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Fetal growth restriction (FGR) and preterm birth are frequent co-morbidities, both are independent risks for brain injury. However, few studies have examined the mechanisms by which preterm FGR increases the risk of adverse neurological outcomes. We aimed to determine the effects of prematurity and mechanical ventilation (VENT) on the brain of FGR and appropriately grown (AG, control) lambs. We hypothesized that FGR preterm lambs are more vulnerable to ventilation-induced acute brain injury. FGR was surgically induced in fetal sheep (0.7 gestation) by ligation of a single umbilical artery. After 4 weeks, preterm lambs were euthanized at delivery or delivered and ventilated for 2 h before euthanasia. Brains and cerebrospinal fluid (CSF) were collected for analysis of molecular and structural indices of early brain injury. FGRVENTlambs had increased oxidative cell damage and brain injury marker S100B levels compared with all other groups. Mechanical ventilation increased inflammatory marker IL-8 within the brain of FGRVENTand AGVENTlambs. Abnormalities in the neurovascular unit and increased blood–brain barrier permeability were observed in FGRVENTlambs, as well as an altered density of vascular tight junctions markers. FGR and AG preterm lambs have different responses to acute injurious mechanical ventilation, changes which appear to have been developmentally programmed in utero.

The volcanic island of Savo, Solomon Islands, hosts an active hydrothermal system discharging unusual alkaline (pH 7-8) sulphate-rich, chloride-poor fluid, with variable admixtures of Ca-Mg-HCO3--rich fluid. Hot springs and their outflow streams precipitate a variety of deposits, including travertine, silica sinter and unusual mixed silica-carbonate rocks. Travertine fabrics are dominated by ray-crystal calcite, associated with rapid abiotic precipitation from a supersaturated solution. Sinter is produced by evaporation of thermal waters, and downstream samples contain preserved traces of micro-organisms, which potentially acted as templates for precipitation. Trace element chemistry of sinters and travertines includes anomalously high levels of Te, indicating a magmatic origin for a component fluid in the hydrothermal system. Springs are close to or at saturation with both calcite and amorphous silica. Increased contributions from the Ca-Mg-HCO3- end-member favours calcite formation; this fluid is of low-temperature origin, and as such is favoured by high rainfall. Mixed samples show cyclical changes between silica and carbonate precipitation, potentially as a result of seasonal variation in rainfall. [ABSTRACT FROM AUTHOR]

The release of activin A in response to intravenous injection of the bacterial cell-wall component lipopolysaccharide (LPS) was investigated in an ovine model of acute inflammatory challenge in newborn and adult sheep, and in non-pregnant and pregnant ewes. Neonatal lambs (<20 days of age) showed a quantitatively similar response in terms of circulating concentrations of activin A, its binding protein follistatin and the cytokine interleukin-6 compared with adult ewes challenged with an equivalent dose (300 ng/kg bodyweight) of LPS. The fever response and plasma tumour necrosis factor-α release in response to LPS, however, were significantly (P< 0.01) less in lambs than in the adult group. Pregnant ewes in the last trimester of gestation had similar responses to LPS, in all aspects measured, compared with their non-pregnant counterparts, apart from an ablated fever response. Although the adult and neonatal sheep responded to LPS, a similar response was not apparent in the fetal circulation, possibly due to a protective effect of the placenta. A 10-fold increase in the dose of LPS (from 300 ng to 3 μg/kg bodyweight) given to neonatal lambs elicited an increase in several cytokine responses measured, with a significant (P< 0.05) increase in follistatin release. In contrast, the amount of activin released by the increased dose of LPS was similar to that invoked by the lower dose. The effect of tolerance to LPS was investigated by giving a second challenge of LPS 5 days after the initial injection. In all animals studied, there was an ablated (P< 0.05) response to the subsequent LPS injection, apart from a similar temperature-response profile. These data provide further evidence that activin A concentrations in the bloodstream are acutely responsive to inflammatory challenge in post-natal life and suggest that the response forms a significant component of the innate immune system.

Developmentally competent oocytes can be collected from xenografted ovarian tissues; however, optimal xenograft conditions need to be established for this technique to be of use in assisted reproduction. In the present study, common wombat ovarian tissue was xenografted under the kidney capsule of nude mice to clarify the role of recipient gonadal status and donor tissue age on graft establishment, follicle development and oocyte recovery. Eighty-nine per cent of all grafts were recovered; of these, 78% contained growing follicles. In female graft recipients, follicle development to the antral stage occurred earlier in ovariectomised recipients compared with intact graft recipients. Similarly, follicle development occurred earlier in recipients of pouch young ovarian tissue grafts when compared with subadult xenografts. Follicle development proceeded to the antral stage in subadult grafts placed under the kidney capsule of male recipient mice, albeit at a slower rate than subadult grafts placed in female recipients. Oocytes were collected from grafts placed in female and male recipients, but no mature oocytes were observed at the time of collection, nor could these oocytes be matured in vitro. The present study demonstrated that common wombat pouch young tissue xenografted to female recipient mice, and subadult ovarian tissue xenografted to male recipient mice, can develop to the antral stage and can therefore facilitate oocyte collection. However, mature oocytes were not obtained using the current protocol.

Cryopreservation procedures generally depend on both the cryoprotectant used and the equilibration conditions to which the material is exposed. The aim of the present study was to examine the effect of cryoprotectants (dimethyl sulphoxide (DMSO) and ethylene glycol (EG)) and equilibration conditions (0, 30 or 120 min at 0°C or 120 min at room temperature) on the fertility of mice receiving cryopreserved mouse ovaries. The study compared the fertility of cryopreserved Day 14 mouse pup ovaries following grafting to adult recipient mice for 4 months. There was no effect of the cryoprotectant or equilibration condition used on the interval to the first plugging/mating or on the interval to the birth of the first litter, the size of litters, the number of litters produced or the total number of offspring produced. Despite this, when compared with control females (untreated, sham and fresh transplant) the cryopreservation and transplantation procedures delayed fertility. However, the size of litters was equivalent for all cryopreserved and control groups (P > 0.05). The results show that, for the equilibration conditions examined, DMSO and EG are equally efficient cryoprotective agents for mouse ovarian tissue.

