THE ANATOMICAL RECORD 237:228-235 (1993) Posterior Pituitary of the Newborn Marsupial Possum, Trichosurus vulpecula R.T. GEMMELL, T. CHUA, R.A.D. BATHGATE, AND C. SERNIA Departments of Anatomical Sciences (R.T.G., T.C.) and Physiology and Pharmacology (R.A.D.B., C.S.), University of Queensland, Brisbane, Australia ABSTRACT The fetal anterior pituitary-adrenal axis is thought to be involved in the initiation of birth in both eutherian and marsupial mammals. Little is known about the structure and function of the posterior pituitary at birth in the marsupial. Immunocytochemistry, high pressure liquid chromatography, and radioimmunoassay were used to identify vasopressin and mesotocin in the posterior pituitary of a newborn marsupial, the brushtail possum, Trichosurus vulpecula. The concentrations of vasopressin and mesotocin in the head of the newborn possum were 0.34 and 0.28 ng, respectively. The concentration of vasopressin was always greater than that of mesotocin, and the amounts of neuropeptides present in the head increased as the possum developed. o 1993 Wiley-Liss, Inc. Key words: Posterior pituitary, Marsupial, Trichosurus vulpecula newborn, Ultrastructure, Immunocytochemistry, Mesotocin, Vasopressin The neurohypophysis of placental mammals secretes the nonapeptide hormones oxytocin and, with the exception of the pig, arginine-vasopressin (Heller, 1974). Both of these hormones as well as mesotocin, a similar peptide to oxytocin, have been reported to be produced in marsupials. In recent studies, mesotocin and vasopressin have been identified in the hypothalamus and pituitary of the brushtail possum (Hurpet et al., 1982; Bathgate et al., 1990). Oxytocin and vasopressin are produced in the hypothalamus and released from the posterior pituitary into the general circulation where their primary roles are in labor, lactation, and water balance. The presence of oxytocin in the ovary (Wathes and Swann, 1982), testis (Guldenaar and Pickering, 1985, Gemmell and Sernia, 1989b1, and adrenal (Hawthorn et al., 1987; Nussey e t al., 1987) suggests a further role for this hormone. Similarly, vasopressin has been identified in testis and adrenal tissue (Nicholson et al., 1984; Hawthorn et al., 1987; Nussey et al., 1987). As previously stated, the primary roles of gonadal oxytocin and vasopressin remain undetermined (Wathes et al., 1984). Suggested roles include stimulation of the smooth muscle in the reproductive tract andlor a n autocrine or paracrine role in the production of steroid hormones (Wathes et al., 1984; Hawthorn et al., 1987; Gemmell and Sernia, 198913). The fetal pituitary-adrenal axis is thought to be involved in the initiation of birth in several eutherian mammals (Liggins et al., 1967; Challis et al., 1977; Thorburn and Challis, 1979). Administration of adrenocorticotrophic hormone to the fetus induces 0 1993 WILEY-LISS, INC birth in various eutherians (Liggins e t al., 1973), suggesting t h a t the anterior region of the pituitary is required for birth. The anterior pituitary and the adrenal glands of various marsupials are capable of secreting hormones a t birth (Gemmell et al., 1982; Gemmell and Nelson, 1988; Gemmell and Rose, 1989), and the pituitary-adrenal axis may play a n important role in controlling the length of gestation in both eutherians and marsupials. Oxytocin and vasopressin are present in the posterior pituitary and hypothalamus of many newborn mammals a long time before birth (Leclerc and Pelletier, 1977). Vasopressin is present in the rat fetus at day 16-17 and oxytocin a t day 20-22 (Whitnall et al., 1985). These neurohormones are synthesised mainly in the supraoptic and paraventricular nuclei of the hypothalamus and released in the posterior pituitary (Laurent et al., 1989). Fetal oxytocin is released during birth and may accelerate labor, whereas fetal vasopressin may regulate fluid balance in the placenta (Swaab et al., 1978). There is no information concerning the functional role of the posterior pituitary in the birth of the marsupial. In this study, the pituitary and hypothalamus of newborn brushtail possums were examined for the Received September 21, 1992; accepted February 18, 1993. Address reprint requests to Dr. R.T. Gemmell, Department of Anatomical Sciences, University of Queensland, St. Lucia, 4072, Brisbane, Queensland, Australia. 