Pituitary-induced lactation in mammary gland explants from the pregnant tammar (Macropus eugenii): a negative role for cyclic AMP

1. alpha-Lactalbumin and casein have been isolated from tammar milk. 2. alpha-Lactalbumin was induced in mammary explants by culture with anterior pituitary. 3. Casein was induced maximally in the presence of a physiological concentration of prolactin alone. 4. Progesterone did not inhibit the prolactin-induced synthesis of casein, alpha-lactalbumin, galactosyltransferase or fatty acids. 5. Both dibutyryl cAMP and a combination of cholera toxin and IBMX did significantly inhibit the induction of casein and alpha-lactalbumin. 6. Progesterone withdrawal is not a component of the lactogenic trigger in this marsupial but cAMP may be a common intracellular signal for negative control of lactogenesis in both marsupials and eutherians.     

Cyclic AMP-dependent tyrosine phosphorylation in tammar wallaby (Macropus eugenii) spermatozoa

Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues were principally located along the tammar sperm flagellum, and occasionally at a small region of the sperm head, adjacent to the acrosome. Associated with the tyrosine phosphorylation of tammar spermatozoa, was a change in sperm head conformation to a T-shaped orientation, further implying the importance of these pathways to normal tammar sperm function. Redox activity, as detected by lucigenin-dependent chemiluminescence, was stimulated by NADPH in caudal sperm preparations but not ejaculated spermatozoa. However, neither sperm population responded to treatment with NADPH with changes in intracellular cAMP or tyrosine phosphorylation. In conclusion, tammar spermatozoa possess the same cAMP-mediated, tyrosine phosphorylation-dependent signal transduction cascade that has been associated with capacitation in eutherian spermatozoa. However in Metatherian spermatozoa we could find no evidence that this pathway was redox regulated.     

Mammogenesis and changing prolactin receptor concentrations in the mammary glands of the tammar wallaby (Macropus eugenii)

All 4 mammary glands of the tammar wallaby showed a steady increase in weight and prolactin receptor concentration during the luteal phase of the oestrous cycle to reach a peak at oestrus. Removal of the corpus luteum abolished this mammogenesis , while pregnancy, which in this species is a day or so shorter than the oestrous cycle, had no effect. This provides an explanation for the previous finding that pregnancy is not a necessary pre-requisite for lactation in marsupials and that nonpregnant animals will lactate very successfully, provided the suckling stimulus is applied at the correct stage of the oestrous cycle. During lactation, only the gland supplying the teat to which the pouch young was attached developed and showed any further increase in prolactin receptors; the other 3 glands remained small and inactive. These results indicate the importance of the suckling stimulus and milk withdrawal on the initiation and maintenance of lactation.     

Structure of the pars distalis in the adult tammar wallaby (Macropus eugenii)

An immunohistochemical, light- and electron-microscopial study was made of the pars distalis in adult tammar wallabies (Macropus eugenii). The pars distalis of this marsupial mammal was divided into three regions, based on the distribution of cell types within the gland. Somatotropic, mammotropic, luteotropic, folliculotropic, corticotropic and thyrotropic cells were identified on the basis of their immunohistochemistry, cytology and ultrastructure. Non-granulated (folliculo-stellate) cells, identified in electron micrographs, were found throughout the pars distalis. Somatotropic cells were predominant in the posterior pars distalis in all animals examined. In the single male specimen and in the non-lactating females examined, small numbers of apparently inactive mammotropic cells were scattered throughout the pars distalis; the same cell type was apparently active and present in considerable numbers in lactating females. Only one morphological type of gonadotropic cell was evident; these cells were scattered throughout the pars distalis, but in largest numbers in the median region. Small numbers of thyrotropic cells were found, most commonly in the anterior pars distalis. Corticotrops were also observed in moderate numbers, predominantly in the anterior regions of the pars distalis.     

