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.

Utilisation of aliphatic compounds by acidophilic heterotrophic bacteria. The potential for bioremediation of acidic wastewaters contaminated with toxic organic compounds and heavy metals.

Acidophilic, heterotrophic bacteria isolated from acidic mine effluent metabolised a range of aliphatic compounds. Aliphatic acids, which are normally thought to be toxic to acidophiles, were utilised as substrates for energy and growth by these bacteria. This biodegradative ability, concomitant with their tolerance of heavy metals, has demonstrated the potential for using these organisms for the bioremediation of multiply contaminated acidic wastewaters.

Mesotocin in the brain and plasma of an Australian marsupial, the brushtail possum (Trichosurus vulpecula).

Oxytocic peptides extracted from the brain and plasma of the brushtail possum, Trichosurus vulpecula, were separated by reverse-phase high pressure lipid chromatography (HPLC) and quantified by specific radioimmunoassays for oxytocin (OT) and mesotocin (MT). The pituitary, hypothalamus and cerebral cortex were found to contain MT only in quantities of 3.9 +/- 0.2 (SE) ug, 17.6 +/- 0.6 ng and 21.0 +/- 2.6 ng respectively. The plasma concentration of MT varied according to the degree of stress of the possum. In anaesthetized animals values of 39.7 +/- 9.7 pg/ml (11 males) and 31.5 +/- 12.9 pg/ml (6 females) were obtained; in four conscious catheterized animals, 9.4 +/- 6.3 pg/ml. Samples taken from three anaesthetized animals during exsanguination contained 271 +/- 102 (SD) pg MT/ml. It was concluded that hypothalamic and extra-hypothalamic MT is present in the marsupial brain and that as in placental mammals, stress stimulates the secretion of mesotocin.

Postnatal development of the skin of the marsupial native cat Dasyurus hallucatus.

Skin development of the Northern native cat was examined from birth to weaning at 150 days post partum. An outer layer of cells, termed the periderm or epitrichium, is present on the epidermis of the newborn. This layer of cells is not discernible at 7 days post partum. Skin development of the native cat differs from that of the eutherian mammal. The periderm of the eutherian is no longer discernible when the developing hairs first penetrate the epidermis. In the marsupial, this loss of the periderm occurs well before the appearance of follicles. Melanocytes and Langerhans cells are seen at day 23 post partum, follicles at day 30, sebaceous glands at day 59, and sweat glands at day 67. Thus, when the mother first leaves her young in the nest at about days 60 to 70 of lactation, the skin is at a stage of development that will assist the young with thermoregulation. The skin continues to develop throughout lactation and attains an adult appearance by day 150 post partum.

The ultrastructure of the pituitary and the adrenal gland of three newborn marsupials (Dasyurus hallucatus, Trichosurus vulpecula and Isoodon macrourus).

The ultrastructure of the pituitary glands of the newborn northern native cat, brushtail possum and the northern brown bandicoot and the adrenal glands of the former two marsupials were examined to determine whether these endocrine glands were functional at birth. The anterior pituitary of all three species was well vascularised and many cells contained electron-dense, membrane bound granules. The adrenal glands were composed of two distinct cell populations. One group of cells possessed dark staining granules, normally observed in catecholamine-secreting cells, and the second group contained large amounts of smooth endoplasmic reticulum and mitochondria with tubulovesicular cristae, indicative of steroid hormone secreting cells. The ultrastructure of the cells of the fetal pituitary and adrenal of all three species would suggest that these glands are functional at birth and, as with many eutherians, the marsupial pituitary and adrenal gland may play an important role in determining the length of gestation.

Ultrastructural identification of Merkel cells around the mouth of the newborn marsupial.

The ultrastructure of the epidermal cells surrounding the mouth of three newborn marsupial species, the Northern native cat Dasyurus hallucatus, the brush tail possum Trichosurus vulpecula and the Northern brown bandicoot Isoodon macrourus were examined. The presence of Merkel cells, highly sensitive touch receptors, would suggest that the sense of touch aids the relatively underdeveloped newborn marsupial to move from the urinogenital sinus to the pouch and to locate the teat.

A comparative study of the corpus luteum.

The corpus luteum (CL) is a transitory organ which has a regulatory role in reproduction. Sharks, amphibians and reptiles have corpora lutea that produce progesterone which influences the rate of embryonic development. The egg-laying monotremes and the two major mammalian groups, eutherian and marsupial, have a CL that secretes progesterone. Most eutherians have allowed for the uterine development of their young by extending the length of the oestrous cycle and the CL or placenta actively secretes progesterone until birth. Gestation in the marsupial does not extend beyond the length of an oestrous cycle and the major part of fetal development takes place in the pouch. Where the extension of the post-luteal phase in the eutherian has allowed for the uterine development of young, the marsupial has extended the pre-luteal phase of the oestrous cycle and has evolved an alternative reproductive strategy, embryonic diapause. The mechanism for the secretion of hormones from the CL has been controversial for many years. Densely-staining secretory granules have been observed in the CL of sharks, marsupials and eutherians. These granules have been reported to contain relaxin, oxytocin or mesotocin, and progesterone. A hypothesis to suit all available data is that all hormones secreted by the CL are transported within such granules. In conclusion, although there are obvious differences in the mode of reproduction in the two main mammalian groups, it is apparent that there is a great deal of similarity in the hormonal control of regression of the CL and parturition.