The aim of the present study was to determine whether activin A concentrations are dependent on feto?placental oxygen availability and to investigate the temporal relationship of activin A with prostaglandin (PG) E2 and cortisol. Nine fetal sheep (six hypoxic and three control) were instrumented and catheterised at 0.8 gestation. Reduced uterine blood flow was used to achieve three levels of hypoxia (mild = fetal SaO2 40?50%; moderate = fetal SaO2 30?40%; severe = fetal SaO2 20?30%), for 4 h on 3 consecutive days. Activin A, PGE2 and cortisol levels were determined in maternal and fetal blood and amniotic fluid. Moderate and severe hypoxia produced a significant (P < 0.05) increase in fetal plasma activin A concentrations. The amniotic fluid activin A concentrations were 15-fold higher than those in the fetal circulation, but were unchanged by hypoxia. The fetal PGE2 response reflected the degree of hypoxia over the 3 days, with moderate and severe hypoxia producing a significant (P < 0.05) increase in PGE2 concentrations. Fetal plasma cortisol concentrations were increased (P < 0.05) during all levels of hypoxia. Fetal arterial activin A was increased in response to moderate and severe hypoxia, but levels were not maintained over the hypoxic period. The increases in activin A and cortisol concentrations preceded the increase in PGE2.

Assisted reproductive techniques, such as in vitro oocyte maturation in conjunction with in vitro fertilisation, may be used as a tool to manipulate reproduction. Using the common wombat as a model for the critically endangered northern hairy-nosed wombat, the present study examined whether oocyte maturation could be achieved under field conditions. At the time of collection, no oocytes were at the metaphase II (MII) stage (0/42). After 60 h culture using the submarine incubation system, 34% of oocytes (24/70) matured to telophase/MII, as indicated by the presence of a polar body. The proportion of oocytes that reached MII was higher for oocytes collected from follicles >2 mm in diameter compared with follicles <2 mm (40% v. 22%, respectively). The presence of cumulus cells alone did not influence the maturation potential. Oocytes without cumulus cells collected from follicles >2 mm in diameter had the highest maturation rate (58%). Maturation was not affected by the reproductive status of the common wombat or a delay of up to 5 h before oocyte collection. In conclusion, the present study demonstrated that oocytes collected from non-stimulated common wombats can mature to MII in culture.

Ovarian tissue xenografting may be applied to increase the population size of rare or endangered animals. However, optimal grafting conditions, such as graft position and recipient hormonal status, are yet to be established. The present study, using common wombat ovarian tissue, showed that development of xenografted ovarian tissue to the antral follicle stage can be achieved irrespective of graft position. However, increased graft recovery rates and follicle survival were evident after grafting under the kidney capsule compared with grafting to subcutaneous sites. No increase in follicle development was observed after placing grafts both under the kidney capsule and subcutaneously in the one recipient compared with grafts placed under the kidney capsule alone or subcutaneously alone. Removal of the recipient's own ovaries at the time of grafting accelerated graft follicle development, with antral follicles seen by Week 12 after grafting compared with by Week 16 in recipients that retained their own ovaries. More oocytes were collected from xenograft recipients receiving hormonal stimulation before collection compared with non-stimulated recipients. No oocytes were mature (extruded a polar body) at the time of collection or after a subsequent period of in vitro maturation. This is the first study to demonstrate that antral follicle development can occur and oocytes can be collected from xenografted common wombat ovarian tissue.

In cases in which ovarian tissue is to be cryopreserved for tissue or gene banking it is important to maintain its integrity and viability. This study examined how delays between the death of an animal and the collection/cryopreservation of its ovarian tissue influenced follicle viability. Mouse ovaries were placed in PBS+antibiotic (in vitro) or left within the body (in situ) at room temperature for 0, 3, 6, 12, or 24 h following the death of the donor. These ovaries were cryopreserved at 1°C/min on dry ice or in a −84°C freezer using a passive cooling device or by conventional slow cooling (0.3°C/min). The ovaries were grafted under the kidney capsule of ovariectomized recipient mice and collected 2 weeks later, and the size and number of follicles were determined. Cryopreserved ovarian tissue grafted immediately after the death of the donor contained numerous viable and healthy follicles independent of the cooling procedure (dry ice, 134 ± 32; −84°C, 165 ± 54; slow, 214 ± 55 follicles per half ovary). Tissues stored in vitrobefore cryopreservation retained viable follicles up to 12 h after death (dry ice, 30 ± 15; −84°C, 86 ± 45; slow, 93 ± 33), whereas tissue left in situhad significantly reduced follicle numbers within 3 h of death (dry ice, 36 ± 12; −84°C, 19 ± 6; slow, 28 ± 7). No significant difference was found between the cooling rates tested, indicating that a passive cooling container which cools at 1°C/min is a suitable alternative to conventional slow cooling. We conclude that ovarian tissues for cryobanking should be cryopreserved as soon as possible after collection or death of the animal to ensure maximal follicular survival.

Ovarian tissues, collected or salvaged from endangered species at the time of gonadectomy or following their death, are being transported to genebanks for storage with the assumption that they will (subsequently) yield sufficient numbers of germ cells to help preserve the species. The present study aimed to quantify the impact of delays in collecting and/or processing ovarian tissue on the number of follicles in this tissue that remained normal after grafting. The study compared the viability of ovarian tissue stored in vitro (in phosphate-buffered saline) versus in situ (in the body) either on ice or at room temperature for 0 (non-stored fresh grafts), 3, 6, 12, 24 or 48 h. The conditions of storage had significant effects on the total number of morphologically normal follicles, with significantly more follicles in grafts developing from in vitro-stored tissue than in situ-stored tissue. Storage temperature and duration of storage, but not the storage temperature alone, influenced follicle survival. Tissue that was grafted immediately after collection (0 h) was best, but normal follicles were recovered in grafts stored in vitro (on ice or at room temperature) or in situ (on ice only) for up to 48 h before grafting. The rate of follicle loss over time was very rapid, with approximately 50% fewer follicles in grafts derived from tissue stored for only 3 h compared with non-stored fresh grafts (0 h). The results show that viable ovarian tissue can be salvaged from animals up to 48 h after death; however, in order to best protect the follicle population, the ovaries should be removed from the animal’s body as soon as possible.

