The aim of the present study in Booroola ewes, either homozygous (BB) or non-carriers (++) of the Fec^B gene, was to test the specificity of the pituitary responses to exogenous hypothalamic releasing hormones by examining the plasma concentrations of FSH, LH, thyroid-stimulating hormone (TSH) and growth hormone (GH) after injecting the animals with different doses of GnRH, thyroid-releasing hormone (TRH) or growth-hormone-releasing hormone, (GHRH) which were administered on separate occasions. The animals (n = 8 per dose) received 0, 3.1 or 12.5 μg of thyroid-releasing hormone and GnRH (i.v.), whereas they (n = 9–13 per dose) received 0, 6.0 or 16.0 μg GHRH (i.v.). For each experiment there were no differences between the genotypes in bodymass or age. Gene-specific differences in the mean pretreatment concentrations of plasma FSH (BB > ++; P < 0.05) but not of LH, TSH or GH were noted. After treatment with GnRH, TRH or GHRH, significant effects of dose were noted for all the hormones; however, a gene-specific effect was observed only for FSH in response to GnRH (BB > ++; P < 0.01) with no genotype × dose interaction (anova). For LH, the effects of genotype and the genotype × dose interaction almost reached significance at the 5% level (genotype, P = 0.055; genotype × dose, P = 0.067). For TSH and GH the respective genotype × dose interactions were not significant. These results support the hypothesis that the Fec^B gene in Booroola ewes influences the release of pituitary hormones in response to hypothalamic releasing hormones only in the case of GnRH, which results mainly in different plasma concentrations of FSH.

Sixteen-day-old fetal mouse ovaries were slowly frozen in 1.5 mol dimethylsulfoxide ml⁻¹ and 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.

The oxytocin receptor antagonist [1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn]-oxytocin (Atosiban) is a specific antagonist of both mesotocin- and oxytocin-induced myometrial contractions in late pregnant tammars in vitro. Continuous intravenous infusion of Atosiban (1 mg kg⁻¹ day⁻¹) for 3 or 7 days from day 24 of the 26.5 day gestation significantly delayed births. In both the 3 day and 7 day infusion groups, all 15 control animals were pregnant and gave birth within the normal time (day 26.75 ± 0.20, mean ± sem), during the infusion of saline. The neonates weighed 387 ± 8 mg. Deliveries were observed in 15 Atosiban-treated animals significantly (P < 0.05) later than in the controls (day 27.85 ± 0.19; neonate weight 413 ± 9 mg). All pouch young were successfully suckled, even in the continued presence of Atosiban. Baseline plasma concentrations of the prostaglandin F metabolite (PGFM) in pregnant tammars were < 200 pg ml⁻¹. A surge in plasma PGFM occurred at birth (811 ± 116 pg ml⁻¹), followed by a rapid fall to baseline concentrations within 1 h after birth. This was observed both in saline- and in Atosiban-treated animals that gave birth during the observation period, and did not differ significantly between the treatment groups. Plasma progesterone concentrations in the control and the Atosiban-treated animals showed the normal pattern of luteolysis immediately after birth. Thus, infusion of an oxytocin receptor antagonist at the end of gestation delays birth, the peripartum surge in prostaglandin release, and the fall in progesterone, suggesting that mesotocin is an important part of the hormonal cascade associated with delivery in this marsupial.

This communication describes a simple technique for obtaining sufficient quantities of pure endometrial fluid (uterine cavitary fluid) for microchemical analysis. We believe this technique has several advantages over other methods of sampling uterine fluid.

Various constituents of endometrial fluid have been measured in flushings of the uterine cavity obtained through the endocervical canal. These measurements are valuable in a qualitative way, but are not indicative of actual cavitary concentrations because the volume of such fluid obtained by flushing is not known. Since endometrial fluid volume may vary from time to time in the same animal, and volume recovery may differ from flushing to flushing, only major variations in measured substances from flushings taken at different times in the same animal may be compared. Direct aspirates of significant volumes of cavitary fluid usually produce a fluid contaminated with blood since catheter insertion and suction traumatizes the endometrium.

Various techniques were tried

The discovery of cell-free fetal DNA (cffDNA) in maternal plasma has enabled a paradigm shift in prenatal testing, allowing for safer, earlier detection of genetic conditions of the fetus. Non-invasive prenatal testing (NIPT) for fetal aneuploidies has provided an alternative, highly efficient approach to first-trimester aneuploidy screening, and since its inception has been rapidly adopted worldwide. Due to the genome-wide nature of some NIPT protocols, the commercial sector has widened the scope of cell-free DNA (cfDNA) screening to include sex chromosome aneuploidies, rare autosomal trisomies and sub-microscopic copy-number variants. These developments may be marketed as ‘expanded NIPT’ or ‘NIPT Plus’ and bring with them a plethora of ethical and practical considerations. Concurrently, cfDNA tests for single-gene disorders, termed non-invasive prenatal diagnosis (NIPD), have been developed for an increasing array of conditions but are less widely available. Despite the fact that all these tests utilise the same biomarker, cfDNA, there is considerable variation in key parameters such as sensitivity, specificity and positive predictive value depending on what the test is for. The distinction between diagnostics and screening has become blurred, and there is a clear need for the education of physicians and patients regarding the technical capabilities and limitations of these different forms of testing. Furthermore, there is a requirement for consistent guidelines that apply across health sectors, both public and commercial, to ensure that tests are validated and robust and that careful and appropriate pre-test and post-test counselling is provided by professionals who understand the tests offered.

