The binding affinities and receptor specificity of sheep uterine cytosol for steroid oestrogens and also for weak plant oestrogens of the isoflavone and coumestan groups and some synthetic compounds were studied. The binding affinities of the weak oestrogens fall within a range which has usually been neglected. Relative molar binding (RMB) affinities for the steroid oestrogens confirmed the importance of the phenolic 3-hydroxyl group and the influence of substitutions at C-16 and C-17, as seen with uterine cytosols from other species. Relative molar binding affinities were very much lower when the oestrogens were present as sulphate esters, glucosiduronate and methyl ether derivatives; acetates showed similar RMB affinities to their parent compounds. Phyto-oestrogens were found to compete with oestradiol for binding sites. Coumestrol and miroestrol had the highest RMB affinities of about 5 (oestradiol-17\g=b\ = 100) when incubated at 25 \ s=deg\ C, and values for genistein, equol, daidzein and O-desmethylangolensin lay between 1 and 0\m=.\05.The mono-methoxy compounds, biochanin A, formononetin and 4\m=' \-methoxy\x=req-\ coumestrol had RMB affinities of less than 0\m=.\01.Incubation at 37, 25 and 4 \ s=deg\ Cshowed that RMB affinities were greater at the lower temperatures.Relative molar binding affinities of the phyto-oestrogens in vitro compared with their oestrogenic potencies in vivo showed that the ranking of most of the compounds by these two criteria was similar. Structure-activity correlations were deduced from the results. A similar relationship of RMB affinity to biological potency was also noted for the steroid oestrogens and a homologous series of stilbenediols. The results obtained are relevant to competitive protein-binding analyses and to the mechanism of action of oestrogens and phyto-oestrogens.

Prostaglandin F2\ g=a\ . (PGF2\g=a\) is luteolytic in the ewe when infused into the ovarian artery (Barrett, Blockey, Brown, Cumming, Goding, Mole & Obst, 1971; McCracken, Baird & Goding, 1971) or the ipsilateral uterine vein (Thorburn & Nicol, 1971). These observations, together with the identification of PGF2\g=a\ in the uterine venous plasma of anaesthetized ewes towards the end of the oestrous cycle (Bland, Horton & Poyser, 1971;McCracken et al. 1971), strongly suggest that PGF2\g=a\ is the uterine luteolytic factor in sheep. This paper describes in detail the changes in PGF concentration in utero\p=m-\ovarian venous plasma of conscious ewes during the oestrous cycle.Polyvinyl catheters (1\m=.\5 mm o.d.) were inserted into the right and left utero\p=m-\ ovarian veins via uterine vein branches in two Merino ewes 10 and 11 days after oestrus. Utero\p=m-\ovarian venous samples were collected every 2-3 h from the day after surgery.Concentrations of PGF were measured by a radioimmunoassay (Thorburn, Nicol, Bassett, Shutt & Cox, 1972) using antisera raised in rabbits. Prostaglandin F2a and PGFloe compete in an almost identical manner with [9-3H]PGF2lx over the analytical range (0-1-3-0 ng) of the assay. Prostaglandin E2 competes only weakly and no crossreaction was observed with a wide range of steroids and lipids tested. Plasma samples (0-5 ml) were extracted with chloroform, then acidified and re-extracted. The second chloroform extracts were chromatographed on silica gel columns to separate the PGF group from other prostaglandins. Method blanks were equivalent to 0-2 ng PGF and recoveries averaged 60%. Identity of the measured substances as PGF or PGF-like compounds was based on the selective extraction procedure, Chromatographie puri¬ fication and the specificity of the antibodies to PGF ; also negligible values were ob¬ tained with peripheral plasma. Data were corrected for blank values and procedural losses. Progesterone was measured by the method of Thorburn & Schneider (1972).With frequent sampling we detected a complex series of peaks in the concentration of PGF between days 13 and 17 of the oestrous cycle (Fig. 1). These peaks were of short duration and increased in frequency as oestrus approached. Very low concen¬ trations were measured at other times except on days 2 and 3 in samples from one ewe (3976). In this same ewe, which had a corpus luteum (CL) in each ovary, there was striking similarity in the concentrations of PGF on each side (Fig. 1). Ewe 4854 had a single CL in the right ovary during the first cycle.