Proteomic analysis of early lactation milk of the tammar wallaby (Macropus eugenii)

We report the successful use of 2D electrophoresis, MALDI MS/MS and chemical derivatisation protocols of guanidination and sulfonation to identify over 100 protein spots present in early marsupial milk (tammar wallaby) at 40 days lactation, where a limited translated genomic database is publicly available for cross species matching and protein identification. Of the proteins identified, 25 matched to 6 existing marsupial milk protein sequences in the NCBI database; another 6 were identified with high confidence to other mammals and have not previously been identified in marsupial milk. By using chemical derivatisation, the reliable identification of a further 81 proteins was achieved. The identified proteins could be grouped into three main functional categories — transport, nutrition and immune protection. All these proteins play a potential role in determining growth and immunological protection of the highly altricial marsupial young at 40 days after birth.     

Cross-fostering of the tammar wallaby (Macropus eugenii) pouch young accelerates fore-stomach maturation

There are two phases of fore-stomach development during the first 200 days of pouch life in tammar wallaby. For the first 170 days, the mucosa displays an immature gastric glandular phenotype that changes to a cardia glandular phenotype, which remains for the rest of the animal’s life. During this 200-day period after birth, the pouch young (PY) is dependent on maternal milk, which progressively changes in composition. We showed previously that PY cross-fostered to host mothers at a later stage of lactation accelerated development. In this study, we investigated whether cross-fostering and exposure to late lactation stage milk affected the transition to cardia glandular phenotype. In fostered PY fore-stomach, there was increased apoptosis, but no change in cell proliferation. The parietal cell population was significantly reduced, and expression of gastric glandular phenotype marker genes (ATP4A, GKN2, GHRL and NDRG2) was down-regulated, suggesting down-regulation of gastric phenotype in fostered PY fore-stomach. The expression of cardia glandular phenotype genes (MUC4, KRT20, CSTB, ITLN2 and LPLUNC1) was not changed in fostered PY. These data suggest that fore-stomach maturation proceeds via two temporally distinct processes: down-regulation of gastric glandular phenotype and initiation of cardia glandular phenotype. In fostered PY, these two processes appear uncoupled, as gastric glandular phenotype was down-regulated but cardia glandular phenotype was not initiated. We propose that milk from later stages of lactation and/or herbage consumed by the PY may play independent roles in regulating these two processes.     

Isolation of major histocompatibility complex Class I genes from the tammar wallaby (Macropus eugenii)

The major histocompatibility complex (MHC) plays an essential role in the adaptive immune system of vertebrates through antigen recognition. Although MHC genes are found in all vertebrates, the MHC region is dynamic and has changed throughout vertebrate evolution, making it an important tool for comparative genomics. Marsupials occupy an important position in mammalian phylogeny, yet the MHC of few marsupials has been studied in detail. We report the isolation and analysis of expressed MHC Class I genes from the tammar wallaby, a model marsupial used extensively for the study of mammalian reproduction, genetics, and immunology. We determined that there are at least 11 Class I loci in the tammar genome and isolated six expressed Class I sequences from spleen and testes cDNA libraries, representing at least four loci. Two of the Class I sequences contain substitutions at sites known to be important for antigen binding, perhaps impacting their ability to bind peptides, or the types of peptide to which they bind. Phylogenetic analysis of tammar wallaby Class I sequences and other mammalian Class I sequences suggests that some tammar wallaby and red-necked wallaby loci evolved from common ancestral genes.     

Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii)

Lactating tammars can provide two different milks simultaneously from adjacent glands to a young newborn (phase 2 of lactation) and an older animal at heel (phase 3 of lactation). Evidence that the two glands are controlled independently is shown by the capacity of mammary explants from these glands to synthesize different whey proteins and DNA and RNA at different rates. Prolactin is essential for the maintenance of milk synthesis, but its role in differential responses of the individual mammary glands remains to be established. Potential mechanisms for the control of milk synthesis are discussed.     

