8th Postgraduate Course for Training in Reproductive Medicine and Reproductive Biology

Oxytocin in reproductive biology

E. Tribollet
Department of Physiology, Geneva University Medical Centre

Oxytocin (OT) is a small peptide of 9 aminoacids well known from clinicians for its potent uterotonic effect. Indeed, administration of OT in the late pregnancy stimulates powerfully the contraction of uterine smooth muscle cells. It is therefore widely used clinically for the induction and augmentation of labour in women. The other well known effect of OT is milk ejection from the mammary gland during lactation.

The traditional concept about OT is that it is a neurohormone produced by neurones located in the hypothalamic paraventricular and supraoptic nuclei. Axons of these neurones end in the posterior pituitary from where they release OT into the general circulation, in response to specific stimuli such as cervix distension or suckling.

The purpose of this lecture is to give a brief overview of new aspects of OT biology related to reproductive functions. In particular, OT has been demonstrated to be synthetized and to act within peripheral reproductive organs in both females and males. OT also exerts neuroendocrine effects on the anterior pituitary and is a neuroactive substance within the central nervous system

PERIPHERAL SYNTHESIS AND EFFECTS

Uterus

Just before the onset of labor, uterine myometrium becomes extremely sensitive to OT because of the large increase in the number of myometrial OT receptors (OTRs). The amount of myometrial OTRs returns to basal levels shortly after birth. The recent cloning of the OT receptor has shown that it belongs to the family of G protein coupled receptors, with seven transmembrane domains. OT binding activates phospholipase C which provokes the hydrolysis of membrane phospholipides into inositol triphosphate and other products. Inositol triphosphate releases Ca²⁺ from intracellular stores, causing a rise of intracellular free Ca²⁺ which is responsible for contraction of smooth muscle cells.

Athough OT has a strong pharmaceutical potential, its physiological role in normal labor has always been equivocal. Indeed, circulating OT is not essential for the initiation or maintenance of labor. There is no evidence that a rise of plasmatic OT actually precedes the onset of labor. Administration of OT antiserum suppresses lactation but does not affect parturition. Moreover, normal parturition has been observed in rats and human in the absence of plasmatic OT in cases of experimental or clinical pituitary gland disfunction.

Recent findings suggest a solution for this paradox. It has been shown both in rat and human that the uterus itself is a major site of OT production during late pregnancy. In the rat, OT is synthetized in the epithelial cell layer of the endometrium. OT mRNA increases more than 150-fold during gestation and at term, exeeds hypothalamic OT m RNA by 70-fold. Expression of the OT gene has also been demonstrated in the human uterus, with the presence of OT m RNA in decidua, and to a lesser extent amnion and chorion. Estradiol stimulates the expression of OT mRNA by choroido-decidual tissue in vitro. In situ levels of OT mRNA increase 3-4 fold at the time of labor.

Thus, OT may act primarly as a local mediator in the uterus at labor time. Endogeneous OT may exert a paracrine control of myometrial contractions both directly by interacting with myometrial OTRs, and indirectly by stimulating the production of prostaglandins F2a (PGF2a ) by endometrial cells where OTRs have also been identified. The rise in uterine OT production, in concert with the rise in uterine OT receptors synthesis may trigger the onset of labor. Dysregulation of uterine OT gene expression may also be an underlying cause of premature or delayed labor. Circulating OT may however still have a role in maintaining labor.

Corpus luteum

OT and PGF2a are the two major factors involved in the regression of corpus luteum (CL) during luteal phase of the ovarian cycle. PGF2a is the luteolytic signal which controls the decrease of progesterone secretion and the degenerescence of CL structures. In human and other primates, PGF2a originates from large luteal cells ; in other mammals, it is produced by the endometrium. In all mammalian species, OT is synthetized by large luteal cells which also possess OTRs. Conflicting results have been reported regarding the role of OT in CL function, due in part to species differences, but also to the age of luteal tissue studied. Indeed, evidence has now been provided that OT is involved in both luteinisation of the young CL and luteolysis of the older CL.

Luteolysis has been most investigated in the sheep. The endometrium begins to produce PGF2a at mid-luteal phase. In the ovary, PGF2a stimulates the release of OT by luteal cells, which in turn stimulates and enhances PGF2a endometrial release. This positive feedback loop between endometrial PGF2a production and OT release by the CL insures the pulsatile character of PGF2a secretion which is required for normal regression of CL. Experimental evidence suggest that initiation of this loop may involve estrogens and OT of central origin. In primates, endometrium is not required for luteolysis ; PGF2a originates from the CL itself where it exerts autocrine and paracrine effects, in particular on OT production.

A luteotrophic effect of OT has recently been demonstrated in several species. In the pig and the cow, in vivo microdialysis studies have shown that local application of OT shortly after ovulation stimulates the production of both progesterone and estradiol. OT also increases progesterone secretion by cultured marmoset granulosa cells. Data obtained with isolated bovine CL suggest that OT acts by enhancing the stimulatory effect of LH on large luteal cells. Immunocytochemical studies of the baboon CL have shown that OT enhances the expression of gap junction protein connexin 43. Thus the effect of OT on CL steroidogenesis may possibly be mediated by facilitation of luteal cells coupling.

