Molecular determinants of a competent bovine corpus luteum: first- vs final-wave dominant follicles.

Reproductive management in cattle requires the synchrony of follicle development and oestrus before insemination. However, ovulation of follicles that have not undergone normal physiological maturation can lead to suboptimal luteal function. Here, we investigated the expression of a targeted set of 47 genes in (a) a first-wave vs final-wave dominant follicle (DF; the latter destined to ovulate spontaneously) and (b) 6-day-old corpora lutea (CLs) following either spontaneous ovulation or induced ovulation of a first-wave DF to ascertain their functional significance for competent CL development. Both the mass and progesterone-synthesising capacity of a CL formed following induced ovulation of a first-wave DF were impaired. These impaired CLs had reduced expression of steroidogenic enzymes (e.g. STAR and HSD3B1), luteotrophic receptors (LHCGR) and angiogenic regulators (e.g. VEGFA) and increased expression of BMP2 (linked to luteolysis). Relative to final-wave DFs, characteristic features of first-wave DFs included reduced oestradiol concentrations and a reduced oestradiol:progesterone ratio in the face of increased expression of key steroidogenic enzymes (i.e. CYP11A1, HSD3B1 and CYP19A1) in granulosa cells and reduced expression of the HDL receptor SCARB1 in thecal cells. Transcripts for further components of the TGF and IGF systems (e.g. INHA, INHBA, IGF2R and IGFBP2) varied between the first- and final-wave DFs. These results highlight the importance of hormones such as progesterone interacting with local components of both the TGF and IGF systems to affect the maturation of the ovulatory follicle and functional competency of the subsequent CL.

A PPAR-independent pathway to PUFA-induced COX-2 expression.

Polyunsaturated fatty acids (PUFAs) induce COX-2 in bovine endometrial stromal cells through activation of peroxisome-proliferator-activated receptor alpha (PPARalpha). We have investigated alternative (PPAR-independent) pathways to COX-2 induction using a reporter construct driven by a COX-2 gene promoter sequence lacking a PPAR response element. This construct was induced by PUFAs, but not by PPAR agonists. PPAR-independent reporter gene expression occurred 6h after PPAR-dependent induction of the endogenous COX-2 gene. In contrast to PPAR-dependent COX-2 induction, which is not affected by NF-kappaB inhibitors, the PPAR-independent pathway was blocked by the NF-kappaB inhibitor MG132 or following deletion of NF-kappaB sites in the COX-2 promoter. The PPAR-independent effect of PUFA was mimicked by the PKC activators 4beta-PMA and prostaglandin F(2alpha), but was not blocked by the PKC inhibitor RO318425. The results demonstrate a pathway to the induction of COX-2 by PUFAs requiring NF-kappaB but not PPAR or PKC.

Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid.

Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.

Ligand-independent activation of steroid receptors.

In several transformed cell lines, the growth factors IGF-I and epidermal growth factor (EGF) activate second messenger systems that cause the phosphorylation of the estrogen receptor (ER). One kinase catalysing receptor phosphorylation is mitogen activated protein (MAP) kinase, and the result of phosphorylation is an increase in receptor transactivation function. EGF and IGF-I, secreted locally and systemically, are involved in uterine-conceptus interactions in early pregnancy, and therefore it is of interest to determine whether these growth factors affect ER function in the uterus. An estrogen response element, chloramphenicol acetyl transferase reporter gene construct (CATERE) was transfected into bovine endometrial epithelial and stromal cells in vitro, and CAT measured during transient expression. Growth factors were added at various times following transfection, and MAP kinase phosphorylation was monitored by western blotting of p42 and p44. The MEK inhibitor U 0126 was used to determine whether the effect of IGF-I on CATERE expression was mediated through MAP kinase, and the anti-estrogen ICI 182780 was used to identify effects involving the ER. In stromal cells, reporter gene activity was increased in a dose dependent manner by IGF-I or hEGF in the presence or absence of estradiol-17beta. In the absence of estradiol the effect of IGF-I was not inhibited by ICI 182780. The effect of IGF-I occurred within an hour, before any detectable increase in cell proliferation, and the activation of CAT expression in response to IGF-I or EGF was blocked by U 0126. In contrast to their effects in stromal cells, neither IGF-I nor EGF affected CAT expression in bovine endometrial epithelial cells. Measurement of phosphorylated MAP kinases p42/p44 by western blotting showed that EGF but not IGF-I activated MAP kinase phosphorylation in both epithelial and stromal cells. In stromal cells, the fact that U 0126 blocked the CAT responses to IGF-I and EGF indicates the involvement of a MAP kinase. But since IGF-I did not activate p42/p44, a different MAP kinase, not detected by the antibody used here, is implicated. As the response was not blocked by ICI 182780, we conclude this effect is independent of ER activation. Therefore in bovine uterine cells in culture effects on MAP kinases p42/p44 can be dissociated from those on ERE-dependent gene expression, and reporter gene expression may be independent of ER activation.