found that there was a relatively high concentration of oestradiol-17\g=b\ in ovarian venous plasma for about 2 days before oestrus, whereas the concentration was extremely low on day 1 and, during the luteal phase, up to day 14 (day 0=onset of oestrus). This paper reports the finding of a postovulatory increase in oestradiol secretion on days 3\p=n-\4of the cycle in conscious ewes.Anastomosis of the utero\p=m-\ovarian vein to the mammary vein (Thorburn & Mattner, 1969, 1971 permitted the timed collection of blood samples from the utero\p=m-\ovarian veins of conscious ewes. The animals demonstrated normal oestrous behaviour on mating with vasectomized rams, normal ovulation and normal cyclical changes in plasma progesterone concentration. Oestradiol-17\g=b\ was measured in 5 ml samples of utero\p=m-\ovarian venous plasma by the method of Shutt (1969), modified by using alumina columns in place of paper chromatography. Micro-columns of i.d. 4 mm were packed with 0-3 g alumina with a water content of 5-0 %. Plasma extracts, dissolved in 7 ml benzene, were run onto the columns; oestrone was eluted with 7 ml 1-2% ethanol in hexane, and then oestradiol with 4 ml 5-0 % ethanol in hexane. The coefficient of variation of replicate analyses on plasma was 13%, water blanks were 15 + 3-0 (s.d.) pg and oestradiol recoveries were 72 + 5-7 (s.d.) %. Results were corrected for procedural losses by measuring the recovery of 5 pg [3H]oestradiol added to each plasma sample. Secretion rates were calculated from the blood flow rate, the haematocrit, and the plasma oestradiol concentration, without correction for any distribution between plasma and red cells. Secretion rates were not corrected for the contribution from afferent arterial blood, since the oestradiol concentrations in five peripheral plasma samples collected on days 1 and 3 were insignificant, being less than 8 pg/ml (30 ml samples of plasma were analysed).Oestradiol secretion rates (Fig. 1 a) increased sharply on day 1, tended to decline by early oestrus and decreased rapidly to very low values on days 1 and 2. Frequent sampling in one sheep showed that the increased secretion on day 1 was maintained for less than 24h (Fig. lo).A further major increase in oestradiol secretion occurred on days 3-4 in all of the

This paper documents the successful development of an artificial insemination (AI) programme for the Koala Phascolurctos cinereus. The protocols for trials involving two methods to induce ovulation and two insemination techniques are described. In Trial 1, interrupted coitus using a ‘teaser’♂ successfully induced ovulation in nine Koalas. Five ♀♀ were inseminated while conscious using a modified ‘foley catheter’ (Cook insemination catheter) resulting in the births of two offspring. The other four ♀♀ were anaesthetized and inseminated using a technique which allowed visualization of the most cranial portion of the urogenital sinus, where semen was deposited using a 3.5 Fr. ‘Tom‐cat catheter’ (urogen‐itoscopic insemination). Three of the four ♀♀ inseminated by this technique produced pouch young. Microsatellite analysis of DNA from the pouch young excluded the teaser ♀♀ as possible sires, confirming that all offspring were sired by donor sperm. In Trial 2, eight ♀♀ were induced to ovulate by injecting them with 250 International Units of human chorionic gonadotrophin (hCG). A luteal phase was confirmed in all eight ♀♀ but only one gave birth following urogenitoscopic insemination. The Koala pouch young in this study are the first of any marsupial to be conceived and born following A1 procedures. Details of the A1 procedures used are presented and the significance of A1 to the conservation biology of P. cinereus discussed.

As an integral part of the development of an artificial insemination programme in the captive koala, female reproductive physiology and behaviour were studied. The oestrous cycle in non-mated and mated koalas was characterized by means of behavioural oestrus, morphology of external genitalia and changes in the peripheral plasma concentrations of oestradiol and progestogen. The mean (+/- SEM) duration of the non-mated oestrous cycle and duration of oestrus in 12 koalas was 32.9 +/- 1.1 (n = 22) and 10.3 +/- 0.9 (n = 24) days, respectively. Although the commencement of oestrous behaviour was associated with increasing or high concentrations of oestradiol, there were no consistent changes in the morphology or appearance of the clitoris, pericloacal region, pouch or mammary teats that could be used to characterize the non-mated cycle. As progestogen concentrations remained at basal values throughout the interoestrous period, non-mated cycles were considered non-luteal and presumed anovulatory. After mating of the 12 koalas, six females gave birth with a mean (+/- SEM) gestation of 34.8 +/- 0.3 days, whereas the remaining six non-parturient females returned to oestrus 49.5 +/- 1. 0 days later. After mating, oestrous behaviour ceased and the progestogen profile showed a significant increase in both pregnant and non-parturient females, indicating that a luteal phase had been induced by the physical act of mating. Progestogen concentrations throughout the luteal phase of the pregnant females were significantly higher than those of non-parturient females. Parturition was associated with a decreasing concentration of progestogen, which was increased above that of basal concentrations until 7 days post partum.