This study was conducted to determine whether early progesterone treatment plays a role in the regulation of messenger RNA (mRNA) expression for oxytocin–neurophysin, oxytocin receptor, prostaglandin G/H synthase (PGHS)-1 and PGHS-2 in the ovine corpus luteum. The expression of ovarian oxytocin, oxytocin receptor, PGHS-1 and PGHS-2 mRNA was investigated in control, progesterone- or RU486-treated ewes. Fifteen ewes were randomly assigned to three groups to receive intramuscular injections of progesterone (12.5 mg; n = 5), RU486, (2.5 mg kg–1 bodyweight; n = 4) or corn oil (1 mL; n =6) twice daily from Day 1 to Day 3 post oestrus. On the morning of Day 4 post oestrus, the corpora lutea were collected and analysed for oxytocin–neurophysin mRNA by Northern blot using a labelled cDNA probe, and for the expressions of the oxytocin receptor, PGHS-1 and PGHS-2 mRNA using the reverse transcription polymerase chain reaction. Administration of progesterone or suppression of progesterone activity with RU486 did not affect expression of oxytocin–neurophysin mRNA (P>0.05). Pretreatment of the ewes with progesterone resulted in the enhancement of luteal oxytocin receptor mRNA expression and suppression of PGHS-1 and PGHS-2 mRNA (P&lt;0.001). These results indicate that early progesterone treatment does not control the expression of oxytocin–neurophysin mRNA in the ovine ovary but may be involved in the regulation of ovarian oxytocin receptor and PGHS expression. It is proposed, on the basis of these results, that progesterone may play a role in premature corpus luteum regression through an intra-ovarian mechanism involving the induction of ovarian oxytocin receptor mRNA expression. Extra keywords: ovary, sheep.

Poly(methylmethacrylate) particles stabilised by poly(12-hydroxystearic acid) in a hydrocarbon medium provide, as a polymer latex, a useful system for investigating the properties of a sterically stabilised concentrated dispersion. In the present work these latices were investigated by small angle coherent neutron scattering and by quasielastic incoherent scattering. The former technique was used to probe the dimensions of the surface layer of poly(12-hydroxystearic acid) and to determine the diameter of the core particle of poly(methylmethacrylate) using dilute dispersions. In concentrated dispersions it was used to determine the structure factor. The latter was compared with that expected for an interacting system of hard spheres and reasonable agreement obtained. The quasielastic technique was used to probe the translational diffusion of the poly(12-hydroxystearic acid) molecules on the surface and the diffusion of the total particles in the concentrated systems.

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•mg•b and 18.9 ± 0.18 ng•mg•12, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p<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<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•mg• 20 min, respectively, p<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 ionopbore A23187 (p<0.05). Addition of prostaglandin Fla (2.8 pM) 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 Fla does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.

Endometrial oxytocin receptor concentrations and oxytocin-induced plasma concentrations of 13,14,dihydro-15-keto prostaglandin F2αwere investigated on days 14 and 17 of the oestrous cycle and on days 14, 17, 25, 65, 85 and 145 of gestation in ewes. Total 13,14,dihydro-15-keto prostaglandin F2αrelease in response to a bolus injection of oxytocin was significantly (P< 0.05) higher at luteolysis (day 17 of the oestrous cycle) than at any other stage of the oestrous cycle or in early gestation. On days 65, 85 and 145 of gestation, total prostaglandin release was significantly (P< 0.05) increased compared with earlier in gestation. Maximum concentrations of 13,14,dihydro-15-keto prostaglandin F2αin response to oxytocin followed a similar pattern. Oxytocin receptor concentrations reflected total oxytocin-induced 13,14,dihydro-15-keto prostaglandin F2αrelease, with increased oxytocin receptor concentrations occurring on day 17 of the oestrous cycle, compared with those observed on day 14 of the oestrous cycle and on days 14, 17 and 25 of gestation. By day 65 of gestation, oxytocin receptor concentrations were again increased. However, on days 85 and 145 of gestation, oxytocin receptor concentrations had decreased to concentrations similar to those observed in early gestation. These results indicate that oxytocin-induced 13,14,dihydro-15-keto prostaglandin F2αrelease during early gestation is minimal despite the presence of endometrial oxytocin receptors. In mid-gestation, oxytocin-stimulated 13,14,dihydro-15-keto prostaglandin F2αrelease is increased with a concomitant increase in uterine oxytocin receptor concentrations. However, by the later stages of gestation, oxytocin receptor concentrations, but not oxytocin-stimulated 13,14,dihydro-15-keto prostaglandin F2αrelease, were again decreased. Thus, there appears to be a dissociation between the response to oxytocin and endometrial oxytocin concentrations at specific stages of the reproductive cycle in sheep.

Dalradian metamorphic rocks, Lower Ordovician meta-igneous rocks (MGS) and Caledonian granites of the Connemara complex in SW Connemara all show intense retrograde alteration. Alteration primarily involves sericitization and saussuritization of plagioclase, the alteration of biotite and hornblende to chlorite and the formation of secondary epidote. The alteration is associated with sealed microcracks in all rocks and planes of secondary fluid inclusions in quartz where it occurs, and was the result of a phase of fluid influx into these rocks. In hand specimen K-feldspar becomes progressively reddened with increasing alteration. Mineralogical alteration in the MGS and Caledonian granites took place at temperatures ~275±15°C and in the MGS Pfluid is estimated to be =1.5 kbar during alteration. The °D values of alteration phases are:-18 to-29‰ (fluid inclusions),-47 to-61‰ (chlorites) and-11 to-31‰ (epidotes). Chlorite d18O values are +0.2 to +4.3‰, while d18O values for quartz-K-feldspar pairs show both positively sloped (MGS) and highly unusual negatively sloped (Caledonian granites) arrays, diverging from the normal magmatic field on a d-d plot. The stable isotope data show that the fluid that caused retrogression continued to be present in most rocks until temperatures fell to 200–140°C. The retrograde fluid had dD ~-20 to-30‰ in all lithologies, but the fluid d18O varied both spatially and temporally within the range-4 to +7‰. The fO2 of the fluid that deposited the epidotes in the MGS varied with its d18O value, with the most 18O-depleted fluid being the most oxidizing. The dD values, together with low (<0‰) d18O values for the retrograde fluid in some lithologies indicate that this fluid was of meteoric origin. This meteoric fluid was probably responsible for the alteration in all lithologies during a single phase of fluid infiltration. The variation in retrograde fluid d18O values is attributed to the effects of variable oxygen isotope shifting of this meteoric fluid by fluid-rock interaction. Infiltration of meteoric fluid into this area was most likely accomplished by convection of pore fluids around the heat anomaly of the Galway granite soon after intrusion at ~400 Ma. However convective circulation of meteoric water and mineralogical alteration could possible have occurred considerably later.