Testicule

Some evidence is available that OT plays a role in testicular function. Concentration of the peptide is higher in the testicule than in the plasma. OT is indeed produced by Leydig cells under the control of LH and of lipoproteins from the seminiferous epithelium. Application of OT within the testicule stimulates the contraction of myoid cells (smooth muscle like cells) which are responsible of the contractile activity of seminiferous tubules. Therefore OT may contribute to the regulation of sperm transport to the epididymis. It should be remembered that spermatozoides are not moving at this stage. It has also been suggested that OT may facilitate conversion of testosterone in dihydrotestosterone by increasing 5a -reductase activity, but this point is still controversial at present.

Prostate

As in the testis, concentrations of OT in the prostate are higher than in the plasma. In addition, OT immunoreactivity and OT mRNA have been demonstrated in the epithelial cells of the acini, which indicates a local synthesis of OT. In vitro investigations have shown that OT can stimulate the contractile activity of prostatic tissue. This suggests that OT could be involved in the maintenance of basal prostatic tone, and in the contractions of the prostate and the resultant expulsion of prostatic secretions at ejaculation.

NEUROENDOCRINE EFFECTS

There is developing credence to the concept that OT may control anterior pituitary function. This was suggested long ago by the observation that some hypothalamic oxytocinergic neurones were projecting their axons towards the median eminence. OT is indeed released in the portal vessels where it is found at a basal concentration approximately one order of magnitude higher than in the peripheral circulation. OTRs have also been characterized in the anterior pituitary, although their cellular localization is still debated.

Prolactine

Prolactine (PRL) is best known as the hormone which controls milk synthesis in the mammary gland during lactation. The fact that hypofertility is often linked to hyperprolatinemia in both males and females suggests that PRL may subserve other functions related to reproduction. In addition, PRL is a "stress" hormone in both sexes, released in large amounts in response to a variety of stressfull stimuli. The function of PRL in stress is however completely unknown.

A stimulatory effect of OT on PRL synthesis and release by lactotrophs has been firmly established on pituitary cell preparations in vitro. The PRL releasing effect of OT can be evidenced only in the abscence of dopamine. Only one subpopulation of lactotrophs respond to OT, which are also responsive to vasoactive intestinal peptide (VIP), but not to thyrotrophin releasing hormone (TRH).

The physiological importance of OT as a PRL releasing factor remains unclear. Passive immunoneutralization and antagonist studies in animal models have demonstrated that endogeneously released OT does indeed contribute to the secretion of PRL in response to suckling and ovarian steroid administration. However, it also appear from these studies that stress-induced PRL secretion (during restraint or inhalation of ether) does not require OT.

Luteinizing hormone

Production of a surge of luteinizing hormone (LH) in pro-estrus is essential to the process of ovulation. In recent years, evidence has accumulated suggesting that OT contributes to the mechanisms modulating LH surge. Concentrations of OT in the portal blood system are higher at pro-estrus than at other times in the ovarian cycle. Additionally, OT in peripheral plasma is also highest at that time. An increase of LH release in response to OT was observed both in animals models and in dispersed cells in culture. The effect of OT was enhanced by estrogens and inhibited by progesterone. Most demonstrative is a recent study conducted on healthy volonteer women, showing that intravenous administration of OT at preovulatory stage of the menstrual cycle markedly advances LH surge and ovulation as compared to infusion of saline. Such an effect was not observed in women in a state with low estrogen or high progesterone levels, nor in males. The site(s) and mechanism(s) of action of OT have yet to be definitely established. Evidence available so far suggest both a direct effect of OT on LH secretion and an indirect effect by sensitization of luteotrophs to GnRH.

CENTRAL EFFECTS

OT fullfills most of the criteria required to be defined as a neurotransmitter. First, OT is not only produced in neurones projecting to the pituitary gland, but also in others whose axons remain and terminate within the central nervous system. Second, in vivo release of OT from these axon terminals have been demonstrated in a number of innervated areas. Third, specific OTRs are detectable in many brain structures, whose activation by OT binding induces neuronal depolarization. And finally, evidence exists that OT is involved in the initiation or regulation of several central functions, in particular functions related to reproduction.

