ABSTRACT

Angiogenesis is the process of generating new capillaries and leads, therefore, to vascularization of tissues. This process occurs during embryological development and during pathological and physiological conditions in adult life, including those involving the reproductive organs. Recent studies, in the field of tumour biology in particular, have led to the identification of several factors responsible for inducing angiogenesis and the elucidation of ways of modulating their activity. This review summarizes our knowledge of angiogenesis in the ovary, testis, endometrium and placenta, and suggests ways in which further research might contribute to a better understanding of the processes controlling reproduction and identify new approaches to the regulation of fertility.

J. Endocr. (1986) 111, 357–366

The binding of three catechol oestrogens, 2-OH-oestradiol-17β, 4-OH-oestrone and 2-OH-oestrone, to the ovine pituitary oestrogen receptor was measured in vitro to establish doses for the assessment of the effects of catechol oestrogens in vivo. Relative to oestradiol (100%) the compounds had receptor affinities of 30, 20 and 5% respectively. A dose of oestradiol sufficient to cause negative-feedback effects on the secretion of LH and FSH in ovariectomized ewes was established by intracarotid (i.c.) injections of 0·625–5·0 μg/dose (n = 3), and by measuring plasma levels of gonadotrophins in jugular venous samples taken at intervals of 20 min from 3 h before until 4 h after injection. A dose-dependent relationship (r = 0·88, P<0·001) was found for oestradiol and plasma LH levels. Plasma FSH was slightly (12–25%) but significantly (P<0·05) reduced by doses of 1·25–5·0 μg oestradiol, but no dose–response relationship was observed.

Ovariectomized ewes (n = 4/group) were given 2·5 μg oestradiol (i.c.) simultaneously with 83 μg 2-OH-oestradiol, 125 μg 4-OH-oestrone or 500 μg 2-OH-oestrone. These doses of catechol oestrogens were chosen as being ten times that of oestradiol, with the relative affinities for oestrogen receptor taken into account. Concurrent administration of such doses of catechol oestrogens had no effect on the negative-feedback action of oestradiol in vivo. We have concluded that catechol oestrogens in the circulation probably do not modulate the action of oestradiol on release of LH or FSH; this does not preclude a possible role for them as locally produced regulators of oestrogen action.

ABSTRACT

The effects of recombinant rat interferon-γ (rRaIFN-γ) and rat IFN (RaIFN, a mixture of IFN-γ and -α) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 × 10⁵ viable cells/well per 0·5 ml) in serumfree medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-γ (10–1000 U/ml) significantly inhibited FSH-stimulated aromatase activity (76·4 ± 2·3% maximum inhibition compared with FSH treatment alone), inhibin (40·4 ± 3·7%), progesterone (47·7 ± 8·6%) and 20α-hydroxypregn-4-en-3-one (20α-OHP) (51·8±1·7%) production in a dose-dependent manner. Furthermore, rRaIFN-γ inhibited FSH- and forskolin (FSK; 30 μmol/l)-induced extracellular cAMP accumulation (46·0 ± 6·6% and 29·1 ± 7·3% respectively). The inhibitory effect of rRaIFN-γ on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20α-OHP production. rRaIFN-γ had no detectable effect on aromatase activity, progesterone production and 20α-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1·5-fold. rRaIFN-γ alone also caused a small but significant increase in basal levels of cAMP. The timecourse studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-γ, FSH-induced progesterone and 20α-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone. There was no difference in DNA content between treatment and non-treatment wells during 5 days of culture. RaIFN had similar effects to rRaIFN-γ. We conclude that IFN-γ can inhibit FSH-induced granulosa cell differentiation and that, in the absence of FSH, IFN-γ stimulated undifferentiated granulosa cells to produce more inhibin. The mechanism of its action is likely to involve changes in cAMP production.

Journal of Endocrinology (1992) 133, 131–139

Oestradiol-17α is the only oestrogen which has been identified in sheep foetal tissues (Velle, 1963). Oestrogens have been detected in blood (Skrzeczkowski, Dobrowolski & Domanski, 1967) and urine (Fevre & Rombauts, 1966) of the pregnant ewe. The oestrogen values reported have been very low relative to those in man. In contrast, this paper reports very considerable oestrogen concentrations in the plasma of the sheep foetus.

Blood was drawn by heart puncture from foetuses within about 15 min. of slaughtering ewes at the city abbatoirs. The blood was collected into ice-cold heparinized bottles, centrifuged, and the plasma stored at −15° until analysed. Gestational ages were estimated from crown-rump lengths.

