Ultrastructural and biochemical evidence for a steroid-containing secretory organelle in the perfused cat adrenal gland.

A correlative study of the ultrastructural and biochemical effects of ACTH on fasciculata cells was carried out on the isolated cat adrenal gland perfused in situ with Locke's solution. The outstanding morphologic feature of cortical cells exposed to microunit ACTH concentrations for 40 min was the abundance of electron-dense granules (0.2-0.4 mum). These organelles were observed in small groups in close proximity to the Golgi region and to the cell membrane. Morphometric and biochemical analysis of control and ACTH-treated glands demonstrated that ACTH stimulation was associated with a fourfold increase in the number of these granules and a comparable increase in the corticosteroid content of the gland. By contrast, ACTH failed to augment cortical lysosomal enzyme activity. These findings, which link steroid release to the appearance of intracellular granules, extend further the parallels between the mechanism of release of newly synthesized steroid and the release of preformed hormones stored in secretory organelles. These results also lend support to the concept that a process related to exocytosis may be the underlying mechanism for extruding steroid from the cortical cell.

Search for a progesterone-binding protein in secretory granules of the ovine corpus luteum.

The hypothesis that electron-dense granules present in the corpus luteum contain progesterone in a protein-bound form was examined using differential and sucrose gradient centrifugation. Ovine luteal tissue was fractionated by differential centrifugation at 1,000 X g (P1 pellet), 10,000 X g (P2), and 82,000 X g (P3; supernatant S3). Samples of P2, P3, and S3 were further fractionated o 20-40% (P2 and P3) or 5-25% (S3) sucrose gradients and examined for progesterone-binding activity by measuring the progesterone content and/or the specific binding of [3H]progesterone of sucrose gradient samples. In addition, saturation binding assays were performed with steroid-free samples of P2 and S3. Saturable binding of progesterone was not found in P2, the fraction containing electron-dense granules. In S3, two progesterone-binding proteins with sedimentation rates of 3.2S and 8.6S and an affinity of 7.1 X 10(5) M-1 for progesterone were detected. The sedimentation behavior of these proteins was distinct from that of ovine plasma transcortin, a 4S protein. The view that a binding protein is released into the interstitial fluid during the exocytosis of granules was examined by measuring the progesterone-binding activity of protein released by slices of corpus luteum in vitro. No binding activity was found. The results of this investigation do not support the hypothesis that putative progesterone-secreting granules observed in luteal tissue contain a binding protein

Plasma concentrations of progesterone, oestradiol-17 beta and 13,14-dihydro-15-oxo-prostaglandin F2 alpha in the pregnant wallaby (Macropus rufogriseus rufogriseus).

The concentrations of progesterone and oestradiol-17 beta in the maternal plasma of Bennett's wallaby, Macropus rufogriseus rufogriseus, were measured daily throughout gestation after reactivation of the diapausing corpus luteum by removal of the suckling pouch young (RPY). Progesterone increased from mean concentrations of 382-424 pmol/l (120-133 pg/ml) during lactation to reach peak concentrations of 908 +/- 172 (S.E.M.) pmol/l (285 +/- 54 pg/ml) (n = 8) 4 days after RPY and 971 +/- 220 and 971 +/- 229 pmol/l (305 +/- 69 and 305 +/- 72 pg/ml) (n = 7) 24 and 25 days after RPY respectively. The mean gestation length (RPY to birth) was 26.8 +/- 0.6 (S.D.) days (n = 6, range 25.75-27.50 days). Immediately after birth the plasma progesterone concentration declined to 299 +/- 51 (S.E.M.) pmol/l (94 +/- 16 pg/ml) (n = 6). Oestradiol-17 beta increased from mean concentrations of 291-553 pmol/l (80-152 pg/ml) during lactation to reach a peak concentration of 967 +/- 331 pmol/l (266 +/- 91 pg/ml) (n = 9) 1 day after RPY. The concentration declined from 7 days after RPY and fluctuated between mean concentrations of 273 and 480 pmol/l before reaching a minimum of 207 +/- 69 pmol/l (57 +/- 19 pg/ml) (n = 6) 19 days after RPY. A transient increase to 542 +/- 207 pmol/l (n = 7) occurred at 22 days after RPY. Plasma concentrations declined to a low of 156 +/- 55 pmol/l (43 +/- 15 pg/ml) (n = 6) 5 days after parturition. The mean concentration of plasma 13,14-dihydro-15-oxo-prostaglandin F2 alpha was less than 2.8 nmol/l (1 ng/ml) for all samples from 13 days after RPY until 4 days after parturition. The results suggest that oestradiol-17 beta may be important in the early stages of blastocyst reactivation to synergize with progesterone in stimulating uterine secretions. 13,14-Dihydro-15-oxo-prostaglandin F2 alpha is unlikely to be involved in the birth process and any luteolytic effect is likely to be from a local production of PGF2 alpha.

