Embryonic mortality in early pregnancy is a frequent occurrence in most mammalian species. It continues to have a major impact on the livestock industry: in cattle, for example, about 35% of all conceptuses die before the third month of gestation. In the human, there is evidence that a high proportion of pregnancy losses are associated with chromosomal anomalies (Simpson, 1980; Angell, Hillier, West et al. 1988). In ruminants, however, chromosomal analysis of early embryos (Berepubo & Long, 1983) coupled with the results of numerous embryo transfer experiments (e.g. Markette, Seidel & Elsden, 1985) indicate that the maternal environment in which the embryo develops is of greater importance.

Many studies have been aimed at understanding what aspects of this 'maternal environment' are crucial, and how these are regulated. This commentary will summarize the information which has been gained historically and will evaluate the additional inputs which are now coming from the

Abstract

Ovarian localization of the IGFs and their binding proteins is species-specific. In the ewe we have demonstrated previously that IGF-II mRNA is found at high concentrations in the theca, whereas IGF-I mRNA is undetectable in ovine follicles at any stage of development, although both are present in the corpus luteum. In this study we have investigated the localization of mRNA for IGF-binding proteins (IGFBPs)-2, -3 and -4 using in situ hybridization to further elucidate the roles of the IGFs and their possible modulation during follicular development. Results were quantified by optical density (OD) measurements of autoradiographs using image analysis. There was intense follicular expression of mRNAs for both IGFBP-2 and -4 although IGFBP-3 mRNA was undetectable. Concentrations of IGFBP-2 mRNA varied significantly (P<0·01) with respect to size, being highest in small (<2 mm) follicles but with no variation attributable to health, whereas expression of IGFBP-4 mRNA was significantly (P<0·01) influenced by health and not size, being higher in healthy than atretic follicles. IGFBP-2, -3 and -4 mRNAs were all present at low concentrations in the corpus luteum (OD 0·04 ± 0·005, n=6; 0·05 ± 0·02, n=5; 0·1 ± 0·02, n=8 respectively), whereas only IGFBP-2 and -4 mRNAs were found in ovarian stroma (OD 0·04 ± 0·006, n=4; 0·04 ± 0·007, n=9 respectively). These data suggest that changes in follicular distribution of IGFBPs-2 and -4 may modulate the actions of the IGFs during follicular growth thus contributing to the process of follicle selection.

Journal of Endocrinology (1996) 151, 241–249

ABSTRACT

Uterine tissue samples were collected from 47 ewes at various stages of the oestrous cycle and early pregnancy (until day 21) and during seasonal anoestrus. Cryostat sections were immunostained to determine the localization of oestradiol and progesterone receptors using specific monoclonal antibodies. Oxytocin receptors were localized by autoradiography in sections from the same ewes using the ¹²⁵I-labelled oxytocin antagonist d(CH₂)₅[Tyr(Me)²,Thr⁴,Tyr-NH₂ ⁹]- vasotocin. Plasma progesterone measurements were made during the preceding cycle up to the time of slaughter.

Oestradiol receptor concentrations were maximal in all regions of the tract at oestrus. Immunostaining of the luminal epithelium, superficial glandular epithelium, stroma and myometrium decreased in the early luteal phase but was maintained for longer in the deep glands. Progesterone receptor immunostaining in the luminal epithelium and superficial glands developed in the early luteal phase (days 1–2) with a somewhat later appearance in the deep glands (days 5–7). Progesterone receptor concentrations in the stroma and myometrium also reached a maximum in the early luteal phase. Myometrial staining was clearly maintained throughout the luteal phase whereas stromal staining was variable between ewes. For both oestradiol and progesterone receptors no differences were apparent between pregnant and non-pregnant ewes between days 2 and 12, but pregnant ewes did not show the general increases in oestradiol receptor staining associated with luteolysis on days 14–15.