229 NEWBORN MARSUPIAL POSTERIOR PITUITARY presence of mesotocin and vasopressin. The presence of these hormones would imply a functional posterior pituitary in the newborn marsupial and a possible role for these hormones in parturition. MATERIALS AND METHODS Morphological Examination TABLE 1. Body weight, concentration of mesotocin and vasopressin within the head of the possum, Trichosurus vulpecula from newborn until day 32 postpartum Days post partum Weight (gms) Mesotocin (ngms) Vasopressin (ngms) Pituitaries from the young of six possums were ex1 0.2 0.26 0.34 2 0.3 0.08 0.44 amined by light and electron microscopy. Two new2 0.3 0 0.20 born, two young at day 3 and two young at day 8 post0.19 3 0.4 0.73 partum, were killed by decapitation and fixed by 0.10 3 0.4 0.38 immersion in 2% paraformaldehyde and 1.25% glutar0.5 0.16 4 0.69 aldehyde in 0.06 M cacodylate buffer, pH 7.2 for 5 min0.5 4 0.01 0.20 utes, followed by immersion in 2% paraformaldehyde 5 0.6 0.04 0.29 and 4% glutaraldehyde in 0.06 M cacodylate buffer, pH 6 0.7 1.95 1.38 7.2, overnight. The heads were subsequently washed 1.1 0.05 0.20 7 for several hours in 0.01 M cacodylate buffer. All tis- 10 1.3 2.80 5.78 1.4 0.94 1.45 sues were postfixed in 1% osmic acid in 0.06 M cacody- 11 3.5 4.90 13.20 late buffer, pH 7.2 for 2 hours, dehydrated, and embed- 23 3.8 3.23 3.64 ded in AralditeIEpon mixture (20 ml Epon 812, 10 ml 24 5.4 7.08 14.82 Araldite, 21 ml DDSA, 12 ml NMA, and 1.0 ml DMP- 27 7.1 4.07 13.26 30, Bio-Rad, Cambridge, UK). Sections 1 pm thick 32 were stained with Azur A and examined with the light microscope. Thinner sections, 50 nanometers thick, were stained sequentially with uranyl acetate and lead citrate and examined in a Zeiss 10 transmission elec- ish peroxidase complex (Amersham, UK, RPN 1051, lot tron microscope. .60), diluted 1 : l O O with PBS for 2 hours at 20°C. The sections were then washed in PBS and incubated in 25 mg 3,3-diaminobenzidine in 50 ml PBS containing 50 lrnrnunocytochemical Localisation of Neuropeptides pl of 10% hydrogen peroxide for 15 minutes at 20°C, before being washed in PBS, dehydrated, and mounted Vasopressin and mesotocin were localised within the in DPX. heads of the young of 16 possums; two newborn, two Sections of adult possum hypothalamus were used as young at day 3, and two young at day 8, and one young positive controls for mesotocin and vasopressin. Coneach at days 11, 12, 13, 14, 19, 22, 25, 54, 67, and 76, trol sections were stained with antisera that were repostpartum. The heads were immersed and fixed over- acted with the corresponding antigen (500 pg/ml for 24 night in 4% paraformaldehyde in 0.1 M phosphate hours at 4°C) prior to use in the staining reaction. Debuffer. The tissues were subsequently washed in phos- tails of this method have been published previously phate-buffered saline (PBS), dehydrated, and embed- (Gemmell and Sernia, 1989a). ded in paraffin wax. Sections 7 pm thick were obtained. Primary antibodies to mesotocin were raised in sheep (SM-42) and those to arginine-vasopressin in Identification of Neuropeptides with rabbits (RA-717). The cross-reactivity of SM-42 with High Pressure Liquid Chromatography isotocin, oxytocin, vasotocin, and vasopressin was 13,1, and Radioimrnunoassay 0.1, and 0.1%, respectively. The cross-reactivity of RA717 to lysine vasopressin was 40%, and 0% to deaminoVasopressin and mesotocin were identified within oxytocin, oxytocin, mesotocin, and isotocin. Details of these antibodies have been published previously (Gem- the heads of the young of 16 possums; a newborn, 2 young at days 2 and 3, and one young at days 4 , 5 , 6 , 7 , me11 and Sernia, 1989a,b; Bathgate et al., 1992a,b). The immunocytochemical technique used in this 10,11,23,24,27, and 32 postpartum and a newborn rat study was basically that of Sternberger (1979) with (in which oxytocin was measured a s mesotocin is not minor modifications. After removal of paraffin, the sec- present in the rat). The young possums were killed by tions were hydrated through descending concentra- decapitation. Extracts of possum brains were separated with high tions of alcohol and washed in PBS. They were treated first with the primary antiserum, diluted 1:500 with pressure liquid chromatography (HPLC) for mesotocin PBS, for 24 hours at 4°C. Sections were then washed and vasopressin measurement by highly specific radiowith PBS and incubated with the secondary antibody, immunoassay (RIA). The procedure for HPLC is a modbiotinylated donkey antisheeplgoat immunoglobulin ification of that of Bathgate et al. (1990).Peptides were (Amersham, UK, RPN 1025, lot 8) following the meso- separated on a 15-35% acetonitrile gradient in 0.1% tocin antibody, and biotinylated donkey antirabbit im- trifluoroacetic acid on a Phenomenex maxsil 10 C-18 munoglobulin (Amersham, UK, RPN 1004, lot 18) fol- reverse-phase HPLC column (250 mm x 4.6 mm, Torlowing the vasopressin antibody. Both secondary rance, CA). The recovery from this procedure was 72%, antibodies were used at a dilution of 1:lOO for 2 hours and all results were adjusted accordingly. RIA for meat 20°C. All sections were again washed with PBS and sotocin and vasopressin have been described previously were incubated in streptavidin-biotinylated horserad- (Bathgate et al., 1992a,b). 