Mesotocin receptors during pregnancy, parturition and lactation in the tammar wallaby

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Abstract

Mestocin receptor concentrations in membrane preparations from reproductive tissues of the tammar Macropus eugenii throughout gestation and lactation were assessed using [3H]-oxytocin as the ligand. There was a single binding site which bound both mesotocin and oxytocin with high and similar affinities. Mesotocin receptor concentrations in the myometrium were low (708±199 fmol mg−1 protein) in early and middle gestation but increased significantly on day 23 of pregnancy of the 26-day gestation period to 1921±552 fmol mg−1 protein. Myometrial receptors reached a peak of 2483±575 fmol mg−1 protein on days 25 and 26 of gestation, but returned to basal levels about an hour after birth. Receptor concentrations in the contralateral non-gravid uterus were much lower (605±75 fmol mg−1) and did not significantly increase throughout the period of gestation but dropped one day before birth. Mesotocin receptors were undetectable in the endometrium, the yolk sac placenta and the lateral, median and anterior vagina of all animals tested. In the lactating mammary gland after birth mesotocin receptors were initially high (588±38 fmol mg−1) but decreased after 200 days and by late lactation were 224±55 fmol mg−1 protein on day 240, close to the time of weaning. Mesotocin receptors in the ipsilateral non-lactating gland were also high in early lactation (430±153 fmol mg−1) and declined in late lactation (62±20 fmol mg−1). The changing concentrations of mesotocin receptors in pregnancy and lactation demonstrate that they are specifically regulated in tammar reproductive tissues. The increase in mesotocin receptors in gravid, but not in the non-gravid myometrium three days before birth may make the uterus responsive to the surge of mesotocin at birth. Since this rise is unilateral and only occurs in the gravid myometrium it must be due to local effects from the ipsilateral ovary or the feto-placental unit. Likewise, the down-regulation of mesotocin receptors in the contralateral, non-gravid myometrium may be due to its proximity to the developing follicle. The changing concentrations in the lactating and the adjacent, non-lactating mammary gland also reflect a differential regulation of mesotocin receptors, probably mediated via the sucking stimulus. Thus, local influences appear to be of primary importance in the regulation of mesotocin receptors during reproduction in this marsupial.

Introduction

Oxytocin receptor concentrations in the uterus and mammary gland change with reproductive status. Oxytocin (OT) induces uterine contractions by a direct action on the smooth muscle cells of the myometrium (reviewed in Giraldi et al., 1990). In eutherian mammals, the levels of oxytocin receptors in the myometrium increase near term. This rise may be important in sensitising the uterus to OT, since plasma OT levels do not increase until after labour has commenced. OT is clearly important for birth since infusion of oxytocin antagonists delays the onset and prolongs labour in rats (Chan and Chen, 1992; Antonijevic et al., 1995) guinea pigs (Schellenberg, 1995), sheep (Owiny et al., 1992) and humans (Goodwin et al., 1996). Thus, a rise in myometrial oxytocin receptor levels appears to be a critical prerequisite for the onset of labour.

In the mammary gland OT acts through specific receptors on the myoepithelial cells surrounding the alveoli, causing contraction and milk ejection (reviewed in Wakerley et al., 1994). As in the uterus, there is a functional change in the sensitivity of mammary gland to oxytocin with reproductive stage. In the rat, the epithelial cells in the mammary gland are most sensitive to OT during lactation (Soloff and Weider, 1983; Soloff, 1985). There is a 2.5-fold decrease in the threshold dose of OT needed for milk ejection between days 1 and 10 of lactation (Sala and Freire, 1974) and the number of OT receptors increases 100-fold between the first day of pregnancy and late lactation (Soloff and Weider, 1983) due to increased numbers of myoepithelial cells (Sala and Wieder, 1983). During postweaning mammary regression the gland becomes less responsive to OT as the receptor numbers fall.

Oxytocic peptides are also important in marsupial birth and lactation. Marsupials have either oxytocin, or the related peptide mesotocin. In the tammar wallaby (Macropus eugenii) the pituitary oxytocic peptide is mesotocin (ile-8-oxytocin) (MT) (Chauvet et al., 1981). Both these peptides cause uterine contractions and milk ejection (reviewed in Tyndale-Biscoe and Renfree, 1987and Bathgate et al., 1996). Tammars are monovular, so only one of the two separate uteri becomes gravid, allowing comparison with the contralateral non-gravid uterus. Similarly, there are four teats and two pairs of associated mammary glands, but only one of these lactates, while the adjacent gland and the contralateral pair regress after birth, again allowing direct comparison within an animal of a lactating and non-lactating gland.