Oxytocin receptors first developed in the luminal epithelium of non-pregnant ewes on day 14 of the cycle and spread to the superficial glands, caruncular stroma, deep glands and myometrium at oestrus before decreasing in reverse order on days 1–2. Specific binding was not detectable on days 5–12 of the cycle or on days 14 or 21 of pregnancy. The appearance of oxytocin receptors in the luminal epithelium on day 14 preceded that of both the oestradiol and progesterone receptors in the epithelial cells and the fall in plasma progesterone. It was followed by the development of oestradiol and oxytocin receptors in the superficial glands, deep glands, caruncular stroma and myometrium, with the two receptor populations showing a significant positive association in these tissues. The loss of oxytocin receptors in all regions occurred as plasma progesterone levels were increasing, but the association between these two variables was only significant in the superficial glands. The development of progesterone receptors in different tissues could not be explained on the basis of either oestradiol receptor content or plasma progesterone. We conclude that all three receptor populations change in a dynamic manner during the oestrous cycle with variations both between days and between different uterine compartments. The complex pattern of receptor formation and loss suggests that, in addition to the circulating steroid hormone concentrations, local paracrine factors are likely to be involved in their regulation.

Journal of Endocrinology (1993) 138, 479–491

ABSTRACT

Oxytocin-binding sites in the endometrium and myometrium of the non-pregnant ewe were characterized. [³H]Oxytocin bound to a single site in both tissues with high affinity; dissociation constants were determined to be 1·96 nmol/l in endometrium and 2·12 nmol/l in myometrium. Oxytocin binding was enhanced by divalent cations with a similar order of potency in both tissues: Co²⁺> Mn²⁺> Ni²⁺> Mg²⁺ > Zn²⁺> Ca²⁺. The endometrial and myometrial binding sites showed the same specificity for oxytocin analogues and related peptides, having high affinity for oxytocin, [Arg⁸]-vasopressin, [Lys⁸]-vasopressin, and the oxytocin-specific agonists [Gly⁷]-oxytocin and [Thr⁴,Gly⁷]-oxytocin. The results suggest that oxytocin receptors present in the endometrium and myometrium of the ewe are similar both to each other and to classical oxytocin receptors.

Journal of Endocrinology (1989) 123, 11–18

ABSTRACT

The morphological and functional characteristics of cultured bovine luteal cells were examined. Dispersed luteal cells were cultured in either Ham's F12 medium or Dulbecco's Modified Eagle's Medium (DMEM), plus 10% adult bovine serum. After 18 h of culture, fibronectin was observed to be associated with some cells showing histochemical 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. In DMEM, cells adopted a fibroblast-like appearance with a doubling time of 34 h while in Ham's F12, cells retained an epithelial-like morphology with a doubling time of 91 h. Dibutyryl cyclic AMP (dbcAMP) but not LH slowed proliferation and altered cell morphology in DMEM. Addition of insulin and dbcAMP was required to maintain 3β-HSD activity in cells cultured for more than 3 days.

Progesterone secretion was significantly greater in Ham's F12 than in DMEM although there was a rapid decline in basal secretion during culture in either medium. Both LH and dbcAMP stimulated progesterone secretion although the effects of dbcAMP were significantly greater than those of LH after 18 h of culture. Addition of insulin significantly enhanced basal, LH-stimulated and dbcAMP-stimulated progesterone secretion after 3 days of culture whereas several antioxidants were without effect. These studies indicate that the morphological and functional characteristics of cultured luteal cells may be best maintained in Ham's F12 plus dbcAMP and insulin.

J. Endocr. (1985) 104, 355–361

ABSTRACT

Blood samples were collected twice daily (09.30 and 17.00 h) via indwelling jugular-vein cannulae from five ewes throughout an entire oestrous cycle. Additional frequent samples were taken at 30-min intervals for 8 h on each of days 3 (early luteal phase), 9 (mid-luteal phase), 12 (late luteal phase) and 0 (day of oestrus). Plasma concentrations of arginine vasopressin and oxytocin were measured in all samples by radioimmunoassay and progesterone was measured in the twice-daily samples only.