Endocrine and developmental changes were examined in Booroola FecB^B/FecB^B (BB, n = 16) and FecB⁺/FecB⁺ (++, n = 20) ewe lambs, and BB (n = 17) and ++ (n = 19) ram lambs from 2 to 53 weeks of age. Blood samples were taken weekly for the measurement of plasma concentrations of FSH, LH, immunoreactive inhibin, progesterone (ewe lambs) and testosterone (ram lambs). Behavioural oestrus in the ewe lambs and testicular volume and the breakdown of foreskin adhesions in ram lambs were recorded. Blood samples were taken from another flock of BB (n = 134) and ++ (n = 109) ram lambs at 20 weeks of age for the analysis of immunoreactive inhibin. In ewe and ram lambs, there appeared to be genotype differences for FSH, LH and immunoreactive inhibin at specific times during the neonatal period. In BB and ++ ewe lambs, respectively, mean FSH concentrations were 4.3 and 2.0 ng ml⁻¹ (sed 0.54) between 4 and 6 weeks, 2.6 and 3.4 ng ml⁻¹ (sed 0.33) between 12 and 28 weeks, and 1.8 and 1.9 ng ml⁻¹ (sed 0.18) between 34 and 53 weeks of age. Mean plasma LH concentrations were lower in BB than in ++ ewe lambs from 26 to 53 weeks of age (P < 0.05) but not earlier. Mean concentrations of immunoreactive inhibin were also lower in BB than in ++ ewe lambs between 2 and 11 weeks (16.0 and 27.4 iu ml⁻¹, respectively; P < 0.01), but thereafter no differences were apparent. In BB ram lambs, FSH concentrations were high for 3–4 weeks longer than in the ++ animals during the first 10 weeks of life. Likewise there were periods between 11 and 20 weeks of age when the plasma LH concentrations were higher in BB than in ++ ram (P < 0.05) lambs. Subsequently, between 19 and 33 weeks of age, the immunoreactive inhibin concentrations were consistently higher (P < 0.05) in BB than in ++ rams and this difference between the genotypes was confirmed in the larger study of 243 ram lambs at 20 weeks of age (BB > ++; P < 0.0005). The endocrine differences, in males and females, could not be attributed to either litter size, livemass or sire. However, limited numbers of sires (two BB and two ++) were used in the present study, so potential sire effects cannot be ruled out. In ewe lambs, the time of onset of puberty did not differ between genotypes. In ram lambs, the onset of puberty was not determined but testicular development, assessed by changes in testosterone concentrations, did not differ between genotypes. Differences in penile development and changes in testicular volume between the genotypes were observed but these were confounded by differences in livemass or sire. The evidence suggests that there are FecB^B-related differences in pituitary and gonadal hormones in neonatal ewes and rams. It is hypothesized that these differences between genotypes are part of a sequence of developmental differences that begin in fetal life.

Summary. To test whether the F gene-specific differences in the plasma concentrations of FSH and LH are due to differences in the pituitary responsiveness to exogenous GnRH, ovariectomized Booroola ewes with hypothalamic–pituitary disconnection (HPD–ovx) were treated with GnRH (250 ng i.v.) once every 2 h for up to 5 weeks. In Exp. 1, jugular venous blood was collected once weekly from 13 FF and 14 + + HPD–ovx ewes for 6 weeks before GnRH treatment and every 2nd, 3rd or 6th day for 5 weeks during treatment. In Exp. 2, jugular venous blood was collected from another 8 FF and 7 + + HPD–ovx ewes at 5- or 10-min intervals over 4 GnRH pulses (250 ng i.v. once every 2 h) on 3 separate occasions after the animals had been subjected to the GnRH pulse regimen for ∼7 days beforehand. Also in Exp. 2, the animals were extensively sampled around a larger (10μg) i.v. injection of GnRH and the pituitary FSH and LH contents assessed after the animals had been re-exposed to the once every 2 h GnRH (250 ng i.v.) pulse regimen for several days following the larger GnRH bolus. In Exp. 3 the distributions of mean plasma concentrations of FSH and LH in individual GnRH-treated HPD–ovx ewes were compared with those in ovariectomized and ovary-intact FF and + + ewes.