The effects of lipopolysaccharide and interleukins-1alpha, -2 and -6 on oxytocin receptor expression and prostaglandin production in bovine endometrium.

Up-regulation of endometrial oxytocin receptor (OTR) expression followed by an increase in pulsatile endometrial prostaglandin (PG) F(2alpha) secretion causes luteolysis in cattle. Inhibition of luteolysis is essential for the maternal recognition of pregnancy but also occurs in association with endometritis. The factors regulating OTR expression at this time are unclear. The OTR gene promoter region contains binding elements for acute phase proteins but their function has not been established. This study investigated the effects of various cytokines on OTR expression and on PGF(2alpha) and PGE(2) production in explant cultures of bovine endometrium. Endometrium was collected in the late luteal phase (mean day of cycle 15.4+/-0.50) or early luteolysis (mean day of cycle 16.4+/-0.24) as determined by the initial concentration of endometrial OTR. Explants were treated for 48 h with: (i) lipopolysaccharide (LPS) and/or dexamethasone (DEX), (ii) ovine interferon-tau (oIFN-tau), or (iii) human recombinant interleukin (IL)-1alpha, -2 or -6. OTR mRNA was then measured in the explants by in situ hybridisation and the medium was collected for measurement of PGF(2alpha) and PGE(2) by RIA. LPS treatment stimulated production of PGF(2alpha), whereas DEX either alone or in combination with LPS was inhibitory to both PGF(2alpha) and PGE(2). Neither of these treatments altered OTR mRNA expression. oIFN-tau reduced OTR mRNA expression but stimulated production of both PGF(2alpha) and PGE(2). In endometrial samples collected in the late luteal phase, IL-1alpha, -2 and -6 all inhibited OTR mRNA expression, but IL-1alpha and -2 both stimulated PGF(2alpha) production. In contrast, when endometrium was collected in early luteolysis, none of the interleukins altered OTR expression or caused a significant stimulation of PGF(2alpha) production but IL-2 increased PGE(2). Neither IL-1alpha nor -2 altered OTR promoter activity in Chinese hamster ovary cells transfected with a bovine OTR promoter/chloramphenicol acetyl transferase reporter gene construct. In conclusion, the action of interleukins on both OTR mRNA expression and endometrial prostaglandin production alters around luteolysis. Pro-inflammatory interleukins suppress OTR expression in the late luteal phase, while LPS stimulates PGF(2alpha) without altering OTR mRNA expression. IL-I and -2 and LPS are therefore unlikely to initiate luteolysis but may cause raised production of PGF(2alpha) during uterine infection.

Acute effects of interferon on estrogen receptor function do not involve the extracellular signal-regulated kinases p42mapk and p44mapk.

Exposure to type I interferons (IFN) increased estrogen receptor (ER) ligand binding and induced protein kinase C (PKC) translocation within 30 min but had no effect on net incorporation of [32P] into ER in Madin Darby bovine kidney (MDBK) cells. Ligand binding was also increased within 30 min by phorbol ester and the protein phosphatase inhibitor okadaic acid. Mitogen-activated protein (MAP) kinase phosphorylation was initially inhibited between 2 and 30 min and subsequently activated between 30 and 60 min after treatment with IFN. The activatory response was blocked by the PKC inhibitor Ro 31-8220. Following transient transfection with an ERE-CAT reporter construct, IFN increased CAT expression after 6 h but decreased ER ligand binding, transcriptional activity and phosphorylation after 48 h, probably as a result of decreased ER concentrations. The results rule out rapid activation of ER ligand binding through phosphorylation at Ser118 by MAP kinase because (1) the increase in ligand binding preceded activation of MAP kinase, and (2) IFN had no short-term effect on [32P]incorporation or ER transcriptional activity. The rapid effect of IFN on ER ligand binding is postulated to reflect phosphorylation of the receptor at Tyr537 by p56lck, a member of the Src family of PKC-activated tyrosine kinases.