Both oxytocin and progesterone showed the expected pattern of plasma concentrations, increasing during the early luteal phase, reaching a plateau and declining either preceding (oxytocin) or at (progesterone) luteolysis. Vasopressin concentrations showed a significant dependence on the day of cycle (P<0·05, analysis of variance) with concentrations lowest at oestrus and minor peaks on days 4 and 8–9. There was no correlation between the concentrations of vasopressin and progesterone. Vasopressin values were significantly higher in the morning than in the afternoon samples (1·3 cf. 0·9 pmol/l; P<0·001). Analysis of the frequent samples showed a significant (P<0·001) dependence on the time of day for vasopressin but not oxytocin. Values were high throughout the morning, declined to a trough at 15.00 h and rose again by 17.00 h.

We conclude that there is a minor variation in the vasopressin concentration during the oestrous cycle, which is not related to the circulating progesterone concentration but could be regulated by oestradiol. We also provide evidence for a diurnal rhythm in the release of vasopressin into the plasma in the ewe. The concentrations of vasopressin measured are unlikely to be sufficient to influence the activity of the reproductive tract in vivo.

Journal of Endocrinology (1992) 134, 107–113

ABSTRACT

Bovine corpora lutea and ovarian stroma were analysed by high-performance liquid chromatography for catecholamine content. High concentrations (up to 102 nmol/g wet weight) were found in both 'central' stroma, containing many blood vessels, and 'peripheral' stroma. Central stroma contained noradrenaline and some dopamine, whereas peripheral stroma contained a higher proportion of dopamine and also significant amounts of 3,4-dihydroxyphenylacetic acid (DOPAC). Occasional samples of stroma had very high amounts of dopamine, suggesting that it is stored in specific regions. Corpora lutea, although devoid of direct innervation, contained dopamine (up to 5·3 nmol/g) and noradrenaline (up to 1·2 nmol/g). The average dopamine: noradrenaline molar ratio was 1·19 : 1 and the concentrations of dopamine and noradrenaline were highly correlated (P < 0·002). The concentration of dopamine was significantly higher in the early luteal phase of the oestrous cycle than during the rest of the cycle or in pregnancy. The levels of noradrenaline and dopamine present in corpora lutea are sufficient to modulate the production of both oxytocin and progesterone by luteal cells in vitro.

Journal of Endocrinology (1991) 129, 221–226

ABSTRACT

Some of the binding characteristics of a novel oxytocin receptor ligand ¹²⁵I-labelled [1-(β-mercapto-β, β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr²,Thr⁴,Orn⁸,Tyr⁹-NH₂]-vasotocin ([¹²⁵I]OTA) have been determined in the sheep uterus. The compound was subsequently used for the autoradiographic localization of oxytocin receptors in the uterus and oviduct of the ewe.

Specific binding of [¹²⁵I]OTA to crude membrane fractions of ovine endometrium was time-dependent and was unaffected by the addition of cations to incubation media. Endometrial membranes contained a single population of saturable, high-affinity binding sites for the iodinated ligand (dissociation constant (K _d) 0·23±0·08 nmol/l) and unlabelled oxytocin competed with [¹²⁵I]OTA for binding sites with high affinity (K _d 1·29±0·4 nmol/l) in the presence of Mg²⁺ In contrast, unlabelled OTA was able to compete with high affinity (K _d 1·13±0·16 nmol/l) in the absence of cation. Competition studies with a number of oxytocin analogues and related peptides and the tissue distribution of [¹²⁵I]OTA binding sites also indicated that [¹²⁵I]OTA bound to the ovine oxytocin receptor. This was further validated by autoradiographic studies which showed specific labelling with [¹²⁵I]OTA to be greater to uterus and oviduct obtained from ewes which had been killed within 2 days of oestrus than to similar tissue from ewes killed during the luteal phase. In both the ampullary and isthmic regions of the oviduct and the myometrium, [¹²⁵I]OTA binding sites were confined to smooth muscle. Endometrial binding sites for [¹²⁵I]OTA were consistently located on the luminal epithelium and epithelial cells lining secretory glands. In addition, in one ewe which had been killed 2 days after cloprostenol treatment, stromal cells were labelled in a caruncular region of the endometrium. The consistency of this observation between similar animals remains to be determined.

