Biochemical and endocrine aspects of oxytocin production by the mammalian corpus luteum

A review of the current state of knowledge of oxytocin production by the preovulatory follicle and corpus luteum is presented. Corpora lutea of a number of mammalian species have been found to synthesize oxytocin. However, the synthesis and secretion of this nanopeptide by the corpus luteum of the ruminant has been most extensively studied because of the potential role of this peptide in facilitating luteal regression. While much information exists relative to various biochemical and endocrine factors that impact on oxytocin gene expression, this aspect about luteal synthesis of this peptide hormone remains enigmatic. Prostaglandin F-2α (PGF-2α) has been shown to be a primary endogenous hormone responsible for triggering luteal secretion of oxytocin. Details are provided regarding the PGF-2α-induced intracellular signal transduction pathway that ultimately results in exocytosis of luteal oxytocin. Evidence is also presented for potential autocrine/paracrine actions of oxytocin in regulating progesterone production by luteal and granulosa cells. Concluding remarks highlight aspects about luteal oxytocin production that require further research.     

Hormonal responses following treatment with different prostaglandin analogues for estrous cycle regulation in cattle

The effect of four different prostaglandin F(2)alpha analogues on secretion of the pituitary hormones prolactin, LH, FSH and oxytocin was evaluated in heifers. In addition, the luteolytic activity was measured by means of progesterone concentrations. For the sake of comparison, the corpus luteum of two heifers was enucleated and the effect on the above hormones determined. There seemed to be no differences among the various analogues concerning their luteolytic action on the corpus luteum. No effect on FSH secretion was observed. Basal concentrations of LH increased about 4 - 5 h after injection of the analogues, concurrently with the decrease of progesterone. Enucleation of the corpus luteum was followed by a similar pattern of hormone secretion. Therefore, the increase in LH concentrations did not appear to be a specific effect of the analogues, but was probably a result of the decrease in progesterone concentration. Within twenty minutes after administration of the analogues a clear increase in plasma prolactin (2-4 h) and oxytocin concentrations (1-2 h) could be demonstrated.     

Secretion of oxytocin and progesterone by ovine corpora lutea in vitro

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.     

The gene for the hypothalamic peptide hormone oxytocin is highly expressed in the bovine corpus luteum: biosynthesis, structure and sequence analysis.

Expression of the vasopressin and oxytocin genes has been described so far only in the hypothalamus. We report here that at least the oxytocin gene is highly transcribed in the bovine corpus luteum during the mid-luteal phase of the oestrous cycle. Luteal cDNA sequence analysis as well as cell-free translation studies showed that the luteal mRNA is essentially similar to that in the hypothalamus, except that in the corpus luteum the poly(A) tail of this mRNA is shorter. When calculating the relative amounts per organ, the active corpus luteum produces approximately 250 times more oxytocin mRNA than a single hypothalamus.     

Inhibition of luteal function by oxytocin antagonist in goats (Capra hircus).

Early corpus luteum development in nonpregnant and pregnant goats was characterized by a steady increase in peripheral plasma concentrations of progesterone and a high release of prostacyclin (PGI-2) but low release of prostaglandin F-2 alpha (PGF-2 alpha). Jugular administration of oxytocin antagonist (OXA) (0.2 microgram/kg/day) on the day of oestrus and for 3 days thereafter to cyclic and mated goats, significantly (P less than 0.001) inhibited progesterone and prostaglandin secretion and reduced conception rate. Co-administration of PGI-2 (200 micrograms/day) with OXA resulted in a steady increase of progesterone and establishment of pregnancy, but co-administration of PGF-2 alpha (175 micrograms/day) with OXA had no effect. It is suggested that oxytocin is required for early development of the corpus luteum and such effects may be mediated via PGI-2 production.     