Structure of an ovine interferon receptor and its expression in endometrium.

A sheep type I interferon receptor (oIFNAR1) cDNA was isolated from a lambda-ZAP library using a reverse transcription (RT)-PCR product probe generated from oestrous endometrial RNA. The oIFNAR1 cDNA was 79, 66 and 95% homologous to human, murine and bovine IFNAR1 cDNAs respectively. The encoded receptor was a 560-amino acid transmembrane protein 80, 66 and 95% similar to human, murine and bovine IFNAR1 respectively. Northern blot analysis of endometrial mRNA revealed the presence of 6.5, 4.3 and 3.7 kb transcripts. Using semi-quantitative RT-PCR the oIFNAR1 mRNA was not found to be down-regulated after 72 h treatment with bovine recombinant IFN-alpha I in in vitro experiments with endometrial explants.

Absence of the oxytocin-induced prostaglandin F2 alpha secretory response in uterus from ovariectomized ewes and activation of the response in vitro.

Oxytocin-induced prostaglandin F2 alpha (PGF2 alpha) responses were measured in explants of uterus from ovariectomized ewes on the day of tissue collection or after culture for 72 h in the presence or absence of oestradiol-17 beta (100 nmol/l). Oxytocin receptor binding activity was 210 +/- 47 fmol [3H]oxytocin bound per mg protein in fresh tissue and 89 +/- 24 and 90 +/- 17 fmol/mg in tissue cultured with control medium or with oestradiol respectively (means +/- S.E.M.). PGF2 alpha production during the hour following oxytocin administration to freshly collected tissue was 272 +/- 77 ng/g/h compared with 193 +/- 35 ng/g/h in the absence of oxytocin. These rates were 2789 +/- 1085 and 353 +/- 135 ng/g/h after culture for 72 h in control medium and 2022 +/- 496 and 342 +/- 134 ng/g/h after culture with oestradiol. Thus oestradiol had no effect on the culture-induced maturation of the PGF2 alpha response. Short-term exposure to arachidonic acid (66 mumol/l) did not increase PGF2 alpha production in fresh tissue but significantly increased basal but not oxytocin-induced PGF2 alpha production after 72 h in culture (P < 0.05). There was an absence of oxytocin-induced inositol phosphate turnover in fresh tissue but after culture concentrations of inositol mono-, bis- and trisphosphates were all significantly increased by oxytocin (P < 0.005). Antisera directed against G-protein alpha sub-units alpha i3, alpha o, alpha q, alpha 11 and the common beta subunit, and prostaglandin H-synthase-1 detected proteins that were present before and after culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of protein kinase C in the inhibitory action of trophoblast interferons on expression of the oxytocin receptor in sheep endometrium.

PhosphoIipid/Ca(2+) -dependent protein kinase C (PKC) and oxytocin receptor were measured in sheep endometrial explants after culture for up to 96 h. Oxytocin receptor binding and PKC activity were reduced by up to 90% in explants exposed to recombinant ovine trophoblast interferon (rolFN-τ), recombinant bovine IFN-α(1) or ovine conceptus secretory proteins (a source of IFN-τ). Inhibition occurred in both caruncular and intercaruncular endometrium taken between days 7 and 10 of the oestrous cycle and in intercaruncular (but not caruncular) endometrium on day 6. Down-regulation of PKC by continued exposure of expiants to 4ß-phorbol myristate acetate, or treatment with PKC inhibitors reduced both oxytocin receptor binding and PKC activity by up to 70%. Tyrosine kinase inhibitors were ineffective. Addition of oxytocin or progesterone, which reduce oxytocin receptor bindingin vivo, also lowered oxytocin receptor bindingin vitro in the absence of any effect on PKC. The data indicate that IFN-τ inhibits oxytocin receptor synthesis by a mechanism involving PKC inhibition, but that a non-PKC pathway also operates to control oxytocin receptor binding in non-pregnant animals. These conclusions were supported by measuring PKC activity and oxytocin receptor binding in endometrium without culture. Prolonged exposure of the endometrium to IFN-τin vivo may lead to PKC down regulation by a mechanism analogous to that involved in the action of continuous activation by agonist, and this may represent one function of the prolonged secretion of IFN-τ over a 10-day period in early pregnancy.