Oxytocin receptors in the ovine corpus luteum.

There is inconclusive evidence that oxytocin acts directly on the corpus luteum and affects steroidogenesis. Since any such action would probably be mediated by oxytocin receptors, these should be present in luteal tissue. In this study, homogenates of corpora lutea from both pregnant and non-pregnant ewes were examined for oxytocin receptors by radioreceptor assay. Specific oxytocin binding was not observed in luteal tissue during the oestrous cycle. However specific binding was found in the corpora lutea of pregnant ewes; appearing at a fetal head length of approximately 0.65 cm (about 30 days of pregnancy) and persisting to a head size of 11 cm, the largest size examined in this study. The affinity (Kd) of the receptor was calculated as 2.9 +/- 0.3 nmol/l (S.E.M.; n = 9), a value similar to that obtained for the uterus. The receptor number ranged from a low of 8.7 +/- 3.2 fmol/mg protein (n = 6) at a head size of less than 0.65 cm, to a maximum of 40.1 +/- 6.5 fmol/mg protein (n = 25) at a head size of 2.5-3.75 cm. These values were lower than our estimate of 588 +/- 39 fmol/mg protein (n = 5) for the uterus. It is concluded that a direct action of oxytocin on the corpus luteum is possible but only after the first month of pregnancy and not in the corpus luteum of the oestrous cycle.

Induction of transient functional luteolysis in cyclic sheep by a 3 beta-hydroxysteroid dehydrogenase inhibitor.

The mechanism by which prostaglandin F2 alpha terminates 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 3 beta-hydroxysteroid dehydrogenase (3 beta-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 3 beta-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 F2 alpha during 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 4h 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 3 beta-HSD activity, per se, is ineffective in bringing about structural luteolysis.

The GH/IGF-I axis in the brushtail possum (Trichosurus vulpecula) pouch young.

Plasma and pituitary GH concentrations and liver GH receptor (GHR), IGF-I and IGF-binding protein-3 (IGFBP-3) mRNA expression were determined in brushtail possum (Trichosurus vulpecula) pouch young aged 12-150 days post-partum and in adults. Mean plasma GH concentrations were highest, measuring around 150 ng/ml, from 12 to 100 days post-partum, and thereafter declined so that by 150 days post-partum levels were not significantly different from those in adults (10.8+/-1.8 ng/ml (S.E.M.)). In contrast to plasma levels, pituitary GH content increased markedly throughout pouch life, with an 87-fold increase between 12 and 150 days post-partum. However, when expressed per gram body weight, pituitary content was relatively constant between 25 and 150 days post-partum, indicating that the decline in plasma GH after 100 days post-partum was not due to decreased synthesis and/or storage of GH in the pituitary gland. Expression of GHR, IGF-I and IGFBP-3 mRNAs was determined by semi-quantitative RT-PCR. Liver GHR and IGF-I mRNA expression were low at 12 and 25 days post-partum and did not show sustained and significant increases (P<0.05) until 125 and 150 days post-partum. IGFBP-3 expression was also low at 12 days post-partum but then increased rapidly to a maximum at 50 days post-partum and thereafter declined. For all three mRNAs, liver expression at day 150 was not significantly different from that in adults. These patterns of gene expression for GHR and IGF-I suggest that the possum liver is resistant to the high plasma GH concentrations during early pouch life and in this way is similar to the fetal liver of some eutherian mammals.

Plasma growth hormone and growth hormone-binding protein during development in the marsupial brushtail possum (Trichosurus vulpecula).