During the 6 weeks before GnRH treatment (Exp. 1), the plasma concentrations of FSH (∼ 1 ng/ml) and LH (⩽0·8 ng/ml) were not different between the genotypes. After GnRH treatment both the mean FSH and LH concentrations increased significantly (P < 0·01) above basal values after 2 days with F gene-specific differences being noted for FSH but not LH (FSH; FF > + +; P < 0·05). Thereafter, the mean FSH but not LH concentrations increased at a faster rate in FF than in + + ewes with the overall mean FSH concentrations between the genotypes being significantly different (P < 0·05).

In Exp. 2 considerable between-animal variation in the pulsatile pattern of FSH but not LH concentrations was seen in ewes of both genotypes during GnRH treatment. The overall mean FSH concentrations were higher in FF than in + + ewes (P < 0·05) and the mean FSH response to each GnRH pulse was significantly higher in FF than in + + ewes (P < 0·05). For LH a trend towards higher mean peak amplitudes and peak areas was noted in FF than in + + ewes but no significant differences were noted. Also, no gene differences were observed in the LH or FSH responses to the 10 μg i.v. GnRH bolus or the pituitary contents of FSH and LH.

In Exp. 3, ≥75% of the FF ewes had higher mean FSH concentrations than in 50% of the + + animals irrespective of treatment (i.e. GnRH–HPD–ovx, ovariectomized or ovary-intact controls; P < 0·05). Likewise, ∼50% of the FF ewes had higher mean concentrations of LH than did 75% of the + + ewes for all treatments.

These findings suggest that an association between the Booroola F gene and GnRH-induced FSH secretion exists, but whether such an association also exists for LH remains uncertain. These data support the hypothesis that at least part of the expression of the F gene is at the level of the pituitary gland to affect its responsiveness to physiological concentrations of GnRH.

Keywords: GnRH; FSH, LH; Booroola ewes; F gene; hypothalamic–pituitary disconnection; ovariectomy

Summary. In Exp. 1 non-pregnant female tammars were injected, on Day 26 (the day parturition would normally occur) after removal of pouch young, with saline, 200 μ ovine prolactin or 5 mg PG and changes in plasma concentrations of progesterone, prolactin, PGF-2α metabolite (PGFM), oestradiol-17β and LH were determined. Luteolysis occurred in females treated with prolactin alone, while treatment with PG first induced a rapid rise in prolactin and subsequently a significant decrease in plasma progesterone. After prolactin treatment the oestradiol peak, oestrus and the LH surge were advanced significantly compared to the saline-treated females.

In Exp. 2 the effects of the same treatments as used in Exp. 1 were determined on Day 23 and again on Day 26 after removal of pouch young in non-pregnant females. On Day 23 both prolactin and PG induced significant elevations in plasma progesterone, but luteolysis did not occur. On Day 26 the treatments initially induced significant elevations in plasma progesterone but these were followed by luteolysis within 8–12 h after treatment. PG treatment induced parturient behaviour in the non-pregnant females within 3–21 min and this persisted during the period that plasma concentrations of PGFM were elevated.

The results show that PG induces birth behaviour and the release of prolactin, while prolactin first induces an elevation of plasma progesterone concentrations and, in the mature CL on Day 26, subsequently induces luteolysis.

Keywords: progesterone; prolactin; LH; oestradiol; PG; tammar

Summary. Administration of epsilon-aminocaproic acid, a fibrinolytic inhibitor, either orally or from an impregnated IUD, had no effect on numbers of implanted embryos, their viability, or their diameters at Day 10 of pregnancy.

Primordial germ cells (PGCs) of the tammar wallaby Macropus eugenii have a distinctive morphology and stain positively for alkaline phosphatase. PGCs are identifiable in embryos with 12 somites, on about day 17 of the 26.5 day gestation period, when they are located in all three germ layers of the developing embryo and in the endoderm of the bilaminar and vascular (trilaminar) yolk sac membranes. PGCs are positive for alkaline phosphatase (ALP) at least between days 17 and 22 of pregnancy. In whole mounts on day 17, three groups of cells positive for ALP occur: about 40 just caudal to the neural tube, and about 20 distributed on either side of the last three somites. By day 21, there are about 150 PGCs in the newly formed gonadal ridges and 275 in the mesenteries. On days 21–22, there are PGCs in the umbilical mesoderm, the dorsal mesentery and the coelomic angles between the dorsal mesentery and the mesonephroi. On day 22, most ALP-positive PGCs are located in the dorsal mesentery, where they occur in groups. They apparently do not migrate through the hindgut endoderm, but occasional PGCs are seen in sites such as the mesonephros, the adrenals, the blood vessels of the yolk sac and in the vicinity of the dorsal aorta and dorsal nerve cord. Between day 23 and day 25, 1 day before birth, most of the 3200–4000 PGCs complete their migration to the gonadal ridges. Although there are marked differences between embryogenesis of tammars and mice, development and the pattern of migration of PGCs in this marsupial mammal are similar to that of eutherian mammals.