The development of rapakivi texture in feldspars from the Ketilidian granitoids of south Greenland has been investigated using Sr, O and H isotopes. A low temperature signature is found in the Sr and O data which seemingly contradicts some textural features that point to a magmatic origin of the plagioclase mantles around the K-feldspar ovoids. An origin for these mantles involving exsolution from an original alkali feldspar solid solution is proposed, which involves growth of mantles over a range of conditions determined by the mobility of the exsolving sodic feldspar. This mobility may be enhanced at high temperatures in the presence of melts or increased fluid pressures and at lower temperatures by the processes responsible for the transformation of K-feldspar to microcline. Rapakivi granites with both white and dark green feldspar occur in south Greenland but show no major isotopic differences, although the dark alkali feldspars contain significantly more fluid. Equivalent fluids in the white alkali feldspars may have escaped during plagioclase exsolution.

A radioimmunoassay for the determination of concentrations of melatonin in the plasma of rhesus monkeys has been developed. Antiserum for the assay was raised against N-acetyl serotonin and there was a 100% cross-reaction with melatonin. Cross-reactivity with closely related indoles, precursors and metabolites was less than or equal to 1%. The lower limit of sensitivity of the assay was 4 pg/tube.The assay has been used for the investigation of diurnal variations and cyclical changes in melatonin concentrations in peripheral plasma of the rhesus monkey. The concentrations of melatonin ranged between 26·6 and 85·3 pg/ml during sampling for 24 h.There was a distinct diurnal variation in the concentration of melatonin in plasma. The concentration during darkness (61·0 ± 7·1 (s.e.m.) pg/ml) was greater (P<0·01) than that during illumination (40·1 ± 6·1 pg/ml). There were no significant differences in the concentration of melatonin in plasma at any time during the 28 day menstrual cycle of the rhesus monkey.

Premature delivery was induced by intrafetal infusion of dexamethasone or (1–24)ACTH, starting between days 124 and 129 of pregnancy in twelve ewes carrying intact (n= 7) or hypophysectomized (n= 5) single fetuses. Concentrations of ovine placental lactogen (oPL) in maternal plasma were measured by specific radioimmunoassay before and during premature delivery. There was no significant change in oPL concentrations before delivery in ewes carrying either intact or hypophysectomized fetuses.The results suggest that a prepartum decline in oPL is not an essential prerequisite for delivery, and indicate that fetal glucocorticoids are not directly responsible for the prepartum fall in oPL concentrations observed at spontaneous delivery.

125I-Labelled murine epidermal growth factor (EGF) was injected or infused into conscious ewes through the jugular vein. Its disappearance from the circulation and the pattern of its distribution in other body tissues and compartments were observed. Single bolus injections of 125I-labelled EGF resulted in a transient peak of radioactive EGF in the circulation which occurred within 1 min of the injection. This was followed by a very rapid fall in radioactivity in the plasma (t½∼ 1 min) and the gradual appearance of 125I-labelled EGF in the urine. Immunoprecipitable 125I-labelled EGF could be detected in urine within 5 min of the start of the experiment. 125I-Labelled EGF accumulated in the urine for several hours following the injection, although with increasing time a substantial amount of non-immunoprecipitable iodide was also found. The rate of disappearance of the 125I-labelled EGF from the plasma of the ewe was found to be faster than the rate of disappearance of free [125I]iodide that had been injected into the ewe.125I-Labelled EGF was also administered by a continuous infusion following an initial bolus injection. This again resulted in a rapid initial fall in radio-activity in blood, followed by a slow rise throughout the period of the infusion. When the infusion was stopped, there was a 15-min period of rapid readjustment, after which the radioactivity in the blood fell at a much slower rate (t½∼70 min) than was seen initially. Again, intact 125I-labelled EGF was transferred to urine throughout the experiment.At autopsy, 125I-labelled EGF was increased in bile, liver, thyroid and kidney. Although most of the 125I found in the thyroid was free iodide, some EGF-like material was also present. There was also EGF-like material found in both the kidney cortex and the kidney medulla. These results indicate that complex multi-compartment pathways for the uptake, distribution and clearance of 125I-labelled EGF exist in the sheep.Journal of Endocrinology(1989) 123,121–130

The granulosa luteal cells of the sheep corpus luteum secrete their hormonal products by exocytosis of granules. Electron micrographs of randomly selected granulosa cells from nine corpora lutea at day 10 of the oestrous cycle were examined to obtain the cellular density of these granules. From the area of the cell, calculated using an x -y digitiser, and the number of granules observed, the number of intracellular secretory granules per µm3 of luteal cell cytoplasm was calculated. There was a large variation in the number of granules per cell within the same corpus luteum and between corpora lutea taken at the same stage of the cycle. The number of intracellular granules in nine corpora lutea varied from 2.12±1.05 granules per µm3 (x, SD, n = 30) cells to 0.36±0.18 granules per µm3 ( x, SD, n = 26). These morphological data suggest that the variation in granule synthesis in individual cells may contribute to the variation in hormone content of corpora lutea at the same stage of the cycle, and the episodic release of hormone into the plasma.