230 R.T. GEMMELL E T AL. Figs. 1-3 NEWBORN MARSUPIAL POSTERIOR PITUITARY 231 Immunocytochemical staining of vasopressin (Fig. 8) and mesotocin was illustrated as dark-staining strands Mesotocin and vasopressin were present within the throughout the posterior pituitary. The nerve termiposterior pituitary of the newborn possum. The concen- nals, which contained dense staining granules observed tration of vasopressin present in the head of the new- in the posterior pituitary of the newborn, were now born was 0.34 ng, which was far less than the 5.0 ng present throughout this region of the pituitary (Fig. 9). Vasopressin and mesotocin were first located in cells found in the head of the newborn rat. A concentration of 4.14 ng of oxytocin was present in the head of the rat, of the presumptive hypothalamus on day 14 postparwhereas 0.28 ng of mesotocin was present in the new- tum, in the region of the supraoptic nucleus. This celborn possum. A higher amount of vasopressin than me- lular staining became more visible and localised from sotocin was always found and the amounts of neuro- day 19 through to day 76 postpartum, when the immupeptides increased as the possum developed (Table 1). nocytochemical localisation of both neuropeptides was The posterior pituitary of the brushtail possum was similar to that reported in the adult possum (Gemmell just forming in the newborn. It was seen as a projection and Sernia, 1989a). from the diencephalon, forming a depression in the roof DISCUSSION of the anterior pituitary. The posterior pituitary was separated from the anterior pituitary by the intermeThe posterior pituitary of the newborn brushtail posdiate part and a cleft between the intermediate and the anterior region (Fig. 1).Examination of the posterior sum contains both mesotocin and vasopressin. Immupituitary with the electron microscope revealed the nocytochemical staining and HPLC and RIA demonpresence of nerve endings containing dense staining strated the presence of these two neuropeptides within granules (Figs. 2,3). These nerve endings were located the posterior pituitary. Thus the possum is similar to i n between the cells forming the posterior region and eutherians in having a functional posterior pituitary at were observed in the neck region of the protruding in- birth, even though the hypothalamus is still forming. As the methods for detecting neurosecretory material fundibular stock from the diencephalon. These nerve endings were not seen in the region of the posterior have improved, oxytocin and vasopressin have been pituitary closest to the anterior region (Figs. 1-3). The shown to be produced in eutherian fetuses earlier than cell bodies of these axons containing the two peptides first thought. Oxytocin was not detected in the pituitary were presumably located in the cellular lining of the of the rat until 1-2 days after birth and in the hypopresumptive third ventricle. thalamus until day 4 postpartum (Choy and Watkins, By day 3 postpartum, the posterior pituitary had in- 1979). In a later study, Whitnall e t al. (1985)observed creased in volume and a ventricle formed in the middle the prohormones neurophysin-vasopressin and neuroof this region, which was in contact with the ventricle physin-oxytocin in the pituitary of the fetal rat at days of the diencephalon (Fig. 4). The nerve endings ob- 16 and 20, respectively. These prohormones were served in the diencephalon close to the posterior region present in the paraventricular and supraoptic nuclei on and in the neck region forming the posterior pituitary day 17, several days before birth. The newborn possum on day 1 were now present in all the regions of the is a t a n overall earlier stage of development than the posterior pituitary adjacent the intermediate zone. Me- newborn rat. The appearance of these neuropeptides in sotocin (Figs. 5,6) and vasopressin were located immu- the newborn possum would suggest a role for these nocytochemically in the posterior pituitary and in the hormones at birth or in controlling a n organ system that diencephalon close to the pituitary. No staining was begins its development as the possum enters the pouch. The hypothalamic nuclei normally involved with the observed in the intermediate or anterior parts of the pituitary, and no cells in the diencephalon were production of these hormones were not observed in the stained. There were no accumulations of neurones in newborn possum. The maturation of the hypothalamothe presumptive hypothalamus that could be termed hypophysial system has been described as having three steps: the differentiation and development of the cell the paraventricular or supraoptic nuclei. By day 8 postpartum, the posterior pituitary had in- bodies of the nuclear neurones, presumably in the hycreased in volume and the ventricle within the posterior pothalamus; the abundance and microscopical appearregion, present at day 