Plasma concentrations of growth hormone (GH) were measured in the brushtail possum (Trichosurus vulpecula) pouch young from 25 through to 198 days post-partum (n=71). GH concentrations were highest early in pouch life (around 100 ng/ml), and thereafter declined in an exponential fashion to reach adult concentrations (10.8+/-1.8 ng/ml; n=21) by approximately 121-145 days post-partum, one to two months before the young is weaned. Growth hormone-binding protein (GHBP), which has been shown to modify the cellular actions of GH in eutherian mammals, was identified for the first time in a marsupial. Based on size exclusion gel filtration, possum GHBP had an estimated molecular mass of approximately 65 kDa, similar to that identified in other mammalian species, and binding of (125)I-labelled human GH (hGH) was displaced by excess hGH (20 microg). An immunoprecipitation method, in which plasma GHBP was rendered polyethylene glycol precipitable with a monoclonal antibody to the rabbit GHBP/GH receptor (MAb 43) and labelled with (125)I-hGH, was used to quantitate plasma GHBP by Scatchard analysis in the developing (pooled plasma samples) and adult (individual animals) possums. Binding affinity (K(a)) values in pouch young aged between 45 and 54 and 144 and 153 days post-partum varied between 1.0 and 2.4 x 10(9)/M, which was slightly higher than that in adult plasma (0.96+/-0.2 x 10(9)/M, n=6). Binding capacity (B(max)) values increased from non-detectable levels in animals aged 25-38 days post-partum to reach concentrations around half that seen in the adult (1.4+/-0.2 x 10(-9) M) by about 117 days post-partum and remained at this level until 153 days post-partum. Therefore, in early pouch life when plasma GH concentrations are highest, the very low concentrations of GHBP are unlikely to be important in terms of competing with GH-receptor for ligand or altering the half-life of circulating GH.

Absorption of 125I-labelled sheep growth hormone in single proximal tubules of the rat kidney.

Techniques of kidney micropuncture and electron microscope autoradiography have been used to study the uptake of 125I-labelled sheep growth hormone (GH) in rat renal proximal tubules. After microperfusion of a proximal tubule with 125I-labelled GH, the transport of label by the tubular epithelium was studied autoradiographically at selected times up to 1 h. The sequential transfer of labelled material from tubule to microvilli, then to small and large apical vacuoles and finally to lysosomes followed the pattern of absorption that has been described for other proteins. Evidence of lysosomal degradation of the transported protein was obtained from studies in vitro; lysosomes isolated from the renal cortex rapidly converted 125I-labelled GH to products of lower molecular weight. In addition to the absorptive pathway through the intracellular vacuolar apparatus is appeared that there was also an alternative pathway, less well defined, whereby GH could be absorbed without being degraded.

Experimental infection of Australian brushtail possums, Trichosurus vulpecula (Phalangeridae: Marsupialia), with Ross River and Barmah Forest viruses by use of a natural mosquito vector system.

Brushtail possums, Trichosurus vulpecula Kerr, were experimentally infected with Ross River (RR) or Barmah Forest (BF) virus by Aedes vigilax (Skuse) mosquitoes. Eight of 10 animals exposed to RR virus developed neutralizing antibody, and 3 possums developed high viremia for < 48 hr after infection, sufficient to infect recipient mosquitoes. Two of 10 animals exposed to BF virus developed neutralizing antibody. Both infected possums maintained detectable neutralizing antibody to BF for at least 45 days after infection (log neutralization index > 2.0 at 45 days). Eight possums did not develop neutralizing antibody to BF despite exposure to infected mosquitoes. These results suggest that T. vulpecula may potentially act as a reservoir species for RR in urban areas. However, T. vulpecula infected with BF do not develop viremia sufficient to infect mosquitoes and are unlikely to be important hosts for BF.

Arginine vasopressin- and oxytocin-like peptides in the testis of two Australian marsupials.

High performance liquid chromatography (HPLC) and specific radioimmunoassay (RIA) for arginine vasopressin (AVP), mesotocin (MT), and oxytocin (OT) were used to identify and quantify these peptides in the testis of the brushtail possum (Trichosurus vulpecula) and the northern brown bandicoot (Isoodon macrourus). Arginine vasopressin (0.092 +/- 0.041 ng/g) and MT (0.198 +/- 0.089 ng/g), but not OT, were found in the possum testis, while the bandicoot testis contained AVP (0.061 ng/g), MT (0.108 +/- 0.024 ng/g), and OT (0.114 +/- 0.053 ng/g). The values correlate well with those reported for AVP- and OT-like peptides in the testis of eutherian mammals. It was concluded that there are neurohypophysial peptides present in the marsupial testis.