To determine the oestrogen conjugates present, plasma was extracted with ether ('unconjugated fraction'), then incubated with molluscan β-glucuronidase (Koch-Light) in the presence of sodium dihydrogen phosphate to inhibit sulphatases (Dodgson & Spencer, 1953), and re-extracted with ether to give the 'glucosiduronate

The effect of 2-bromo-α-ergocryptine (bromocriptine) on the induction, initiation and maintenance of milk secretion was studied in post-parturient ewes and in ovariectomized ewes artificially induced to lactate by treatment with oestrogen plus progesterone and then dexamethasone. Treatment with bromocriptine (about 0·4 mg/kg every 3 days) lowered and maintained the plasma concentration of prolactin at < 12 ng/ml. Ewes receiving bromocriptine concurrently with oestrogen plus progesterone during the priming phase had a significantly lower (P< 0·05) mean cumulative milk yield than control ewes, although the milk of the treated ewes contained normal amounts of fat, protein and lactose. Administration of bromocriptine during dexamethasone-induced lactogenesis had no significant effect on the mean cumulative milk yield but significantly (P<0·05) increased the milk fat and protein content. In established lactation, bromocriptine markedly reduced the milk yield in both intact and ovariectomized ewes. The concentration of protein was not significantly affected although the milk fat content was higher in the bromocriptine-treated than in the control ewes. The effects of bromocriptine on milk yield and composition during galactopoiesis could be reversed by concurrent infusion of prolactin and the results suggest that prolactin is an important hormone during mammogenesis and galactopoiesis in the sheep.

The ability of oestradiol-17β (OE₂) and 2-hydroxyoestradiol (2OH-OE₂) to translocate pituitary oestrogen receptors to the nuclear compartment and to affect plasma concentrations of LH, FSH and prolactin was studied in ovariectomized ewes. Mean (± s.e.m.) nuclear oestrogen receptor levels (% of total pituitary oestrogen receptors) after intracarotid injections of 1·25 μg OE2, 400 μg 2OH-OE2 or vehicle were 8·7±2·3, 16·9 ± 3·2 and 0·8±0·1% respectively. Whereas 1·25 or 12·5 μg OE₂ significantly lowered plasma LH and FSH, 400 μg 2OH-OE₂ did not affect plasma LH or FSH levels. Injection of 4000 μg 2OH-OE₂, however, significantly affected plasma LH and FSH. Plasma prolactin levels were not significantly affected by the treatments. These data indicate a discrepancy between oestrogen receptor occupancy and effects on gonadotrophin and prolactin secretion after injection of catechol oestrogen.

ABSTRACT

Epidermal growth factor (EGF) is a potential intra-ovarian modulator of gonadotroph action on differentiated follicular cells. Specific binding sites have been identified in the ovary and functional differentiation in cultured granulosa cells can be modulated by treatment with EGF. The aim of this study was to determine if EGF was capable of altering ovarian function in vivo during the follicular phase of the sheep oestrous cycle. Fourteen cross-bred ewes with ovarian autotransplants were treated with progestagen pessaries for 12 days. Three ewes were infused with murine EGF (mEGF) via the jugular vein (75 μg/kg bodyweight per 12 h) during the 12 h preceding progestagen pessary withdrawal, and received an injection of a prostaglandin analogue at 0 h to induce luteolysis. Over the same time-period, two doses of EGF were administered to other groups of ewes by infusion into the ovarian artery (low: 6 μg/12 h, n = 3 and high: 60 μg/12 h, n = 3). The remaining five ewes were not infused with EGF (controls). Jugular and ovarian venous blood samples were taken at 10-min intervals at two stages during the follicular phase (21–27 h and 38–42 h after pessary withdrawal) and every 2 h from 44 to 76 or 86 h. mEGF, LH, FSH, inhibin, androstenedione, oestradiol-17β and progesterone concentrations in plasma were determined using radioimmunoassays. The secretion rates of androstenedione, oestradiol, progesterone and inhibin by the ovary were calculated.

EGF acted directly on the ovary in a dose-dependent manner. Oestradiol secretion was inhibited following treatment with EGF but androstenedione secretion was unaffected. EGF appears therefore to act within the granulosa cells to inhibit aromatization. Inhibin secretion was also suppressed by treatment with EGF, though it was not possible to determine if this was caused by a direct or indirect action of EGF on granulosa cells. The rate of progesterone secretion increased in ewes receiving systemic (i.e. via the jugular vein) and high-dose intra-arterial infusions of EGF, even though a preovulatory LH surge was not observed in these animals during the entire experimental period. Concomitant increases in both LH and FSH secretion were associated with these effects of EGF on ovarian function.

In conclusion, EGF appears to act directly on the granulosa cells of the follicle to inhibit aromatization and also to inhibit inhibin production. The low levels of oestradiol and inhibin in the presence of high levels of gonadotrophin indicate that atresia may have been induced in medium to large antral follicles. The increase in progesterone secretion following high doses of EGF may be derived from a luteinized follicle. FSH-stimulated functions cease when a follicle luteinizes and progesterone secretion commences. EGF treatment inhibited both oestradiol and inhibin secretion whilst enhancing progesterone which suggests that EGF may also be involved in the induction of functional luteinization. EGF or an EGF-like substance may therefore be an important factor in the induction of functional luteinization, with atresia occurring in antral follicles which are exposed to EGF too early in their development.