Milk-ejection reflex

The survival of new-born mammals requires an adequate supply of milk from their mother. During nursing, maternal oxytocin is released by posterior pituitary axons in response to suckling by the new-borns, stimulates the contraction of myoepithelial cells of the mammary gland, and milk is thus ejected. In the rat, this neuroendocrine reflex is characterized by the intermittence of milk ejections which occur at few minutes intervals despite constant sukling stimulus. Each pulse of OT released during nursing is preceded by an increase of the firing rate of OT-secreting hypothalamo-neurohypophysial neurones. There is strong evidence that OT itself, by a central action, determines the interval between each milk ejection : intracerebroventricular (icv) administration of OT increases markedly both the frequency and the amplitude of the reflex while administration of an OT antagonist temporarly slows the milk ejections, suggesting that endogeneous OT is normally invoved in their timing. The site of action of OT may be the supraoptic and paraventricular nuclei themselves, where a local dendritic release of OT has been demonstrated and where OTRs have been localized. Thus OT might facilitates its own release by exerting autocrine and paracrine effects on OT neurones. Alternatively, OT may activates neurones of a limbic structure, the bed nucleus of the stria terminalis, where OTRs are also present, and which is involved in the transformation of the constant suckling stimulus into a sequence of intermittent burst of action potentials by hypothalamic OT neurones.

Maternal behavior

OT has been shown to facilitate maternal behavior in rat and sheep. The mother rat builds a nest prior to parturition, crouches over her young once born, and if one of the pups is displaced, carries it back in the nest. Injection of OT into the cerebral ventricles can induce these and other aspects of maternal behavior in virgin female rats, provided their estrogens levels are high. Moreover, intracerebroventricular administration of an OT antagonist following parturition was shown to impair spontaneous maternal behavior. These data support a role for endogeneous OT in stimulating the onset of maternal behavior in rat. A similar demonstration has been made in the sheep which display a markedly different behavioral pattern. While rats are born immature and helpless, the young sheep is precocious and can follow his mother soon after birth. A specific bond is formed between the mother and her young, provided she can smell him within the 2 hours following birth. Central administration of OT, either in the cerebral ventricles or in specific areas of the brain, stimulates a rapid onset of maternal behavior and induces the female sheep to stay with her lamb and to feed him.

Sexual behavior

The effects of OT on sexual behavior have been studied mainly in the rat. The receptive female rat displays a typical posture, lordosis, which enables mounting and intromission by the male. Numerous studies have established that lordosis behavior is facilitated by infusion of OT in the cerebral ventricles or directly within some hypothalamic areas, whereas it is impaired by infusion of an OT antagonist. As for maternal behavior, OT is effective only in the presence to high estrogen levels. In the male rat, OT appears to be the most potent agent discovered so far to induce penile erection. A similar response can be induced in rabbits and monkeys.

The concentration of plasmatic OT increases by roughtly 10-fold during orgasm both in man and woman. Accordingly, milk ejection during orgasm has been reported to occur occasionally in lactating women. Thus, circulating OT probably play a role in events occuring in genital tract organs during orgasm. Since both OT and its receptors are present in several brain areas of both man and woman, it is not unreasonable to speculate that central OT may also be involved in behavioral aspects of the reproductive function in humans.

CONCLUDING REMARKS

One of the current strategies in attempting to evaluate the essential role of a given endogeneous compound is to manipulate the expression of the gene coding for this compound at early stages of development. Using homologous recombination in embryonic stem cells, deletion of the coding region of the OT gene has been recently successfully achieved, yielding OT deficient mice. Mice lacking OT were both viable and fertile. Males did not have any reproductive behavioural or functional defects. Similarly, females lacking OT had no obvious deficits in fertility or reproduction, including gestation and parturition. However, although OT-deficient females demonstrated normal maternal behavior, all new borns die shortly after birth because of the dam’s inability to nurse. Postpartum injections of OT to the OT-deficient mothers restored milk ejection and rescued the offspring.

Thus, OT appears to play an essential role only in milk ejection in the mouse ! This however does not mean that the multiple reproductive activities that have been attributed to OT are pure experimental artefacts ! The major drawback of the method used in the experiment just reported is that deletion of the OT gene occurs prior to development of the network and mechanisms involved in the control of reproductive functions. Since this control is obviously a finely tuned multifactorial proccess, the original lack of OT may well be compensated during development by other compounds. An objective evaluation of the role of OT would require the possibility to switch off the OT gene acutely in adult animals after normal development, which the development of new technologies should allow in a near future.

REFERENCES

1. Dreifuss J.J. (1993) Oxytocin in reproductive biology : newly discovered sites of production and action. In : Reproductive Health (Ed A. Campana), Frontiers in Endocrinology 2, 71-74, Ares-Serono Symposia Publications.
2. Gainer H. and Wray S. (1994) Cellular and Molecular biology of Oxytocin. In : The Physiology of Reproduction, 2^nd edition, Edts Knobil and Neill, 1099-1128, Raven Press, N.Y.
3. Oxytocin in Maternal, Sexual, and Social Behaviors (1992) Annals of the New York Academy of Sciences 652, Edts Petersen C.A., Caldwell J.D., Jirikowski G.F. and Insel T.R.
4. Oxytocin, Cellular and Molecular Approaches in Medicine and Research (1995) Advances in Experimental Medicine and Biology 395, Edts Ivell R. and Russell J.A., Plenum.
5. Nishimori K. Young L.J., Guo Q., Wang Z., Insel T.R. and Matzuk M.M. (1996) Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc. Natl. Acad. Sci. USA 93, 11699-11704.