Maturation of the growth axis in marsupials occurs gradually during post-natal life and over an equivalent developmental stage relative to eutherian species.

The separation of a nutrition-responsive insulin-like growth factor (IGF) system and a growth hormone (GH) responsive IGF system to control pre- and post-natal growth of developing mammals may originate from the constraints imposed by intra-uterine development. In eutherian species that deliver relatively precocial young, maturation of the GH regulatory system is coincident with the time of birth. We measured the hepatic expression of the four key growth axis genes GH-receptor, IGF-1 and -2, and IGFBBP-3, and plasma protein concentrations of IGF-1 from late fetal life through to adult stages of a marsupial, the tammar wallaby. The data clearly show that maturation of GH-regulated growth in marsupials occurs gradually over the course of post-natal life at an equivalent developmental stage to that of precocial eutherian mammals. This suggests that the timing of GH-regulated growth in marsupials is not related to parturition but instead to the relative developmental stage.

Early onset of ghrelin production in a marsupial.

Ghrelin regulates appetite in mammals and can stimulate growth hormone (GH) release from the pituitary. In rats and humans, ghrelin cells appear in the stomach during late fetal life. Nevertheless, the role of ghrelin in early mammalian development is not well understood. Marsupials deliver highly altricial young that weigh less than 1g so they must feed and digest milk at a comparatively immature stage of development. Since they complete their growth and differentiation while in the pouch, they are accessible models in which to determine the time course of ghrelin production during development. We examined the distribution of gastric ghrelin cells, plasma ghrelin concentrations and pituitary expression of the ghrelin receptor (ghsr-1alpha) and GH in the tammar wallaby, Macropus eugenii. There were ghrelin immunopositive cells in the developing mesenchyme of the stomach from day 10 post partum (pp) to day 150pp. Subsequently ghrelin protein in the fore-stomach declined and was absent by day 250pp but remained in the gastric cells of the hind-stomach. Ghrelin was detected in the developing pancreas from day 10pp but was absent by day 150pp and in the adult. Pituitary ghsr-1alpha expression and plasma concentrations of ghrelin increased significantly up to day 70-120pp while GH expression was also elevated, declining with GH to reach adult levels by day 180pp. These results demonstrate an early onset of gastric ghrelin expression in the tammar in concert with a functional stomach at a relatively earlier stage than that of developmentally more mature eutherian young.

Exon 3 of the growth hormone receptor (GH-R) is specific to eutherian mammals.

Growth hormone receptor (GH-R) plays a critical role in the control of growth and metabolism in all vertebrates. GH-R consists of 9 coding exons (2-10) in all eutherian mammals, while the chicken only has 8 coding exons, and does not have an orthologous region to exon 3 in eutherians. To further understand the evolutionary origins of exon 3 of the GH-R in eutherians we cloned the full-length GH-R sequence in a marsupial, the tammar wallaby to determine whether exon 3 was present or absent in marsupial liver cDNA. There was no evidence for the presence of an exon 3 containing mRNA in sequence of tammar pouch young and adult livers. We next examined the genomes of the platypus (a monotreme mammal) and the grey short-tailed opossum (another marsupial). Like the tammar, the GH-R gene of neither species contained an exon 3. GH receptor can obviously function in the absence of this exon, raising speculation about the function of this domain, if any, in eutherians. A comparison of exon 3 protein sequences within 16 species of eutherian mammals showed that there was approximately 75% homology in the domain but only 3 of the 21 amino acids were identical (Leu12, Gln13 and Pro17). Interestingly, we detected greater evolutionary divergence in exon 3 sequences from species that have variants of GH or prolactin (PRL) in their placentas. These data show that exon 3 was inserted into the GH-R after the divergence of the marsupial and eutherian lineages at least 130 million years ago.

Perturbed growth and development in marsupial young after reciprocal cross-fostering between species.

