed that further results in Experiments 3 and 4 were obtained when OT receptors were blocked. Concentrations of uterine OT receptors in cattle is negatively correlated with progesterone concentration in peripheral blood, and its highest concentration is measured at the time of esttus (37). Nevertheless, on Day 17 of the estrous cycle, small increase in the number of OT receptors occurs (33,37). Therefore, the CAP infusion in our studies was started just before this critical time (Day 15) and lasted beyond the time of spontaneous luteolysis. However, CAP infusion affected neither the time of luteolysis nor duration of estrous cycle compared with that of the control animals. These results differ from those of earlier studies (9,47,49), which underscored the importance of uterine OT receptors for initiation and the course of luteolysis in ruminants. However, most of data considered by the above authors were obtained in sheep, whereas the results from other species, including ruminants (3,5,12,28), have not consistently supported their thesis. Therefore our findings on ovarian OT need to be considered in light of reports that often are not adequately considered when the role of ovarian OT is discussed. It has been shown that naproxen, an inhibitor of endogenous prostaglandin synthesis, suppressed uterine contractile response to OT in the rat (4). In this study the increase of uterine OT receptor concentration, that normally occurred on Day 23 of pregnancy, was delayed in naproxen treated animals. Flint and Sheldrick (8) observed that continuous infusion of OT in sheep markedly reduced uterine OT receptors. However, when those ewes were OT receptor concentration was similar to that of control animals. treated with PGF2,, Furthermore, Sheldnck (45) reported that OT infusion followed by synthetic progestin and

Theriogenology

1293

17

18

*”

20

9

21

u&

20

W

2 cl.

60

s

40 20

f 2 4

.^^

F15

2 % .r

0

16

17

18

19

20

I

21

3

Time (days of estrous cycle)

Figure 3. Concentrations of progesterone, oxytocin, estradiol, and PGFM in blood plasma samples collected every hour in four CAP-treated heifers between Days 15 and 22 of the estrous cycle or until signs of estrus occurred (arrow). Heifers received a 30 min infusion of 8 mg of CAP every 4 h.

1294

20 200 100 80

1.5

60

10

40

5

20 0 400 300

I6 #

0

17

16

200 100 1

80 60 40 20 0

16

17

18

19

20

Time (days of estrous

Figure

4.

Concentrations of progesterone, oxytocin, samples collected every hour in four control estrous cycle or until signs of estrus occurred

cycle)

estradiol, and PGFM heifers between Days (arrow).

in blood plasma 15 and 22 of the

1295

-90

-60

-30

0

30

60

90

Time (minutes) Figure

Table

5.

2.

Effect of noradrenaline (NA; 4 mg130 min) infusion into the abdominal heifers pre-treated with (a) saline (SAL; n=4) or (b) CAP (8 mg/30 min; peripheral plasma concentrations of oxytocin and PGFM (mean f SEM).

Influence of noradrenaline oxytocin and progesterone curve (relative units)

(4 mg infused into the abdominal secretion (mean f SEM) measured

of on

aorta for 30 min) on by area under the

Treatment

Oxytocin

Saline (n=4)

2768 k480

260.9 f30,2

CAP (n=4)

2287 +298

250.9 +24.5

PGFZ, analogue treatment the prevailing hypothesis progesterone or OT rather

aorta n=4)

Progesterone

did not diminish uterine OT receptors. These data do not. support that regulation of uterine OT receptors is dependent upon than on the PGF*, or a PGFpainduced substance.