3 (Fig. 4), had disappeared (Fig. ance of the neurosecretory material; and the hormone 7). The cleft located between the intermediate and an- content of the neural lobe (Heller and Lederis, 1959). terior regions of the pituitary was prominent (Fig. 7). The paraventricular nucleus of the fetal rat was first observed on day 16-17 of gestation with thymidine radiography (Altman and Bayer, 1979). Thus the paraventricular nucleus begins to produce oxytocin and vasopressin soon after its formation in the rat. The Fig. 1. Frontal section through the anterior (AN), intermediate (I), hypothalamo-neurohypophysial system of mammals and posterior (P)pituitary of a newborn possum stained with azur A. with a relatively long gestation, such as the guinea pig, The arrows (+) denote the position of the nerve endings that contain sheep, and human, is functional before birth (Donev, secretory granules. x 250. 1970). In contrast, the supraoptic and paraventricular Fig. 2. Area of posterior pituitary indicated by a n arrow in Figure 1. nuclei of the golden hamster formed 3-5 days postparNerve endings (N), containing dense staining granules, are located tum (Auer, 1951). Further study is required to verify between the cell bodies of the presumptive posterior pituitary. this result for the golden hamster. In the possum, the x 3,500. neuropeptides were observed in the forming posterior pituitary in advance of the formation of the hypothaFig. 3. Higher magnification of the dense staining granules (-1 lamic nuclei. shown in Figure 2. x 15,000. RESULTS 232 R.T. GEMMELL ET AL. Fig. 4. Frontal section through the anterior (AN), intermediate (I), and the posterior pituitary (PI of the possum a t 3 days postpartum. x 200. Fig. 5. Immunocytochemical localisation of mesotocin (+) in a paraffin section of the posterior (P) pituitary of the possum a t day 3 postpartum. No staining was observed in the intermediate (I) or anterior (AN) regions of the pituitary. x 75. Fig. 6. Higher magnification of the intermediate (I) and posterior pituitary (P) shown in Figure 5. Dark lines of precipitation indicate the immunocytochemical localisation of mesotocin (+) in this paraffin section in which the nuclei of the cells have also been stained with haematoxylin. x 200. Fig. 7. Frontal section through the anterior (AN), intermediate (I), and the posterior pituitary (P) of the possum at 8 days postpartum. x 100. nuclei of the cells have also been stained with haematoxylin. The staining was confined to the posterior (P) and no staining was observed in the intermediate (I) or anterior pituitary. x 200. Fig. 8. Dark lines of precipitation indicate the immunocytochemical localisation of vasopressin (-j in this paraffin section in which the Fig. 9. Dense staining granules ( + j in nerve endings located within the posterior pituitary of a possum at 8 days postpartum. x 15,000. 234 R.T. GEMMELL ET AL. The fetal pituitary-adrenal axis has been shown to be involved with the initiation of birth in several eutherian mammals (Liggins et al., 1973; Challis et al., 1977; Thorburn and Challis, 1979). Hormonal secretions from the fetal anterior pituitary stimulate the adrenal cortex to produce cortisol, which in t u r n crosses the placenta to produce the hormonal cascade that initiates birth in eutherians. Although the newborn marsupial is a t a far less developed stage that any eutherian, a n active anterior pituitary and adrenal axis exists. Secretory granules have been observed in the anterior pituitary, and morphological and biochemical evidence for the production of steroid hormones by the cortex of the newborn marsupial adrenal gland has been obtained from all species of marsupial examined (Gemmell and Rose, 1989; Sherman and Krause, 1990). The fetal pituitary and adrenal glands are functional at birth and are thus capable of initiating parturition (Walker and Gemmell, 1983; Gemmell and Nelson, 1988; Gemmell and Rose, 1989). The presence of hormones within the posterior pituitary of the newborn possum suggests that this part of the pituitary may also be involved with parturition. The posterior pituitary of the eutherian newborn has been suggested to influence labour and regulate fluid balance in the placenta (Swaab et al., 1978). Secretory granules have been observed in the posterior pituitary of other newborn marsupials. Hughes e t al. (1989) reported two types of membrane-bound granules in nerve axonal terminals within the posterior lobe of the newborn tammar wallaby, Macropus eugenii. Similar granules were reported in nerve terminals in the posterior pituitary of the newborn bandicoot, Isoodon macrourus (Hall and Leslie, 1990). Vasopressin is involved in the regulation of fluid balance. In all marsupials so far studied, the mesonephros is the functional urinary unit at birth. 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