Cross-fostering of marsupial young between species can potentially facilitate propagation of endangered or rare marsupial species by artificially increasing the number of progeny produced. The present study compares the growth and development of normal and cross-fostered tammar and parma wallabies. Tammars cross-fostered into the pouches of parmas grew at a similar rate to naturally reared tammar young and had developmental milestones at a similar age. However, parma young cross-fostered between the day of birth and 15 days post-partum into tammars that were carrying young of equivalent developmental stages did not grow normally and were lost from the pouch. Parma young cross-fostered at 30 days survived, but had significantly reduced growth rates and their developmental milestones were delayed compared with normally reared parma young. Thus, growth can be affected by cross-fostering, even between species like tammars and parmas that are of similar size and have similar lactation lengths. The results of the present study suggest that maternal milk regulates the timing of development of each species and a mis-match in the time that each young receives critical milk components can have a marked effect on their growth and development.

Cycle of the seminiferous epithelium in a marsupial species, the brushtail possum (Trichosurus vulpecula), and estimation of its duration.

We investigated the cycle of the seminiferous epithelium in a marsupial, namely the brushtail possum (Trichosurus vulpecula), using semithin sections of seminiferous tubules embedded in Spurr's resin. Using 14 steps of spermatid development as markers, we were able to class tubular cross-sections into 10 well-defined stages of the seminiferous epithelial cycle. The duration of one cycle was 13.5 days, as determined by injections of [(3)H]-thymidine and autoradiographic examination of the most advanced sperm cells at 2 h and 17 days after injection. The durations of stages I-X were 21.4, 66.4, 54.1, 47.0, 29.8, 28.5, 25.3, 25.0, 12.0 and 15.9 h, respectively, estimated by the relative percentage of occurrence of each stage. It was estimated that the life spans of the main germ cells were as follows: type B spermatogonia, 5.4 days; primary spermatocytes, 16.7 days; secondary spermatocytes, 0.7 days; and spermatids, 21.4 days. The results suggest that the kinetics of spermatogenesis in marsupials show a similar pattern to that in eutherians.

Hypophyseal-portal somatostatin (SRIH) and jugular venous growth hormone secretion in the conscious unrestrained ewe.

Somatostatin (SRIH) release into hypophyseal portal blood varies reciprocally with growth hormone (GH) pulse generation in the male rat. However, few studies have directly evaluated this relationship in the female of any species. To address this issue, we carried out intensive (5 min) and extended (240 min) simultaneous monitoring of hypophyseal portal SRIH and internal jugular GH secretion in 7 unanesthetized ewes. Bihormonal synchrony was assessed by three statistically independent but complementary analyses: (i) cross-approximate entropy (X-ApEn) analysis to appraise the conditional regularity of SRIH/GH release patterns; (ii) cross-correlation analysis of paired sample SRIH and GH release rates, and (iii) probability analysis of random versus nonrandom SRIH and GH discrete pulse concordance. From a one-variable perspective, ApEn analysis documented consistently more irregular patterns of SRIH than GH release (94 +/- 4.3 and 72 +/- 8.1%, respectively, of the mean irregularity of 1,000 individual random-shuffled cognate series, p = 0.034). From a two-variable perspective, X-ApEn analysis revealed a nearly mean random relationship between SRIH and GH release patterns (group mean +/- SEM, 94 +/- 4.5% of the mean asynchrony of 1,000 randomly shuffled SRIH/GH pairs). Cross-correlation analysis disclosed highly variable linkages between SRIH and GH secretion; viz, negative cross-correlations in 5 sheep, positive relationships in 4, and both positive and negative SRIH/GH associations in 2 animals, wherein changes in SRIH secretion either preceded or followed those of GH. Peak detection by model-free cluster analysis quantified a total of 28 SRIH and 31 GH release episodes. Corresponding interpulse intervals (min) were comparable (37 +/- 4 (SRIH) and 43 +/- 12 (GH)), but the mean fractional (%) amplitude of SRIH peaks was 3.5-fold lower (60 +/- 10%) than that for GH (225 +/- 50%) (p = 0.024). Pulse-concordance probability testing showed that discrete peaks of SRIH and GH secretion coincided only 33% of the time, although this value exceeded chance expectation (p < 10(-4)). In summary, the present analysis applies intensive (5 min) and extended (240 min) simultaneous sampling of hypophyseal-portal and jugular venous blood to quantitate the degree of coordinate SRIH and GH secretion in the unanesthetized ovariectomized ewe. Thereby, we unmask highly irregular SRIH release patterns, and nearly random SRIH and GH associations. We conclude that, to the extent that in vivo sampling reflects physiological SRIH/somatotrope activity, the female sheep maintains complex time-varying interactions between SRIH and GH release.