Prostaglandin F2 alpha and oxytocin interactions in ovarian and uterine function

The oxytocin-neurophysin gene is expressed in several nontraditional sites within the endocrine system. In the ovary its expression in the corpora lutea is initiated by ovulation. Ovarian oxytocin concentrations reach maximal levels around day 11 of luteal cycle and fall to a nadir at estrus. PGF2 alpha has the capacity to release oxytocin from the corpus luteum, and oxytocin in turn releases PGF2 alpha from the uterine endometrium or decidua. This positive feedback loop between the ovary and the uterus ensures the completion of luteolysis in species that depend on the presence of the uterus for the termination of luteal lifespan. Immunization against oxytocin has been shown to disrupt this loop, resulting in much-prolonged luteal cycles. In primates and other species in which luteal life span is independent of the uterus, an oxytocin PGF2 alpha interaction may take place within the ovary itself. At parturition a related interaction takes place which ensures the expulsion of the fetus and placenta in an orderly manner. Oxytocin of both pituitary and ovarian origin reaches the uterus via its blood supply and binds to two types of receptors: one on myometrial cells, the occupation of which initiates contractions, and the other on decidual cells, the occupation of which initiates prostaglandin generation. This prostaglandin diffuses into the adjacent myometrium and augments the oxytocin-induced contractions. In conjunction with a direct softening effect by prostaglandins on the cervix the augmented contractions achieve the force needed to dilate the cervix and expel the fetus. An additional source of oxytocin during labor may be the placenta, another non-traditional site for the occurrence of oxytocin.     

In vivo desensitization of a high affinity PGF2 alpha receptor in the ovine corpus luteum.

The corpus luteum (CL) of the sheep exhibits a differential sensitivity to PGF2 alpha in vivo in terms of an increase in oxytocin (OT) secretion and a decrease in progesterone secretion, pointing to the presence in vivo of both high and low affinity receptors for PGF2 alpha. The presence of the high affinity PGF2 alpha receptor was assessed by monitoring the secretion rate of OT from the ovine CL in response to subluteolytic infusions of PGF2 alpha. Rapid desensitization to PGF2 alpha occurred after only one hour of infusion, while a minimum rest period of six hours was required to restore sensitivity. The possibility that these findings could be explained by the depletion and resynthesis of OT was excluded by demonstrating an increase in OT secretion rate with supra-physiological levels of PGF2 alpha two hours after desensitization. Collectively, these results indicate the presence of a high affinity receptor for PGF2 alpha in the ovine CL which exhibits desensitization and recovery in vivo. The temporal nature of the desensitization and recovery of the high affinity PGF2 alpha receptor controlling luteal OT secretion may contribute to the pulsatile nature of PGF2 alpha release from the ovine uterus.     

A mathematical model of pregnancy recognition in mammals

In this paper we develop a mathematical model of the luteal phase of the reproductive cycle in mammals with the aim to generate a systems understanding of pregnancy recognition. Pregnancy recognition is initiated by the production of interferon tau (IFNτ) by the growing conceptus. This ensures that the maternal corpus luteum (CL) remains viable to secrete progesterone, which is critical for providing a uterine microenvironment suitable for embryonic growth. Our mathematical model describes the interactions among the CL, the reproductive hormones and the hormone receptors in the uterus. It also characterises the complex interactions amongst the uterine oestrogen, progesterone and oxytocin receptors that control the sensitivity of the uterus to oestrogen, progesterone and oxytocin, respectively. The model is represented by a dynamical system and exhibits qualitative features consistent with the known experimental results in sheep. A key factor identified was a time-dependent threshold for the IFNτ signal below which the presence of the embryo might not be recognised and thus pregnancy would likely fail. Furthermore, the model indicated that if the IFNτ signal is later than around day 13 of the cycle, then pregnancy will not be recognised irrespective of the IFNτ concentration. The thresholds in the concentration and time of the IFNτ signal is a screening mechanism whereby only embryos of sufficient quality are able to prevent luteolysis (i.e. regression of the CL). The effect of progesterone secretion rate from the CL on pregnancy recognition was investigated. The model suggests that if the secretion rate is low then the initiation of the IFNτ signal is delayed, which in turn compromises the likelihood of a pregnancy being recognised by the CL. Furthermore, pregnancy recognition does not occur below a critical threshold in the progesterone secretion rate. In summary, the model can be used to identify the most favourable conditions for pregnancy recognition.     