Ontogeny of thyroid hormone receptors in the brushtail possum (Trichosurus vulpecula).

Newborn marsupials do not have a thyroid gland at birth. The gland develops while the young marsupial is in the mother's pouch. The young brushtail possum initiates secretion of thyroid hormones from its own thyroid at about Day 65 post partum. However, during the first three weeks of pouch life thyroxine is passed from the mother to the young via the milk. To determine if this maternal thyroxine can effect organ development in the young possum before it initiates secretion of thyroxine from its own thyroid, the ontogeny of thyroid hormone receptors was determined in nuclear extracts of lung, liver and kidney by radioreceptor assay, using (125)I-labelled tri-iodothyronine as ligand. Receptor density was calculated for tissues removed from young possums at Days 25 (n = 5), 50 (n = 4), 100 (n = 3) and 150 (n = 4) and from adults (n = 5). Receptors were found in possums of all age groups, including the small 25-day pouch young. Significant differences were not found in the receptor density between different tissues or at various ages. The association constant Ka (4.0+/-2.6 L nmol[-1] for lung) was similar in different tissues and at the various ages examined. The passage of thyroid hormones from the mother to the developing marsupial via the milk may have a role in the slow development of organ systems early in pouch life by acting on thyroid receptors in the pouch young. However, the functional maturation of the thyroid gland of the young possum, not an increase in receptors, appears to coincide with the rapid increase in the rate of growth and development which occurs in later pouch life.

Plasma concentrations of thyroxine and growth hormone in the developing marsupial bandicoot, Isoodon macrourus.

Plasma concentrations of thyroxine (T4) and growth hormone (GH) were measured in the developing bandicoot from 21 through to 69 days post partum. Thyroxine concentrations increased from 7 ng mL(-1) at 21 days post partum to reach a maximum of around 40 ng mL(-1) approximately 40 days post partum. After this time, plasma T4 concentrations decreased until about 50 days post partum, when levels were not different from those in the adult (9.2 +/- 0.7 ng mL(-1), n = 10). In contrast, GH concentrations were greatest early in pouch life, with a maximum concentration of 88.2 ng mL(-1) at 24 days post partum, and thereafter declined to adult levels (4.9 +/- 0.9 ng mL(-1), n = 7) by about 60 days post partum. The temporal relationship between T4 and GH in the developing bandicoot is similar to that seen in developing eutherian mammals, but in the latter species, peak plasma T4 and the decline in GH occur before or soon after parturition, whereas in the bandicoot these events occur more than one month post partum. This comparison between eutherian mammals and a marsupial indicates that the timing of these endocrine changes correlates with key developmental or maturational changes rather than the time of parturition.

Comparative aspects of oxytocin-like hormones in marsupials.

Unlike eutherian mammals which secrete only oxytocin (OT), marsupials secrete the typically reptilian mesotocin (MT) and/or OT as their oxytocic peptides. Our laboratories have been conducting research on various aspects of the roles of OT-like peptides in three marsupials, the brushtail possum, the northern brown bandicoot and the tammar wallaby. By providing information on the functions of OT-like peptides in these species we hope to provide some clues as to the evolution of neurohypophysial hormones in marsupials. Brain and peripheral distributions of OT-like peptides have been studied in the possum and bandicoot. As in eutherian mammals, OT-like peptides are distributed throughout the brain and are present in the testis, corpus luteum, prostate and adrenal glands. Studied on the regulation of release of MT into plasma in the possum show that it is regulated by similar mechanisms to OT release in eutherian mammals. OT receptors have been characterized and localized in the possum and the tissue distributions and pharmacological characteristics of the receptor are similar to both the sheep and rat OT receptors. The marsupial OT receptor shows no pharmacological specificity for MT over OT which is reflected in the similar potency of these peptides in eliciting contractions of the uterus of the tammar wallaby in vitro. MT seems to play an important but not essential role in parturition in the tammar. MT concentrations are increased immediately after delivery in the tammar but infusion of an OT antagonist before expected birth delays but does not prevent parturition. The presence of OT receptors in the marsupial mammary gland and the sensitivity of the gland to exogenous OT and stimulation of mesotocinergic neurones demonstrates that these peptides are important for marsupial lactation. Our data suggest that the presence of MT with or without OT in marsupials is a result of a neutral mutation rather than functional evolution.

Some effects of tick infestations on juvenile northern brown bandicoot (Isoodon macrourus).

The effect of tick infestations on body weight and various blood parameters was monitored in juvenile northern brown bandicoots (Isoodon macrourus) after release into tick-infested or tick-free enclosures. Three species of ticks were observed in the enclosures, Haemaphysalis humerosa, Ixodes tasmani and Ixodes holocyclus. Bandicoots released into tick-infested enclosures showed a reduced growth rate (1.8 versus 2.5 g/day increase in body weight), a reduced haematocrit value (27.4 versus 40.0%) and an increased number of white blood cells when compared with bandicoots released into tick-free enclosures. These results suggest that tick infestations may influence the health of juvenile I. macrourus.