Oxytocin receptor binding activity in cultured ovine endometrium.

Oxytocin receptor binding activity in explants of caruncular and intercaruncular endometrium collected from luteal phase ewes increased during culture. An initial rise in binding activity occurred during the first 24 h of culture in both tissues; binding activity in intercaruncular endometrium continued to increase until day 6, remained unchanged on day 8 and had decreased by day 10 of culture. The maximum concentration of receptors in caruncular endometrium was significantly lower than that in intercaruncular endometrium (P < 0.001) and did not change significantly between days 4 and 10 of culture. Apparent dissociation constants and maximal binding of oxytocin receptor in cultured caruncular and intercaruncular endometrium were 3.09 and 2.72 nmol l-1 and 249 and 459 fmol [3H]oxytocin bound mg-1 protein, respectively. Concentrations of oxytocin receptor remained constant in myometrium during 96 h of culture. The rise in endometrial oxytocin receptor concentration did not result from exposure to fetal calf serum, phenol red or insulin in the culture medium. Substituting fetal calf serum with sheep serum or BSA did not block the rise in receptor binding activity. Actinomycin D and cycloheximide inhibited the rise in receptor concentration in both tissues. Co-culture of lung or kidney with endometrium had no effect on binding activity, whereas co-culture with luteal tissue effectively reduced the rise in oxytocin receptor concentration. To establish whether synthesis of functional oxytocin receptors occurred during culture, the effect of oxytocin on prostaglandin F2 alpha (PGF2 alpha) production was assessed in fresh tissue and after 48 h in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ovarian hormones on oxytocin receptor concentrations in explants of uterus from ovariectomized ewes.

The effects of oestradiol, progesterone, oxytocin and combinations of these hormones on oxytocin receptor binding in explants of uteri from ovariectomized ewes were determined. Receptor binding remained unchanged after 96 h in culture in control medium. Oestradiol at concentrations of 1 pmol-10 mumol l-1 did not alter receptor binding activity in tissue cultured for 96 h, but at 100 mumol l-1 oestradiol significantly reduced (P < 0.01) receptor binding activity. Progesterone and oxytocin significantly reduced receptor binding activity in explants cultured for 96 h (P < 0.05). Explants cultured in medium containing progesterone and oestradiol or oxytocin and oestradiol showed receptor binding characteristics similar to those found in tissue cultured with progesterone or oxytocin alone. When explants were cultured for 72 h in medium containing oestradiol followed by 24 h in medium containing oestradiol alone, oestradiol with oxytocin, oestradiol with progesterone, oxytocin alone, progesterone alone, or in medium with no added hormones, receptor binding activity was always reduced in the presence of progesterone and oxytocin whether or not oestradiol was present in the medium. Receptor binding activity in explants cultured for the final 24 h in medium containing oestradiol or no added hormones were similar to those in tissue cultured in control medium for a total of 96 h. These data show that progesterone and oxytocin reduce oxytocin receptor binding activity in cultured uterine tissue and, in contrast to its effect on the rat uterus, that oestradiol is not a potent stimulator of oxytocin receptor synthesis in uterine tissue of ovariectomized ewes in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin delays oestradiol-induced luteolysis but does not affect oestradiol-induced luteinizing hormone secretion in the ewe.

Circulating concentrations of progesterone, the prostaglandin F2 alpha (PGF2 alpha), metabolite 13,14-dihydro-15-keto PGF2 alpha (DHKF2 alpha) and luteinizing hormone (LH) have been measured in cyclic ewes receiving a continuous infusion of oxytocin, in order to investigate the effect of maintained concentrations of circulating oxytocin on the luteolytic action of oestradiol-17 beta. Oxytocin (3 nmol h-1 intravenously) was given from Day 7 until Day 17 after oestrus with oestradiol-17 beta (2.76 mumol, intramuscularly in sesame oil, 0.5 mL-1) administered on Days 9 and 10. Control ewes, given a continuous infusion of saline (154 mmol L-1, 3 mL-1 h-1) and sesame oil on Days 9 and 10, underwent luteal regression at the expected time, with mean concentrations of plasma progesterone falling to below 1.5 nmol L-1 on Day 16 (0900 hours). Mean plasma progesterone concentrations fell to less than 1.5 nmol L-1 on Day 13 (0900 hours) in ewes receiving saline and oestradiol-17 beta. Oxytocin infusion delayed luteolysis in all treated ewes; those receiving oxytocin infusion and sesame oil failed to undergo luteal regression during the period studied and in animals receiving oxytocin and oestradiol-17 beta, luteolysis was significantly delayed when compared to ewes treated with saline and oestradiol with progesterone concentrations falling to < 1.5 nmol L-1 on Days 14 (n = 1 ewe), 15 (n = 1 ewe), 16 (n = 2 ewes) and > 17 (n = 2 ewes) (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of continuous infusion of oxytocin on ovarian function and uterine oxytocin receptor concentration in the cyclic ewe.