Uterine blood supply as a main factor involved in the regulation of the estrous cycle--a new theory

The paper presents a new theory on the physiological mechanism of initiation of luteolysis, function of endometrial cells and protection of corpus luteum. This theory is based on previous studies published by the authors and their coworkers on the retrograde transfer of PGF2alpha in the uterine broad ligament vasculature during the estrous cycle, early pregnancy and pseudopregnancy. The studies were focused on cyclic changes in uterine blood supply and the apoptosis of endometrial cells. Moreover, the results of many other authors are cited. The statements of the theory are as follows: 1. The initiation of luteolysis is a consequence of regressive changes in the endometrium which are due to the reduction of the uterine blood supply below the level necessary to provide for the extended needs of active endometrium. 2. During the luteal phase, both a considerable increase in uterine weight and a decrease in blood flow through the uterine artery, resulting from increasing progesterone concentration, reduce the uterine blood supply. In comparison to the volume of blood flowing to the porcine uterus during the estrus period, only 30-40% of the blood volume is determined on day 12 of the estrous cycle. The uterine weight at that time is 40-60% larger than that in the early luteal phase. Thus, due to the considerable constriction of uterine blood vessels, there is a discrepancy between the requirement for oxygen and other factors transported by blood and the possibility of supplying the uterus with these substances. After reaching the threshold of uterine blood supply level, which in pigs takes place around day 12 of the estrous cycle, regressive changes and PGF2alpha release from endometrial cells occurs. 3. Estrogens and progesterone are the major factors affecting blood flow in vessels supplying the uterus. The factors that modulate, complement and support vasodilation and vasoconstriction are: PGE2, LH, oxytocin, cytokines, neurotransmitters and other local blood flow regulators. In some animal species these modulators, especially those of embryonic origin, may be crucial for the status of uterine vasculature. 4. During early pregnancy, the action of embryo signals (estrogens, cytokines), endometrial PGE2 as well as LH results in the relaxation of the uterine artery (pigs: day 12) and, consequently, in an increase in uterine blood supply. This reaction of the maternal recognition of pregnancy effectively prevents regressive changes in well developed endometrial cells to occur. 5. Local uptake and retrograde transfer of PGF2alpha into the uterine lumen during early pregnancy protects corpus luteum from PGF2alpha luteolytic action. 6. During the period of regressive changes resulting from the limited uterine blood supply, endometrial cells restrain PGF2alpha synthesis. They are, however, still capable of releasing prostaglandin when uterine blood supply is improved after the embryo appears in the uterus. This potential capability for PGF2alpha synthesis was demonstrated in in vitro studies when endometrial cells collected during its regressive phase were incubated in medium and stimulated by LH and oxytocin. 7. Prostaglandin F2alpha pulses in venous blood flowing from the uterus do not confirm pulsatile secretion of PGF2alpha. The pulses may result from the pulsatile excretion of PGF2alpha with venous blood according to the rhythmic uterine contractions associated with oxytocin secretion. 8. The results supporting this concept are presented and discussed in due course. The critique of Bazer and Thatcher's theory on exocrine versus endocrine secretion of prostaglandin F2alpha during the estrous cycle is also depicted.     

Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep

Jugular venous concentrations of oxytocin and progesterone changed in parallel during the oestrous cycle in the ewe, falling at luteal regression and rising with formation of the new corpus luteum. These fluctuations in the circulating concentration of oxytocin were not caused by changes in its metabolic clearance rate. On Days 6-9 of the cycle circulating oxytocin concentrations exhibited a diurnal rhythm, peaking at 09:00 h; this rhythm was absent on Days 11-14. Although there was no evidence for increased production of oxytocin at or preceding luteal regression in samples taken daily, more frequent sampling revealed that two thirds of detected surges of uterine secretion of prostaglandin (PG) F-2 alpha were accompanied by raised levels of oxytocin. This oxytocin was not of pituitary origin. Luteal regression induced with cloprostenol on Day 8 after oestrus caused a decrease in circulating progesterone level followed after 24 h by a fall in oxytocin. Measurements of oxytocin in the ovary and other organs before and after treatment with cloprostenol identified the corpora lutea as a major potential source of oxytocin, and suggested that 98% of luteal oxytocin was available for secretion in response to prostaglandin stimulation. The data are consistent with a role for ovarian secretion of oxytocin in response to uterine release of PGF-2 alpha in the control of luteal regression.     

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum

Summary The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.     