Initial studies on inhibin, activin and follistatin focussed on their role as regulators of adult gonadal function via feedback regulation of anterior pituitary hormones and via intragonadal control of steroid hormones. The discovery of isoforms of follistatin which are either secreted or retained on the cell surface and which bind activin and, to a lesser extent, inhibin adds a further dimension to the regulation of these peptides. More recently, the cloning of inhibin and activin, and the observation of their close homology to the transforming growth factor-beta family of peptides, has led to an interest in their possible role as growth and differentiation factors. Activin, inhibin and follistatin are expressed in embryonic and fetal tissues, as well as in the placenta. However, although activin is a potent regulator of growth and differentiation in a number of cell types, their role in embryonic and fetal development has yet to be established. High concentrations of inhibin have been observed in the fetal gonads, particularly the testes, and in the fetal adrenals of a number of species and a sex difference in fetal plasma concentrations has also been observed. Although the stimulus for high concentrations of inhibin in the fetus is not know, they are associated with decreased testicular testosterone and a decrease in the concentration of circulating follicle-stimulating hormone (FSH); this suggests that, as in the adult, inhibin may be involved in the regulation of fetal testicular androgen and pituitary FSH secretion during late gestation. The recent reports of elevated concentrations of inhibin and, particularly, activin in amniotic fluid during late gestation and its ability to stimulate the production of prostaglandin E2 by fetal membranes provides yet another potential role for this hormone in the regulation of events leading to parturition.

The pulsatile release of oxytocin from the corpus luteum in the sheep is responsible for the pulsatile release of prostaglandin F2 alpha (PGF2 alpha) from the uterus at luteolysis. It has been proposed that PGF2 alpha also reinforces this process by stimulating the release of oxytocin from the corpus luteum. It is, however, unlikely that PGF2 alpha is the major stimulus for oxytocin release at this time. Although the stimulus for the pulsatile release of oxytocin from the corpus luteum appears to reach the ovary from the peripheral circulation, the nature of the stimulus is unknown. Pulses of oxytocin originating from the corpus luteum have also been observed during early pregnancy, but the release of PGF2 alpha, in response to this signal, is abrogated in some way by ovine trophoblast protein-1 (oTP-1). This protein has been shown to inhibit endometrial prostaglandin production and to decrease the amount of PGF2 alpha released in response to oxytocin. Reduction of uterine oxytocin receptor concentrations by conceptus secretory proteins or by interferons related to oTP-1 remains equivocal. Inhibition of uterine oxytocin receptors is, however, probably the major mechanism that prevents luteal regression during early pregnancy. In cyclic sheep the specific inhibition of uterine oxytocin receptors by 1-deamino-2-D-Try (oET)-4-Thr-8-Orn-oxytocin (CAP), a synthetic oxytocin receptor antagonist, inhibits luteal regression and suppresses pulsatile, but not basal, secretion of uterine PGF2 alpha. Thus, the effects of CAP directly parallel the endocrinological changes that occur in early pregnancy in the sheep.

Concentrations of progesterone, prolactin, LH and 13,14 dihydro-15-keto-prostaglandin F2α(PGFM) were measured in plasma of eight tammar wallabies at 8-hourly intervals during the end of pregnancy and post-partum oestrus initiated by removing the pouch young, and during the end of the oestrous cycle, similarly initiated. In the non-pregnant cycle oestrus occurred 29·7 ± 0·7 (mean ±s.e.m.) days after initiation of the cycle, was preceded by a slow decline in progesterone concentration from 1·6 nmol/l to less than 0·64nmol/l and was followed by a preovulatory peak of LH 5·3± 3·9 h later. In the pregnant cycle birth occurred 26·1±0·2 days after removing the pouch young and was followed 8·0 ± 2·1 h later by oestrus and 16·0± 2·5 h by an LH peak. The latter events thus occurred 3·2 days earlier in the pregnant than in the non-pregnant cycle. Parturition coincided with a very rapid decline in progesterone and a transient high peak of prolactin. In two females sampled less than 25 min after parturition there was a transient peak of PGFM but in all others the concentrations of PGFM remained basal throughout. It is suggested that the fetus and/or placenta is involved in both the premature decline in progesterone and the initiation of parturition and that onset of oestrus and ovulation, being a consequence of a decline in progesterone, are therefore also determined by the fetus.

The concentrations of inhibin and follistatin in amniotic fluid and in tissue extracts from the placenta, gonads and adrenals of fetal sheep were measured using radioimmunoassays. These tissue extracts were from whole fetuses from days 16 to 45 and from the individual organs from day 46 to 145 (term) and were assayed at multiple dilutions. The capacity of these extracts to alter FSH production of rat anterior pituitary cells in culture was also assessed at multiple dilutions.Immunoactive inhibin concentrations in amniotic fluid from both sexes increased during gestation and levels were significantly greater in males than females. Peak concentrations of immunoreactive inhibin of 11·2±1·9 ng/ml were found in males at 116–125 days of gestation. Follistatin concentrations did not change throughout gestation and no significant difference was noted between sexes. Mean follistatin levels throughout gestation were 3·0±0·9 ng/ml for males and 3·7±0·9 ng/ml for females.Despite the potential for FSH inhibition by inhibin and follistatin, amniotic fluid from both sexes at all stages of gestation stimulated FSH secretion in the pituitary cell bioassays, suggesting the presence of activin which was confirmed by the measurement of immunoactive activin (13·3±2·5 ng/ml) in a specific radioimmunoassay.Maximum concentrations of immunoactive and bioactive inhibin in placental extracts were observed in late gestation (2·2 ±0·6 and 3·8±1·6 ng/g respectively) and there was no significant difference between sexes. Follistatin concentrations in placental cotyledons ranged from 11·5 to 27·1 ng/g with no significant difference between sexes. In view of the higher follistatin concentrations compared with inhibin, it is likely that the capacity of placental extracts to suppress FSH production by pituitary cells in culture is due predominantly to follistatin.Immunoactive inhibin was observed in high concentrations in the fetal testis throughout gestation; with concentrations increasing to a maximum of 1993·0± 519·7 ng/g at 126–135 days of gestation with a ratio of bioactive: immunoactive inhibin of 1:20. Although bioactive and immunoactive inhibin was also observed in fetal ovaries and adrenals from both male and female fetuses, concentrations were lower than those observed in fetal testes. Follistatin concentrations in the fetal testis were elevated between 70 and 95 days (97·6 ng/g) and then declined. Similar concentrations were found in the adrenal glands of both sexes (males 83·5–103·3 ng/g: females 55·3–95·8 ng/g). In both males and females, immunoactive inhibin concentrations in fetal adrenals increased during gestation peaking at levels of 34·4±16·5 and 27·8± 9·0 ng/g respectively. These data suggest that the capacity of adrenal extracts to suppress FSH production by pituitary cells is due to both inhibin and follistatin.These studies demonstrated that significant concentrations of immunoactive inhibin and follistatin are present in amniotic fluid, and the fetal gonads, adrenal glands and placenta in sheep. The role of these proteins during fetal development requires further study.Journal of Endocrinology(1994) 141,219–229