The uterus is refractory to the effects of exogenous oxytocin in the first half of gestation. After day 23 of gestation both OT and MT are about equipotent in inducing contractions of tammar myometrial strips (Shaw, 1983; Renfree et al., 1996; Parry et al., 1997a). This coincides with an increase in MT receptor concentrations in the gravid uterus (Parry et al., 1997a). The increase of receptors in the gravid myometrium precedes birth by 3–4 days suggesting that receptor synthesis does not provide the trigger for birth. However, the action of mesotocin is apparently important for birth since the oxytocin-receptor antagonist, atosiban, inhibits the action of mesotocin and oxytocin on myometrium in vitro, and infusion of atosiban in late gestation delays, but does not prevent, birth (Renfree et al., 1996), and MT rises precipitously a few minutes before birth (Parry et al., 1996).

Post-natally the sensitivity of mammary glands to oxytocin and mesotocin also changes with stage of lactation. Macropodid marsupials can give birth to a new young whilst still suckling a large young at foot. This results in concurrent asynchronous lactation, where adjacent mammary glands produce milk of a very different composition (reviewed in Tyndale-Biscoe and Renfree, 1987). In the agile wallaby, Macropus agilis, the threshold doses of oxytocin needed for milk ejection increase about 8-fold between days 50 and 250 of lactation (Lincoln and Renfree, 1981a, Lincoln and Renfree, 1981b) in striking contrast to rats, where threshold doses decrease as gland sensitivity increases with lactation. Change in gland sensitivity with lactation is also apparent in adjacent glands of females suckling two young of different ages (Lincoln and Renfree, 1981a, Lincoln and Renfree, 1981b), so it was suggested that this change in sensitivity was due to a change in mesotocin receptor concentrations as lactation progressed (Renfree, 1983). In the brushtail possum, the only marsupial in which mammary mesotocin receptors have been so far reported, the concentration of oxytocin receptors in the lactating mammary gland decreases during lactation (Sernia et al., 1991). However, in macropodid marsupials mammary gland mesotocin receptor concentrations are not known.

In a previous study using an iodinated oxytocin antagonist as tracer, MT receptors were observed to be up-regulated in the gravid myometrium and down-regulated in the contralateral non-gravid myometrium of the tammar, suggesting a differential effect of local paracrine factors (Parry et al., 1997a). Similarly, it is likely that the mammary gland sensitivity to the milk-ejecting effects of oxytocin during concurrent asynchronous lactation is controlled by local induction of mesotocin receptors (Lincoln and Renfree, 1981a). This study therefore measured the concentration of mesotocin receptors through gestation and lactation to investigate their role in the differential regulation in adjacent tissues in the reproductive tract and the mammary gland of the tammar, M. eugenii.

Section snippets

Animals

Tammar wallabies were maintained in our colony at Monash University, Clayton, as previously described (Renfree et al., 1989). Wallabies were housed in open grassy enclosures, and provided with water, lucerne cubes and fresh vegetables ad libitum. All experiments were approved by the University of Melbourne Animal Experimentation Ethics Committees and the animal handling and husbandry were in accordance with National Health and Medical Research Council of Australia (1990)guidelines.

Females known

Assay validation

Saturable binding of [3H]-OT was found in myometrium and mammary tissue (Fig. 1) and the amount of binding increased with time reaching a plateau after 2–3 h (data not shown). Specific binding was proportional to the amount of membrane preparation added to the incubation mixture. The extra centrifugation step during preparation of the uterine membranes removed some non-receptor protein which had coagulated in the freeze–thaw cycle. This effectively concentrated the supernatant, which led to a

Discussion

This study has confirmed observations of differential regulation of myometrial mesotocin receptors during gestation in the tammar wallaby, and extends these observations to show that mestocin receptors are also differentially regulated in the mammary gland. In both these tissues, local factors are clearly important in modulating receptor content to provide physiologically important changes in sensitivity to mesotocin during gestation and lactation.

The dissociation constant and cross

Acknowledgements

We are grateful to Professor RJ Fairclough and Dr TM Lau for advice on the receptor assay and helpful discussions. We also thank Dr Carl Rudd, Anne Duns and Richard Moyle for their invaluable assistance with the animals. We are especially grateful to Dr Per. Melin and Ferring Pharmaceuticals for the gift of the oxytocin antagonist atosiban. Animals were held under permit number RP-92-099 from the Department of Conservation and Natural Resources, Victoria, Australia. This research was supported

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