Effect of day of the oestrous cycle, side of the reproductive tract and heat shock on in-vitro protein secretion by bovine endometrium

Endometrial explant cultures were prepared from 16 Brahman x Angus cows killed on Days 0, 2, 5 or 8 after oestrus. Cultures proceeded for 24 h at 39 degrees C (homeothermic) or 43 degrees C (heat shock) in a modified Eagle's minimal essential medium supplemented with 50 microCi L-[4,5(-3)H]leucine. Analysis by two-dimensional polyacrylamide gel electrophoresis of de-novo synthesized proteins secreted into the medium indicated that the major types of secreted polypeptides did not change over Days 0-8. Nevertheless, overall endometrial secretion of protein (incorporation of [3H]leucine into non-dialysable radioactivity in culture supernatants) was greatest at Day 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable material in tissue homogenates was also greatest at Day 0. For tissue cultured at 39 degrees C, several individual polypeptides were secreted at greater rates by endometrium from the horn of the uterus ipsilateral to the corpus luteum, with side differences tending to be greatest at Day 0 or Day 2. Overall, secretion of de-novo synthesized protein by endometrium was significantly elevated by heat shock at Day 0, but not affected thereafter. Nonetheless, heat shock reduced secretion of several individual proteins and exhibited interactions with day of the oestrous cycle and with side of the uterus. Secretion of 7 polypeptides was reduced by heat shock in tissue from the ipsilateral horn of the uterus but not in endometrium from the contralateral horn. We suggest that endometrial protein secretion changes quantitatively during the early oestrous cycle. In addition, there is a local influence of the ovary bearing the corpus luteum on endometrial function that may be disrupted by heat shock.     

Ultrastructural localisation of oxytocin and neurophysin in the ovine corpus luteum

Summary Polyclonal rabbit antisera raised against oxytocin, bovine neurophysin I and vasopressin were used, together with an immunogold complex, to localise the peptides in ultrathin sections of ovine corpus luteum. The only organelle which consistently showed gold labelling was the secretory granule of the large luteal cell. In non-consecutive sections of the same large luteal cell all the granules showed a similar level of labelling after oxytocin or neurophysin I antisera: however no immunolabelling was detected for vasopressin. Oxytocin and neurophysin seem to be rapidly lost after secretion since exocytosed granule cores showed no labelling above background levels.     

Progesterone production by dispersed luteal cells of non-pregnant cows: effects of oxytocin and oestradiol

The effects of oxytocin and oestradiol on progesterone production by dispersed luteal cells of non-pregnant cows were studied. In acute incubation (3 h), oxytocin, at a concentration of 800 mIU/ml, significantly inhibited the production of progesterone induced by HCG (10 IU/ml). Suppression of basal progesterone production was evident in some corpora lutea. Lower oxytocin concentrations (4 and 40 mIU/ml) had no effect. At a concentration of 400 mIU/ml, oxytocin may be inhibitory to basal and HCG-induced progesterone production. Oestradiol (1 μkg/ml) had no effect on basal progesterone production but may suppress the production of progesterone induced by HCG. However, incubation with oxytocin (400 mIU/ml) plus oestradiol (1 μg/ml) resulted in a significant inhibition of HCG-induced progesterone production. These data provide evidence for an inhibitory effect of oxytocin on the corpus luteum of non-pregnant cows. Oestradiol may interact with oxytocin to inhibit the bovine corpus luteum function.     

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein is associated with bovine luteal oxytocin exocytosis

The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F(2alpha) (PGF(2alpha)) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF(2alpha) on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.     

Regulatory effect of steroid hormones and fetal tissues on expression of oxytocin receptor in the endometrium of late pregnant ewes.

Oxytocin receptors play an important role in the establishment of pregnancy and parturition in ruminants. Previous studies in cyclic and early pregnant ewes have indicated that receptor concentrations are regulated by steroid hormones and fetal secretory products. This study investigated the effect of oestradiol and progesterone, or co-culture with placenta or corpus luteum on oxytocin receptor expression. Endometrial explants from late pregnant ewes were cultured for up to 96 h in various treatment combinations. After culture, tissues were subjected to in situ hybridization and autoradiography with 125I-labelled oxytocin receptor antagonist to localize and measure the expression of oxytocin receptor mRNA and protein. Results were quantified as absorbance units from autoradiographs. Oxytocin receptors were confined to the endometrial luminal epithelium and both mRNA and 125I-labelled oxytocin receptor antagonist binding were upregulated spontaneously in basic serum-free medium. Upregulation occurred earlier in the presence of oestradiol (0.1 mumol l-1) but the final receptor concentration was similar to that found in the basic medium. Continuous progesterone treatment (1 mumol l-1) and co-culture with corpus luteum both delayed the increase in oxytocin receptor mRNA, but a short initial (4 h) period in progesterone-free basic medium resulted in loss of the inhibitory effect. Co-culture with placental tissues had no effect. In conclusion, oxytocin receptor expression in the luminal epithelium increased immediately on removal from the maternal environment. This occurred regardless of treatment and did not require the presence of steroid hormones, but could be accelerated or delayed by oestradiol and progesterone, respectively. There may be an additional inhibitory factor present in the corpus luteum.     