Journal of Endocrinology (1993) 137, 253–264

ABSTRACT

Acid extracts of bovine preovulatory granulosa cells and corpora lutea (CL) were subjected to high-performance liquid chromatography (HPLC) and found to contain two peaks of immunoreactive (ir) oxytocin (OT), one corresponding to authentic OT and the second eluting 8 min later. The second peak was more abundant than authentic irOT in preovulatory follicles and in the early CL, but became less abundant as the CL matured (mid luteal) and was close to the limit of detection in the late CL. This peak could be detected only by an OT antiserum which recognized both the biologically active form of OT, as well as the post-translational processing intermediate Gly¹⁰-extended oxytocin. A second more specific OT antiserum (OT-933) did not recognize the second peak as strongly. Further analysis of the second peak revealed a complex of OT bound to its neurophysin (NP-I) which could be dissociated under denaturing conditions. Furthermore, we were able to create this complex in vitro by combining the two materials together under acid conditions, similar to the pH predicted in secretory granules, but not under neutral conditions. Measuring irNP-I by radioimmunoassay showed a single peak with a similar retention time to the OT/NP-I complex, confirming the identity of the unknown peak. Incubation of CL slices in culture showed a time-related release of both OT and NP-I, with OT having a greater rate of release in the mid luteal CL.

These data suggest the presence of an OT/NP-I complex in the bovine preovulatory granulosa cells and CL, as well as the unbound peptide presumably within the secretory granules. The ratio of OT/NP-I complex and free peptide changes with ageing of the the CL, perhaps indicating regulated differences in the post-translational processing of the prohormone.

Journal of Endocrinology (1991) 128, 305–314

Growth hormone was measured by radioimmunoassay in blood samples collected from ewes during the hormonal induction of lactation and during hand-milking post partum. Ovariectomized ewes were induced to lactate with injections of progesterone + oestradiol benzoate every 3 days for 30 days (priming phase) and then daily injections of dexamethasone for 5 days (trigger phase). The ewes were then milked daily. Immunoreactive GH levels fluctuated considerably but were generally in the range 1–15 ng/ml during all phases of lactation and were unaffected by bromocriptine treatment. Milk yield was unrelated to GH levels.

Growth hormone was also measured in blood plasma taken at frequent intervals around the time of milking in lactating ewes approximately 4 weeks post partum. Although a clear prolactin response to milking was observed, there was no indication of a GH response.

Although there is probably a minimum requirement for GH for lactation, a relationship between immunoreactive GH levels and milk yields was not established, perhaps because of limitations of the radioimmunoassay to detect all the biologically active GH.

Abstract

Previous studies have demonstrated that endothelin is present in the ovine endometrium and increases at around the expected time of implantation. To characterize further uterine endothelin at the time of establishment of pregnancy in sheep, endothelin was measured by radioimmunoassay in uterine flushings obtained during the oestrous cycle and in pregnant ewes up to the time of implantation (day 16). During the oestrous cycle, the highest amounts of endothelin were present in uterine flushings on day 14 (1·1 ±0·2 ng endothelin/uterus). During early pregnancy, basal levels of endothelin (0·5–0·6 ng endothelin/uterus) were present in uterine flushings for the first 10 days and then increased on day 14 to levels similar to those found at the equivalent stage of the oestrous cycle. On days 15 and 16 of pregnancy, endothelin content in the uterine lumen increased to significantly (P<0·05) higher concentrations (2·9±0·4 ng endothelin/uterus) when compared with the non-fertile cycle. The principal isoform present in flushings at the time of implantation was endothelin-1, as determined by reverse-phase HPLC. Endothelin was released principally by purified endometrial epithelial cells in culture, with barely detectable amounts released by endometrial stromal cells or conceptus tissue, which is consistent with the epithelium being the principal source of endothelin in the uterine lumen. Endothelin binding sites were present in endometrium and myometrium, as demonstrated by specific binding of ¹²⁵I-labelled endothelin-1, which was saturable and displaced by endothelin-1. Both endothelin_A and _B sub-types of receptors were present as demonstrated by the biphasic displacement of ¹²⁵I-labelled endothelin-1 binding by the specific endothelinB agonist BQ3020. These were localised principally on luminal and glandular epithelium and in the vasculature of the endometrium and myometrium as shown by autoradiography. Endothelin receptors were also present on the conceptus obtained at the time of implantation. In the day 20 conceptus, endothelin immunostaining was localised principally in the heart, in trophoblast in uninucleate but not in binucleate cells, and in fetal membranes. This immunostaining of the conceptus may represent binding to receptor sites. It is concluded that endothelin-1 is present in the uterine lumen and may play an important role in the paracrine regulation of the conceptus and endometrium at the time of rapid embryo development, implantation and early placentation.

Journal of Endocrinology (1995) 147, 235–244