Immunological aspects of gestation in the tammar wallaby, Macropus eugenii.

Sensitization to male histocompatibility antigens and repeated pregnancy to the same male were found to have little effect on fertility or length of gestation in the tammar wallaby, M. eugenii. However, in some sensitized females a long interval occurred between removal of pouch young and the next birth. In addition to studies on fertility, the immunological response of female tammars to their mate has been examined by one-way mixed leucocyte culture (MLC) carried out at the beginning and end of one breeding season. In virgin females, examined at the beginning of the breeding season, the MLC response to the prospective mate peaked on day 6. In contrast, by the end of the season, MLC responses were much lower and peaked earlier, on day 3 to day 5.     

Role of androgens in survival of spermatozoa in epididymis of tammar wallaby (Macropus eugenii).

Studies of undiluted micropuncture samples of luminal fluid from the cauda epididymidis of the tammar indicated that spermatozoa are immotile in situ and spontaneously activate during collection or subsequent incubation in vitro. The suppression of sperm motility was related to the androgen status of the tammars and when this was increased by the use of Silastic implants of testosterone propionate, the spontaneous activation of samples was delayed for up to 2 h during incubation in vitro. Spermatozoa survived for up to 9 weeks when isolated in the cauda epididymidis between ligatures around the ductus. However, even after isolation for 3 weeks their viability was reduced compared with samples from the contralateral, unligated duct. Isolation of a length of ductus between ligatures also reduced the concentration of spermatozoa in the lumen of the duct and reduced the concentration of some proteins in the epididymal plasma. However, it did not affect the electrophoretic pattern of detergent extracts of spermatozoa. A study of the effects of orchidectomy and testosterone therapy indicated that sperm survival in the epididymis is androgen dependent. Orchidectomy reduced the concentration of spermatozoa in the luminal fluid and the volume of luminal fluid, and resulted in an increase in the concentration and a change in the electrophoretic pattern of protein in the fluid. The effects of orchidectomy were reduced or prevented by testosterone therapy. It is concluded that the cauda epididymidis of the tammar is at least as well adapted for sperm storage as it is in the eutherian mammals that have been studied.     

Recycling of urea nitrogen to the gut of the tammar wallaby (Macropus eugenii)

• 1.1. The transfer of urea from plasma to the gut of tammar wallabies (Macropus eugenii) was studied using infusions of [¹⁵N]-urea and [¹⁴C]-urea in animals given either a high nitrogen chopped lucerne hay or a low nitrogen chopped oaten hay diet.
• 2.2. The proportion of urea entering the plasma pool which was degraded in the gut was similar on both diets (74–86%). Incorporation of urea into non-ammonia nitrogen in the gut was 1.1–1.7 g N/day in tammars given the high nitrogen diet, equivalent to 34–53% of dietary N intake, and 0.75–0.78 g N/day in tammars given the low nitrogen diet, equivalent to 103–112% of dietary N intake.
• 3.3. On the lucerne diet 63% of bacterial nitrogen in the midstomach was derived from ammonia.
• 4.4. It appears that blood urea is transferred into the stomach of tammars given lucerne more readily than into the rumen of sheep given lucerne.

     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

Characterization and expression of Peroxiredoxin 1 in the neonatal tammar wallaby (Macropus eugenii)

Peroxiredoxin 1 (PRDX1) is a ubiquitously expressed antioxidant with vital roles in basal metabolic functions. In addition PRDX1 is involved in cell differentiation and proliferation, apoptosis and innate immunity. In this study, we have characterized PRDX1 from the tammar wallaby (Macropus eugenii). Tammar PRDX1 has high conservation of functional residues and motifs, and demonstrates a close homology with eutherian and vertebrate orthologues. Stimulation of adult tammar leukocytes with lipopolysaccharide and lipoteichoic acid suggests a role for PRDX1 in innate immune defences. PRDX1 expression in the organs of tammar pouch young was mildly elevated early in life possibly reflecting its role in basal metabolic processes. Later increases in PRDX1 expression correlated with functional maturation of several immune organs or with preparation for increased oxidative stress of emergence. The findings of the study are reflections of the complex integrated roles that PRDX1 has in regulation of oxidative stress, apoptosis, cell differentiation and proliferation, and innate immunity.     