The autoradiographic technique demonstrated appears sufficiently sensitive to allow further studies into the distribution of the endometrial oxytocin receptor throughout the oestrous cycle, and into its regulation at luteolysis and during the establishment of pregnancy.

Journal of Endocrinology (1991) 128, 187–195

ABSTRACT

The presence of oxytocin receptors in ovine oviduct has been investigated. High-affinity binding sites for [³H]oxytocin were detected in crude membrane fractions prepared from the oviducts of ewes killed during the oestrous period. The dissociation constant calculated for these sites in competition studies was 1·7 nmol/l. Similar dissociation constants were calculated for [Arg⁸]-vasopressin and the oxytocin-specific agonists [Gly⁷]-oxytocin and [Thr⁴, Gly⁷]-oxytocin, indicating that these sites represent oxytocin receptors. At least one additional site of lower affinity and undetermined identity was present.

The relative concentration of oxytocin-binding sites in preparations of oviduct membranes were estimated in ewes killed at different stages of the oestrous cycle using a single concentration of [³H]oxytocin. Binding was low during the luteal phase of the cycle but increased to a maximum at oestrus (77·7 fmol/mg protein). Binding fell after ovulation, reaching what appeared to be basal concentrations by the early luteal stage of the cycle. Binding to oviductal membranes from prepubertal, anoestrous and pregnant ewes was also low, but in anoestrous animals which had been treated with progesterone and oestrogen it was similar to values measured in ewes at oestrus. These results are consistent with the existence of oviductal oxytocin receptors which are regulated by ovarian steroids.

We conclude that oxytocin receptors are present in the oviduct of the ewe around the time of ovulation. The significance of oxytocin to events taking place in the oviduct at this time remains to be determined.

Journal of Endocrinology (1990) 124, 353–359

ABSTRACT

The present study was designed to determine the localization of the endometrial oxytocin receptor during the ovine oestrous cycle, particularly on day 14, the time of initiation of luteal regression in the ewe. Samples were obtained from 29 ewes at different stages of the oestrous cycle (several during the luteal phase and on every day between day 14 (− 2) and day + 3 of the oestrous period).

Oxytocin receptors were localized autoradiographically in sections of uterine tissue, using the ¹²⁵I-labelled oxytocin receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr²,Thr⁴,Orn⁸,Tyr⁹-NH₂]-vasotocin (¹²⁵I-labelled OTA). There was some variation in the pattern of ¹²⁵I-labelled OTA labelling between different uterine tissue samples from the same ewe and also between samples obtained from different ewes thought to be at the same stage of the oestrous cycle. A clear overall pattern did, however, emerge with ¹²⁵I-labelled OTA-binding sites distributed between luminal epithelial cells, glandular epithelial cells and caruncular stromal cells to varying extents on different days of the cycle.

During the luteal phase (days 5–12) clear specific labelling of endometrial tissue was generally absent. On day 14 labelling was evident on the luminal epithelium, but only in nine tissue samples out of a total of 18 studied, indicating that the entire luminal surface did not contain oxytocin receptors at this time. Between the day before oestrus and day 3 of the oestrous cycle the luminal epithelium was consistently labelled. The most extensive labelling of the remaining endometrial tissue was observed on the day of oestrus, with ¹²⁵I-labelled OTA-binding sites clearly present on the stromal cells within caruncles and on a large proportion of secretory epithelia. This contrasted with the day before and the day after oestrus when labelling of glandular tissue was confined to the superficial endometrium, and labelling of caruncular stromal cells, although sometimes evident, was never as intense as on day 0. On days 2 and 3 labelling varied between being similar to that found on day 1 and being confined to the luminal epithelium and very few superficial secretory glands.

The results of this study lead us to conclude that the oxytocin receptor shows a differential distribution between stromal cells, epithelial cells lining secretory glands and luminal epithelial cells during the oestrous cycle; that the steroidal regulation of the oxytocin receptor differs between endometrial cell types; and that control of the luminal epithelial oxytocin receptors is probably of particular importance to the regulation of prostaglandin F_2α release at luteal regression and during the maternal recognition of pregnancy.

Journal of Endocrinology (1991) 130, 199–206