The effectiveness of trilostane and azastene as inhibitors of adrenal steroidogenesis was compared by in-vitro and in-vivo methods. A radioimmunoassay was developed for the measurement of cortisol in ovine plasma, incubation medium and tissue extract using a specific antiserum raised against cortisol 21-acetate,3-carboxymethyloxime : bovine serum albuTrilostane (20 μmol/l) decreased cortisol synthesis and release both in unstimulated and in ACTH-stimulated adrenal tissues in vitro. The same concentration of azastene had a lesser effect on unstimulated adrenals and was completely ineffective in blocking the stimulatory action of ACTH. In vivo, trilostane suppressed adrenal steroidogenesis in pregnant and cyclic ewes but the suppression in pregnant ewes was over a longer period, and after lower doses.It is concluded that trilostane had an inhibitory effect on ovine adrenal steroidogenesis both in vitroand in vivo.

Immunoactive inhibin (ir-inhibin) concentrations were determined in chronically catheterized sheep between 120 and 145 days gestation. Maternal plasma ir-inhibin concentrations remained basal (0.19 ± 0.05 ng/ml) throughout the period of study. Immunoactive inhibin concentrations in male and female fetal plasma were elevated above those observed in maternal plasma, with the concentrations in plasma of male fetuses (7.38 ± 0.04 ng/ml) being significantly greater (p< 0.001) than those in female fetuses (1.07 ± 0.14 ng/ml). The concentrations of ir-inhibin in amniotic fluid of ewes bearing male fetuses (10.93 ± 1.55 ng/ml) were significantly greater than in ewes bearing female fetuses (2.81 ± 0.32 ng/ml; p< 0.05) but not significantly different from the concentrations in male fetal plasma. Immunoactive inhibin concentrations decreased following surgery in gonadectomized fetuses, to 3.25 ± 0.99 ng/ml within 6 h, and remained at a mean value of 0.75 ± 0.38 ng/ml from 24 h after gonadectomy. The half-life of circulating inhibin in fetal plasma, estimated from the decay curve during the first 6 h after surgery, was 3.94 ± 0.88 h. There was a significant (p< 0.05) decrease in the concentration of ir-inhibin in amniotic fluid after gonadectomy; however, this decrease occurred gradually over 7 days, and ir-inhibin concentrations did not fall to those concentrations observed in fetal circulation at any time after gonadectomy. It is concluded that the major source of circulating ir-inhibin in male fetal plasma, but not in amniotic fluid, is the gonads.

The effects of exogenous oxytocin (OT) and estradiol-17β (E2) on plasma concentrations of prostaglandin (PG) E2and 13, 14-dihydro-15-keto-PGF2α(PGFM) were investigated on Day 14–15 (NP) of the estrous cycle and Days 14–16 (PI) and 21–25 (EP) of pregnancy in the ewe. Basal concentrations of PGFM were significantly elevated in utero-ovarian venous (UOV) plasma on Day 14 of pregnancy (4.05 ± 0.81 nM, mean ± SEM) compared to that observed on Day 14 of the cycle or Days 21–25 of pregnancy (2.29 ± 1.3 nM and 1.06 ± 0.56 nM, respectively). PGFM release increased significantly following intera-arterial bolus injections of 50, 500, and 5000 mU OT at 2-h intervals in all experimental groups. There was no significant difference in area and peak height of the PGFM response between the 3 groups studied. The time to peak PGFM response was, however, significantly longer in the PI group. No significant changes in concentration of PGFM were observed in any experimental group following 1-h infusions of E2at 5, 50, and 500 pmol/min. Long-term (15– 18 h) infusion of E2at 83 pmol/min increased the peak height of the OT-induced PGFM response at both stages of gestation studied. PGE2concentrations in UOV plasma were less than 0.05 nM in all samples studied. These results demonstrate that PG release can be induced in response to OT during the period in which ovine trophoblastic protein-1 (oTP-1) is released by the conceptus. During pregnancy, oTP-1 does not appear to inhibit the E2induction of uterine OT receptors.

Summary.In sheep the basal concentration of LH in jugular vein plasma was significantly higher during the first 50 days of gestation than in late pregnancy or at parturition. The pituitary response to a single i.v. injection of 200 μg synthetic LH-RH was determined at different stages of gestation and compared with that of anoestrous and cyclic sheep. Pituitary response to LH-RH decreased progressively with advancing gestation: by 56 days after mating the response had declined to 35% and by parturition to 14% of the value in anoestrous sheep. The pituitary response to LH-RH increased after parturition and the pattern of recovery differed in non-lactating and lactating sheep. By 63 days post partumthe response to LH-RH in non-lactating and lactating animals had returned to values similar to those in sheep during anoestrus and sheep during the luteal phase of the oestrous cycle.A decrease in pituitary responsiveness during pregnancy was associated with a decrease in pituitary content of LH. The quantity of LH released in response to a standard injection of LH-RH was linearly related to pituitary LH content.

Pregnenolone sulphate, pregnenolone, progesterone and 20α-hydroxy-4-pregnen-3-one concentrations in peripheral plasma of normal cyclic ewes were measured by radioimmunoassay. The concentrations of these steroids were correlated with that of progesterone. The concentrations of all the steroids measured in peripheral plasma varied in a cyclic manner and showed a significant (P<0·05) positive correlation with the concentration of progesterone.Peripheral plasma concentrations of these steroids in ovariectomized and ovariectomized, dexamethasone-treated ewes were also determined. The plasma concentration of progesterone in ovariectomized ewes was undetectable but the concentrations of pregnenolone sulphate, pregnenolone and 20α-hydroxy-4-pregnen-3-one remained similar to those observed at oestrus. Administration of dexamethasone to ovariectomized ewes had no effect on pregnenolone sulphate or pregnenolone concentrations but 20α-hydroxy-4-pregnen-3-one concentrations, which were already very low, decreased further.It is proposed that the ovary, probably the corpus luteum, secretes pregnenolone sulphate, pregnenolone and 20α-hydroxy-4-pregnen-3-one; however, pregnenolone sulphate and 20α-hydroxy-4-pregnen-3-one may also arise from the metabolism of circulating pregnenolone and progesterone.J. Endocr.(1987) 113,231–237.