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii)

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.     

The extracellular matrix locally regulates asynchronous concurrent lactation in tammar wallaby (Macropus eugenii)

Asynchronous concurrent lactation (ACL) is an extreme lactation strategy in macropod marsupials including the tammar wallaby, that may hold the key to understanding local control of mammary epithelial cell function. Marsupials have a short gestation and a long lactation consisting of three phases; P2A, P2B and P3, representing early, mid and late lactation respectively and characterised by profound changes in milk composition. A lactating tammar is able to concurrently produce phase 2A and 3 milk from adjacent glands in order to feed a young newborn and an older sibling at heel. Physiological effectors of ACL remain unknown and in this study the extracellular matrix (ECM) is investigated for its role in switching mammary phenotypes between phases of tammar wallaby lactation. Using the level of expression of the genes for the phase specific markers tELP, tWAP, and tLLP-B representing phases 2A, 2B and 3 respectively we show for the first time that tammar wallaby mammary epithelial cells (WallMECs) extracted from P2B acquire P3 phenotype when cultured on P3 ECM. Similarly P2A cells acquire P2B phenotype when cultured on P2B ECM. We further demonstrate that changes in phase phenotype correlate with phase-specific changes in ECM composition. This study shows that progressive changes in ECM composition in individual mammary glands provide a local regulatory mechanism for milk protein gene expression thereby enabling the mammary glands to lactate independently.     

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C(16:0), which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.     

Qualitative and quantitative changes in milk fat during lactation in the tammar wallaby (Macropus eugenii)

There are major quantitative and qualitative changes in the milk lipids during lactation in the tammar wallaby, Macropus eugenii. The crude lipid content of the milk is relatively low during the first 10 weeks of lactation; between 10 and 26 weeks post partum the lipid content increases gradually but after that it increases rapidly. The triglyceride fraction of the lipid at early stages of lactation contains a large amount of palmitic acid and relatively little oleic acid whereas mature milk exhibits little palmitic and much oleic acid. In the early stages of lactation fat represents 15% of the total solids and carbohydrate 55%; around 26-30 weeks post partum the carbohydrate moiety falls sharply to a level less than 2% of the solids while lipids increase to c. 60% of the solids. These changes coincide with increases in milk solids, emergence of the young from the pouch, ingestion of herbage, and fermentation of cellulose in the stomach.     

Control of parturient behaviour by prostaglandin F-2 alpha in the tammar wallaby (Macropus eugenii)

Nulliparous female tammar wallabies during the non-breeding season and adult male wallabies were treated with PGF-2 alpha at doses of 0.008, 0.04, 0.2 and 1.0 mg/kg. All the male and female wallabies responded to the three highest doses by showing parturient behaviour. At the lowest dose 4/4 males and 1/4 females responded. The peak concentrations of PGF-2 alpha metabolite (PGFM) in the peripheral plasma after administration of 0.008, 0.04 and 0.2 mg PGF-2 alpha/kg were 0.70 +/- 0.08, 3.02 +/- 0.37 and 8.48 +/- 0.76 ng/ml (mean +/- s.e.m.). Since the peak plasma concentrations of PGFM at normal parturition are reported to be 2.5 +/- 0.9 ng/ml, parturient behaviour can be induced by physiological concentrations of exogenous PGF-2 alpha. The effectiveness of PGF-2 alpha in males indicates that parturient behaviour is probably a result of a direct action of PGF-2 alpha on the brain, rather than a response to uterine or vaginal contractions. These experiments confirm that PGF-2 alpha is an important behavioural hormone in the tammar wallaby.