Effect of intra-ovarian infusion of oxytocin on plasma progesterone concentrations in pregnant ewes.

The function of oxytocin receptors in the corpus luteum of pregnant ewes was investigated by infusing saline or oxytocin (100 ng/min) into the utero-ovarian artery of pregnant ewes (62 +/- 5 days, n = 12). During a 4-h infusion, plasma oxytocin (OT) concentration increased to 268 +/- 80 pg OT/ml in the OT-infused group and remained unchanged at 2.5 +/- 1.5 pg OT/ml in the saline-infused group. Progesterone concentration in jugular venous plasma (17 +/- 9 ng/ml) rapidly decreased during oxytocin infusion to 59 +/- 10% and 26 +/- 9% of control at 1.5 and 2 h, respectively; the utero-ovarian venous concentration of 64 +/- 38 ng/ml decreased by a similar magnitude during oxytocin infusion. Electron microscopy of corpora lutea, removed at the end of the experiments, showed no indication of luteolytic changes following oxytocin infusion. It was concluded that oxytocin markedly and rapidly reduces progesterone secretion in pregnant ewes.     

Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro

Slices of porcine endometrium and corpus luteum tissue obtained from mature sows throughout the luteal phase of the oestrous cycle were incubated in culture medium which was analysed at regular intervals over a period of 8 hours for prostaglandin F and progesterone. Prostaglandin F secretion was greatest by endometrium obtained during the mid III to late I luteal stage of the cycle and the increased levels secreted by this tissue were paralleled by high levels of secretion from corpus luteum tissue. The addition of indomethacin (10 μg/ml) to the culture medium completely abolished prostaglandin F secretion by both endometrium and luteal tissue indicating that the high levels of the prostaglandin were due to synthesis. Progesterone secretion by the corpus luteum was maximal from early luteal tissue and had declined to considerably lower levels by late stage tissue when prostaglandin secretion was greatest. The possible physiological significance of luteal prostaglandin F secretion is discussed.     

Secretion of neurophysins by the ovary in sheep

Measurements of venoarterial concentration differences across the ovary in anesthetized sheep have demonstrated that the ovary secretes ovine neurophysin I/II (oNP I/II) and that this process is stimulated by the prostaglandin F2 alpha analogue, cloprostenol. A parallel increase in the secretion of oxytocin (OT) was observed in response to cloprostenol, and the mean molar ratio of oNP I/II to OT secreted was 1.2. There was no detectable ovarian secretion of oNP III. Secretion of oNP I/II and OT was absent after hysterectomy. The data support other evidence indicating that the corpus luteum synthesizes OT, and confirm that the neurophysin associated with OT in the sheep is oNP I/II.     

Counter current transfer of PGF2 alpha in the mesometrial vessels as a mechanism for prevention of luteal regression in early pregnancy

A new concept has been presented on the mechanism protecting the corpus luteum during oestrous cycle, early pregnancy and pseudopregnancy induced by oestrogens. The concept is based on the recently discovered mechanism of back transfer of prostaglandin F2 alpha from the broad ligament vasculature into the uterus and on the participation of oestrogen in this process. The morphological facilitates for counter-current transfer of PGF2 alpha in the area of mesometrial vasculature and ability the uterus to bind PGF2 alpha were presented. It has been concluded that the process of PGF2 alpha back-transfer from mesometrial vasculature into the uterus may reduce in uterine venous blood the amplitude of PGF2 alpha pulses and by this way may reduce the penetration of prostaglandin into subovarian area and from there to the corpus luteum.