The concentration of prolactin in samples of fetal plasma and amniotic fluid collected from sheep with indwelling catheters has been measured by radioimmunoassay. There was considerable variation between animals in the concentration of prolactin in fetal plasma which ranged from <2 μg/l to >50 μg/l. In five out of eight fetuses which were sampled at intervals throughout the last 30 days of pregnancy, there was an increase in the concentration of prolactin in the plasma before parturition. The concentration of prolactin in amniotic fluid was consistently low (approximately 1 μg/l). Induction of premature parturition by intrafetal infusion of dexamethasone was accompanied by an increase in the concentration of prolactin in only one of three fetuses infused.These results indicated that prolactin is present in fetal plasma but that an increase in prolactin in fetal plasma before the onset of parturition does not always occur. The low concentration of prolactin in amniotic fluid contrasts with the high concentrations of this hormone found in the amniotic fluid of the primate.

Sixteen-day-old fetal mouse ovaries were slowly frozen in 1.5 mol dimethylsulfoxide ml−1and subjected to one of two thawing procedures – fast thaw or slow thaw. Fresh and frozen–thawed fetal ovaries were transplanted orthotopically (to the bursal cavity) to either bilaterally or unilaterally ovariectomized adult female recipients. Fresh fetal ovaries were also transplanted heterotopically (under the kidney capsule) to intact, bilaterally or unilaterally ovariectomized adult females. Transplantation of fetal ovaries to bilaterally ovariectomized adult recipients resulted in restoration of cyclic activity within 20.5 ± 4.7 (mean ± sem) days or 23.4 ± 0.8 days in orthotopic and heterotopic groups, respectively. Developing follicles and corpora lutea were observed within 4 weeks after transplantation of fetal ovaries to heterotopic sites and within 6 weeks after transplantation to orthotopic sites. After orthotopic transplantation, 33% of the recipients became pregnant. Orthotopic or heterotopic transplantation to intact of unilaterally ovariectomized recipients resulted in quiescence of the fetal ovary. After cryopreservation, transplantation of fetal ovaries to bilaterally ovariectomized recipients resulted in restoration of cyclic activity within 19.3 ± 2.1 days and 23.4 ± 5.1 days after transplantation in slow thaw and fast thaw groups, respectively. Fertility was restored to 86% of fast thawed and 25% of slow thawed fetal ovary transplants to bilaterally ovariectomized adult recipients. No ovarian tissue was observed on the side of the fetal graft in unilaterally ovariectomized recipients that received frozen–thawed fetal ovaries. These results demonstrate that cryopreserved fetal ovarian tissue can be transplanted to adult recipients with subsequent restoration of fertility and that this process is dependent on the absence of the ovaries of the recipients.

Summary.Pituitary responsiveness to 200 μg synthetic LH-RH and to 10 μg TRH was determined in anoestrous sheep before and after treatment for 3 weeks with progesterone (100 mg/day), or oestradiol (250 μg/day) plus progesterone (100 mg/day). There was a marked decrease in pituitary responsiveness to LH-RH after progesterone (P< 0·01 ), or oestradiol + progesterone (P< 0·01) treatments, and an increase in response to TRH after oestradiol + progesterone (P< 0·01) treatment. The results demonstrate selective and simultaneous feedback effects of oestradiol and progesterone on pituitary responsiveness to two hypothalamic releasing hormones.

The subcellular distribution of progesterone and oxytocin within the ovine corpus luteum was investigated using differential and density gradient centrifugation. Progesterone and oxytocin were associated with particles which sedimented to a density of 1·049–1·054 g/ml and 1·054–1·061 g/ml respectively. Particle-associated progesterone did not, however, display physical or biochemical characteristics consistent with its storage within secretory granules. When particle-associated progesterone was incubated in HEPES buffer at 37 °C, 70% of the total progesterone was recovered in the incubation medium. The remaining stable particle-associated progesterone was not affected by treatments which stimulated oxytocin release and which have been shown to cause the release of peptides and biogenic amines from secretory granules. These results suggest that particle-associated progesterone represents the intercalation of progesterone into cell membranes and they do not support the hypothesis that progesterone is stored, in a protein-bound form, in luteal secretory granules.J. Endocr.(1986) 108,109–116

The mechanism by which prostaglandin F2αterminates luteal function in the sheep is unclear even though it is used extensively in animal husbandry. At the time of luteal regression, a decrease in 3β-hydroxysteroid dehydrogenase (3β-HSD) activity is apparent in the corpus luteum, but it is not known whether the decrease in enzyme activity is the primary cause of structural luteolysis. The effect of trilostane, a 3β-HSD inhibitor, on luteal function and morphology has therefore been investigated. Intravenous injection of trilostane in the mid-luteal phase of the oestrous cycle caused a decrease in ovarian tissue progesterone content. A transient decrease in peripheral and utero-ovarian vein plasma progesterone was observed but there was no significant effect on the length of the luteal phase of the cycle. There was no significant change in plasma 13,14-dihydro-15-oxo-prostaglandin F2αduring the period when plasma progesterone was depressed. Morphological examination of the corpora lutea revealed a decrease in the concentration of electron-dense granules without any other features of impending luteal regression. When plasma progesterone was reduced for more than 10 h by two injections of trilostane 4 h apart, there was again no subsequent effect on the length of the oestrous cycle or on the return to oestrus. Plasma progesterone returned to preinjection levels within 24 h of injection. This evidence suggests that competitive inhibition of 3β-HSD activity, per se, is ineffective in bringing about structural luteolysis.J. Endocr. (1984) 100,61–66