Intact cyclic ewes have been used in experiments designed to examine the mechanism by which uterine oxytocin receptor synthesis is controlled during the oestrous cyclic. Previous experiments have shown that the prostaglandin F2 alpha analogue cloprostenol is luteolytic in ewes receiving oxytocin by continuous intra-venous infusion. When ewes receiving oxytocin are given cloprostenol uterine oxytocin receptor concentrations are raised, whereas in animals receiving oxytocin alone, they remain low. To investigate whether inhibition of oxytocin receptor binding activity by oxytocin is either dependent on elevated plasma progesterone concentrations or over-ridden by oestrogens secreted by ovarian follicles maturing as a result of cloprostenol treatment, ewes were given oxytocin by continuous intravenous infusion (3 nmol h-1) between Days 12 and 17 after oestrus and one of the following: no further treatment; cloprostenol [125 micrograms intramuscularly (i.m.)] on Day 15; progesterone, by subcutaneous implant, from Day 14 with cloprostenol on Day 15; medroxyprogesterone acetate (MPA; 6 mg depot i.m.) on Day 14 followed by cloprostenol on Day 15; or oestradiol-17 beta (2.75 mumol i.m.) on Days 14 and 15. Concentrations of oxytocin receptor were measured at autopsy on Day 17 in caruncular endometrium, intercaruncular endometrium and myometrium. Ovarian follicles and corpora lutea were examined to determine the effect of treatment on these tissues. Treatment with oxytocin alone resulted in the maintenance of corpora lutea, reduced follicular development and a low concentration of uterine oxytocin receptor. Cloprostenol initiated luteolysis in oxytocin-treated ewes. This was associated with a high level of oxytocin receptor binding activity in all ewes except those receiving exogenous progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin-induced secretion of oxytocin and prolactin in red (Cervus elaphus) and Pere David's (Elaphurus davidianus) deer hinds: evidence for oxytocin of luteal origin.

Peripheral plasma concentrations of oxytocin in female red deer during the luteal phase of the oestrous cycle (9.3 +/- 2.1 fmol/ml) exceeded those in the follicular phase (3.1 +/- 1.4) or during seasonal anoestrus (3.2 +/- 1.3). In both red and Père David's deer hinds during the mid-luteal phase of the cycle, systemic administration of a luteolytic dose of the prostaglandin F2 alpha analogue, cloprostenol, caused the concentration of oxytocin in the peripheral circulation to rise. Mean (+/- SEM) concentrations increased from 8.1 +/- 0.7 to 97 +/- 8 fmol/ml in red and from 6.2 +/- 0.7 to 153 +/- 30 fmol/ml in Père David's hinds within 5 min of treatment. During seasonal anoestrus oxytocin secretion in response to cloprostenol was reduced to less than 10% of that during the breeding season, in both species. Cloprostenol treatment raised circulating concentrations of prolactin in both species during the breeding season, and during anoestrus in red deer only. The concentration of oxytocin in a single corpus luteum removed at laparotomy from one red deer hind at the mid-luteal phase of the cycle was 66 nmol/g wet wt; identification was authenticated by HPLC. These results suggest that the corpus luteum secretes oxytocin in the Cervidae, as established previously in the Bovidae, and that luteal oxytocin secretion is stimulated by prostaglandin.

Effect of continuous infusion of oxytocin on prostaglandin F2 alpha secretion and luteolysis in the cyclic ewe.