1296

Theriogenology

infusion of OT from Day 10 of the cycle prolongs the duration of the estrous cycle and prevents the rise of uterine OT receptors in cattle (13). In this study it was assumed that OT down-regulates its own receptors and this way prevents luteolysis. However, it is possible that OT can stimulate progesterone production directly, acting upon CL receptors (40) mainly during early and mid-luteal phase in cattle, as reported by Miyamoto and Schams (38). This would explain why infusion of OT in heifers from Day 15 affects neither uterine OT receptors nor length of the cycle (24). In support of our interpretation, when OT administered systemically, it prevented OT receptor formation in luminal epithelium, but not when OT was infused into the uterine lumen (1). This suggest that to affect its own receptors, OT needs to be delivered directly to the ovary. Because the highest amount of OT in the bovine CL was found in mid-cycle (IO), we assume that OT may play an autoctine or paracrine role in the bovine early and middle CL, and thus it may be involved in the progesterone synthesis/secretion. But to demonstrate this, the CL has to be in intact (2122.42). Dispersed luteal cells are not a suitable model for the such studies (2,42). Oxytocin is suggested to be a trigger for the luteolytic pulses of PGF*,. However, in frequently collected blood samples in sheep, it was found that uterine PGFza surges preceded the release of ovarian OT and OT-associated neurophysin during luteolysis (39). The same results were obtained in well-designed experiments in goat (5). In our recent study on heifers (28) approximately 50% of OT or PGFM pulses were in absence of each other in blood samples collected every 15 min for 12 h on Days 16 to 19 of the cycle. This is comparable to the data obtained in our present study (Table 1). Thus, our findings differ from those by Hooper et al. (16) in ewes, who reported that 96% of PGFza pulses coincided with a pulse of OT; on the other hand, 45% of OT pulses did not produce a pulse of PGF2, from the uterus, which is similar to that of our observations (Table 1). Further, OT is assumed to be responsible for the pulsatile secretion of PGF2, as a way of down-regulating its own uterine receptors (35). However, to demonstrate this female cows were treated with up to 100 IU of exogenous OT (18,19,30,32). This increased plasma concentrations of OT to over 2000 pg/ml (18) whereas physiological pulses of OT in the peripheral blood during luteolysis in cattle ranged from only 30 to 80 pg/ml (28,44, present study). Finally, reduction of ovarian OT content by about 80% affects neither luteolysis nor the duration of the estrous cycle (27). Moreover, PGF analogue given to heifers stimulates temporary concomitant increase Of progesterone, PGFM, and OT. When, however, CAP was administered 30 min before PGF analogue, then in frequently collected blood samples both the height and area under the curve of PGFM was lower by 22 and 35%, respectively, compared with that of the salinepretreatment heifers (D. Skatzynski and J. Kotwica, unpublished observations). Therefore we assume that ovarian OT acts mainly inside the ovary as an autocrlne or paracrlne hormone; however, this does not exclude its a possible involvement in a luteolytic process already underway. This conclusion leads back to the key question about the mechanism of IUteOlySiS initiation. It was showed that the high doses of exogenous progesterone administered to heifers within a few hours after ovulation (mimicking an earlier luteal phase) hastened luteolysis and to shortened estrous cycle duration (11,12,23). It should be emphasized that from the time of first progesterone injection until first pulse of PGF there was 10 to 13 d depending on the duration of the luteal phase. Similar time of progesterone dominance is observed during the normal estrous cycle in cattle (i.e., from Days 4 to 6 until Days 15 t0 18 after estrus). In oophoretomized sheep, Silvia and Raw (46) observed the pulsatile release of PGF after treatment with progesterone only. Thus it is possible the length Of progesterone exposure/dominance during the estrous cycle is a key factor in regulating the Onset and

Theriogenology magnitude of pulsatile in the ewe (41).

1297

PGF

secretion

from

the uterus

in cattle

(30)

similar

to that as suggested

Although NA stimulates concomitant secretion of progesterone and OT, progesterone concentration was also increased after NA challenge, even though OT receptors were blocked by CAP. Recently we discovered that NA stimulates progesterone secretion, affecting both p1 and p2-receptors; whereas OT is secreted from bovine isolated CL after P2-receptor activation only (G. Miszkiel and J. Kotwica, unpublished observations). It is concluded that NA can affect ovarian OT and progesterone secretion independently of each other. In conclusion, data obtained in this study do not support the concept that ovarian OT is important for the luteolysis in cattle, as has recently also been reported by Hansel and Blair (14) and by Blair et al. (3). who found that luteal regression can occur in the absence of OT release from the luteal cells. Thus, if ovarian OT is involved in CL regulation (both in steroidogenesis and luteolysis) it plays a more facilitating than a mandatory role.

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