The development of uterine oxytocin receptors is an important regulatory step in the initiation of labour. Paracrine production of oxytocin by uterine and placental tissues may also be involved in some species. Placentome, intercotyledonary endometrium, myometrium and fetal membranes were collected from 3–5 ewes each, at regular intervals throughout pregnancy and from eight ewes during labour. Localization of mRNA encoding oxytocin and its receptor was by in situhybridization; oxytocin peptide concentrations were measured by radioimmunoassay and oxytocin receptor concentrations were measured by autoradiography and radioreceptor assay. In the intercotyledonary endometrium, mRNA encoding the oxytocin receptor was located in the luminal epithelium only. Both the epithelial and myometrial receptors were detected at low concentrations from the fourth week of gestation onwards, with a major increase associated with the onset of labour. In the placentomes, oxytocin receptors were localized to a stromal capsule surrounding the placental villi. Expression in this region was maximal in mid-gestation, declining in the second half of pregnancy and remaining low during labour. Cervical oxytocin receptors were detected at low concentrations in the epithelium and the muscular/connective tissue layers from day 22 of pregnancy onwards. There was no evidence for the local uterine production of oxytocin in the ewe; mRNA encoding oxytocin was undetectable and oxytocin concentrations were always <23 pg g−1wet mass of tissue. These results suggest that regulation of the timing of oxytocin receptor development varies between the different tissue types, despite a similar steroidal background. The receptors in the luminal epithelium are probably associated with the ability of exogenous oxytocin to induce the release of PGF2αthroughout most of pregnancy. The increase in receptors in both the intercotyledonary endometrium and myometrium at term suggest an involvement in labour, whereas their role in caruncular stroma in mid-pregnancy is unknown.

Summary.Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitroin a dose-dependent manner, but the dose—response lines were not parallel. The addition of cycloheximide blocked the stimulation by interstitial fluid but not that of hydroxycholesterol. Use of the compounds SU 10603 and cyanoketone (which inhibit 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase respectively) or aminoglutethimide (which acts on the cholesterol side-chain cleavage enzyme) showed that the stimulatory factor(s) in interstitial fluid stimulated steroidogenesis at the cholesterol side-chain cleavage enzyme, before the conversion of pregnenolone. This enzyme is rate-limiting in the synthesis of testosterone by Leydig cells and a site of action of LH; therefore, these results support the view that an interstitial fluid factor may be involved in the paracrine regulation of testicular steroidogenesis.

Summary.Rhythmic contractions occur in the sheep uterus at oestrus and during pregnancy from about 65 days to term (145 days). To define factors responsible for these contractions we have examined and quantified the degree of synchronization of electrical activity in the uterus and isolated segments of myometrium in 3 types of experiments. In the first, a segment of myometrium was totally separated from the uterus. After a period of 9–16 days the isolated tissue developed a typical pattern of uterine activity which showed no significant degree of synchronization with EMG bursts in the body of the uterus. During labour, the isolated tissue showed changes in activity similar to those observed in the uterus. In the 2nd experiment, the tubal end of one of the uterine horns was severed from the uterus, but a connection was retained with the uterus via the oviduct and ovarian blood vessels. Activity in the partly isolated segment remained in synchrony with the uterus. In a 3rd experiment, impulse propagation through nerves and smooth muscle to the tip of a horn was disrupted by severing 'the tip' from the uterus while its blood supply from the ovarian vessels was retained. The blood vessels were momentarily frozen, and denervation confirmed by monoamine histofluorescence. In 5 out of 6 animals the operated tissue showed activity that was not synchronous with the rest of the uterus. These data indicate that: (1) isolated uterine muscle in vivohas rhythmicity resembling that of intact myometrium and (2) systemic or local circulating factors are not responsible for synchronizing uterine activity before parturition, although circulating factors do play a major role in increasing the uterine activity which occurs at parturition and at oestrus, and (3) hydraulic continuity between different regions of the uterus is not essential for maintaining co-ordinated activity.

Summary.With advancing pregnancy in the ewe there was a marked decline in plasma LH concentrations and pituitary LH-RH responsiveness (integrated LH release) and a marked increase in plasma prolactin values and pituitary TRH responsiveness (integrated prolactin release). In lactating ewes plasma LH levels and pituitary LH-RH responsiveness had returned to values found in the luteal phase of the normal cycle by 21 days post partum, whereas at 42 days post partumprolactin levels were still high. No interaction between TRH and LH-RH on prolactin and LH release in dioestrous ewes was detected. In non-pregnant ewes plasma prolactin levels were significantly higher in June than in January but TRH responsiveness was similar. It is concluded that, in sheep, pituitary LH secretion recovers more rapidly from the chronic negative feedback effect of oestrogens and progesterone in pregnancy than prolactin secretion recovers from the chronic positive feedback effects of oestrogens. This finding may be a contributory factor in the resistance to resumption of breeding activity.

Summary.The concentration of progesterone in maternal plasma of the bandicoot increased from 3·9 to 12·6 ng/ml between 11 and 4 days before parturition and remained at this level for at least 7 days. Concentrations of 13,14-dihydro-15-keto-prostaglandin F-2α increased from basal levels of 288 pg/ml (4 days before birth) to maximal levels of 2534 pg/ml immediately after parturition, then decreased progressively. It is suggested that PGF-2α, but not progesterone, influences the initiation of parturition in this species.

The concentration of ovine placental lactogen (oPL) was measured by radioimmunoassay in plasma samples from chronically catheterized ewes and their fetuses from day 110 of gestation to term (about day 145).Concentrations of oPL in the plasma of the mother and fetus were raised after surgery, and remained raised for 3–5 days after the operation. Concentrations of oPL were greatest in the fetus at days 120–124 of gestation, and then declined until delivery. Mean concentrations of oPL in the fetus in late pregnancy for single, twin and triplet pregnancies were 101±6 (s.e.m.), 100±11 and 117±59 ng/ml respectively and were not significantly different.Mean concentrations of oPL in the mother in late pregnancy for single, twin and triplet pregnancies were 718±227, 1387±160 and 1510±459 ng/ml respectively; the difference between these means was significant (P<0·05). Peak concentrations were noted at days 130–139 of gestation after which concentrations fell and were significantly lower on the day of delivery (P<0·01). Concentrations of oPL in the mother showed no circadian rhythm. The mean concentrations of oPL in maternal plasma during late pregnancy was significantly correlated to the combined fetal weight at birth (r= 0·624, P<0·01).