Circulating concentrations of 13,14-dihydro-15-keto PGF2 alpha (DHKF2 alpha), 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 alpha (PGF2 alpha) secretion but had no detectable direct effect on LH or PRL. Oxytocin (3 nmol h-1 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 +/- 3.2 (mean +/- s.e.m.; cycle length of control ewes 16.7 +/- 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 alpha (measured as DHKF2 alpha) were unchanged, but there was only one episode of PGF2 alpha 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 greater than 25 days while the corpora lutea were maintained. Circulating PRL concentrations were high in ewes undergoing delayed luteolysis but there was not discrete episode of PRL secretion associated with the pre-ovulatory LH surge in these animals. To investigate the possibility that the pattern of PGF2 alpha 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 less than 10% of that seen in ewes similarly treated on Days 11-13 after oestrus. Low levels of luteal oxytocin may therefore reduce PGF2 alpha secretion in ewes undergoing delayed luteolysis.

Post-translational processing of oxytocin-neurophysin prohormone in the ovine corpus luteum: activity of peptidyl glycine alpha-amidating mono-oxygenase and concentrations of its cofactor, ascorbic acid.

Peptidyl glycine alpha-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of oxytocin synthesis, was measured in extracts of ovine corpora lutea throughout the oestrous cycle. Activity of PGA was low early in the cycle but increased between days 2 and 10 (from 2.3 to 9.0 pmol/mg protein per h) and remained high until day 15. Thereafter, activity declined rapidly at structural luteolysis and was low in corpora albicantia collected 18 and 20 days after ovulation (1.28 and 1.07 pmol/mg protein per h respectively). Luteal concentrations of ascorbic acid, a cofactor for PGA, were high (4.7 mumol/g wet wt tissue) by day 4 after oestrus; concentrations fell rapidly after day 15 (to 2.1 mumol/g on day 16). Concentrations of ascorbic acid were also high in the pituitary gland and in the adrenal medulla and cortex. Concentrations of oxytocin in luteal tissue, which were low (0.3 nmol/g wet wt) on day 2 after oestrus, were highest (2.73 nmol/g) on day 6 and declined thereafter (0.56 nmol/g on day 10, 0.08 nmol/g on day 15 and not detectable on days 18 and 20). Concentrations of oxytocin, progesterone, PGA and protein were measured in subcellular fractions obtained after density gradient centrifugation of extracts of corpora lutea collected on days 6, 7 and 12 of the oestrous cycle, and on day 7 from an anaesthetized ewe before and after treatment with the prostaglandin F2 alpha analogue, cloprostenol. PGA colocalized with particle-associated oxytocin in fractions of density 1.049-1.054 g/ml. Exogenous [3H]oxytocin and [3H]progesterone and endogenous progesterone localized in fractions of density 1.035 g/ml. Oxytocin and PGA were depleted from fractions of density 1.049-1.054 g/ml following cloprostenol treatment in vivo. Fractionation of extracts of ovine corpora lutea by high-performance liquid chromatography (HPLC) followed by radioimmunoassay and radioreceptor assay for oxytocin demonstrated the presence of at least two cross-reacting substances with elution characteristics distinct from oxytocin. Concentrations of these peptides increased as the cycle progressed. These compounds differed from the putative C-terminally extended post-translational processing intermediates, oxytocinyl-glycine, oxytocinyl-glycine-lysine and oxytocinyl-glycine-lysine-arginine, as indicated by their elution positions on HPLC and the specificities of the assays used to detect them, and no conclusions could be drawn on which post-translational processing step was rate-limiting in oxytocin synthesis. These data are consistent with the suggestion that post-translational processing of oxytocin-neurophysin prohormone takes place in secretory granules in luteal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Mesotocin and luteal function in macropodid marsupials.

Pituitary glands and corpora lutea collected at various stages of the reproductive cycle of the tammar wallaby (Macropus eugenii), were extracted and fractionated by high-performance liquid chromatography, and specific radioimmunoassays were used to measure mesotocin ([Ile8]-oxytocin) and oxytocin. Mesotocin, but not oxytocin, was identified in extracts of pituitary; the mean concentration of mesotocin in this tissue was 0.75 nmol/g wet weight. Neither mesotocin nor oxytocin was detected in extracts of corpus luteum. In female Bennett's wallabies passively immunized against mesotocin during seasonal reproductive quiescence, there was no significant effect on peripheral progesterone concentrations and there were no births, matings or changes in vaginal smears in the 2 months following treatment. Thus mesotocin is unlikely to act as a systemic luteostatic agent during seasonal quiescence.