Actin polymerisation during morphogenesis of the acrosome as spermatozoa undergo epididymal maturation in the tammar wallaby (Macropus eugenii)

In the tammar wallaby ( Macropus eugenii ), post-testicular acrosomal shaping involves a complex infolding and fusion of the anterior and lateral projections of the scoop-shaped acrosome into a compact button-like structure occupying the depression on the anterior end of the sperm nucleus. The present study has generated cytochemical and histological evidence to demonstrate that the occurrence of actin filaments (F-actin, labelled by Phalloidin-FITC) in the acrosome of tammar wallaby spermatozoa is temporally and spatially associated with the process of acrosomal shaping in the epididymis, through a pool of monomeric actin (G-actin, labelled by Rh-DNase I) present in the acrosome throughout all stages of epididymal maturation. F-actin was not detected in the acrosome of testicular spermatozoa, but was found in the infolding and condensing acrosome of caput and corpus epididymal spermatozoa. When the spermatozoa completed acrosome shaping in the cauda epididymidis, F-actin disappeared from the acrosomal area. The strong correlation between the occurrence of F-actin and the events of acrosomal shaping suggested that the post-testicular shaping of the acrosome might depend on a precise succession of assembly and disassembly of F-actin within the acrosome as the spermatozoa transit the epididymis. Thus, actin filaments might play a significant role in the acrosomal transformation, as they are commonly involved in morphological changes in somatic cells.     

Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula)

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated ‘scoop’-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the ‘scoop’. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.     

Spermiogenesis and spermiation in a marsupial, the tammar wallaby (Macropus eugenii)

Fourteen steps of spermatid development in the tammar wallaby (Macropus eugenii), from the newly formed spermatid to the release of the spermatozoon into the lumen of the seminiferous tubules, were recognised at the ultrastructural level using transmission and scanning electron microscopy. This study confirmed that although the main events are generally similar, the process of the differentiation of the spermatid in marsupials is notably different and relatively more complex than that in most studied eutherian mammals and birds. For example, the sperm head rotated twice in the late stage of spermiogenesis: the shape of the spermatid changed from a T-shape at step 10 into a streamlined shape in step 14, and then back to T-shape in the testicular spermatozoa. Some unique figures occurring during the spermiogenesis in other marsupial species, such as the presence of Sertoli cell spurs, the nuclear ring and the subacrosomal space, were also found in the tammar wallaby. However, an important new finding of this study was the development of the postacrosome complex (PAC), a special structure that was first evident as a line of electron dense material on the nuclear membrane of the step 7 spermatid. Subsequently it became a discontinuous line of electron particles, and migrated from the ventral side of the nucleus to the area just behind the posterior end of the acrosome, which was closely located to the sperm–egg fusion site proposed for Monodelphis domestica (Taggart et al. 1993). The PAC and its possible role in both American and Australian marsupials requires detailed examination. Distinct immature features were discovered in the wallaby testicular spermatozoa. A scoop shape of the acrosome was found on the testicular spermatozoa of the tammar wallaby, which was completely different to the compact button shape of acrosome in ejaculated spermatozoa. The fibre network found beneath the cytoplasm membrane of the midpiece of the ejaculated sperm also did not occur in the testicular spermatozoa, although the structure of the principal piece was fully formed and had no obvious morphological difference from that of the epididymal and ejaculated spermatozoa. The time frame of the formation of morphologically mature spermatozoa in the epididymis of the tammar wallaby needs to be determined by further studies.     

The detection of mature T- and B-cells during development of the lymphoid tissues of the tammar wallaby (Macropus eugenii)

The distribution of T- and B-cells in the developing lymphoid and immunohaematopoietic tissues of the tammar wallaby were investigated using antibodies to the mature cell surface markers, CD3, CD5 and CD79b. In the thymus, CD3- and CD5-positive T-cells were first observed at day 12 postpartum whilst rare B-cells were first detected at day 23. Both T- and B-lymphocytes were first stained on day 21 postpartum in the spleen and day 24 in lymph nodes. In one sample from a 7-day-old animal, rare CD79b-positive (CD79b+) lymphocytes were observed in the gut-associated lymphoid tissues. However, CD3+ cells were not apparent until day 12 and CD5+ cells were not detected until day 74 postpartum. No lymphocytes were detected in liver or bone marrow samples and no bronchus-associated lymphoid tissues were observed. The pattern of development and the distribution of T- and B-cells in the lymphoid and immunohaematopoietic tissues were similar to those observed in eutherian mammals and in limited studies of other metatherians. However, the detection of apparently mature T- and B-cells in the thymus and gut-associated lymphoid tissues (GALT) at the same postnatal age highlights the need for a more substantial study of the development of GALT. This is, at present, limited by availability of marsupial-specific antibodies.     

In search of neutrophil granule proteins of the tammar wallaby (Macropus eugenii)

Two approaches have been used to isolate and identify proteins of the granules of neutrophils of the tammar wallaby, Macropus eugenii. Stimulation with PMA, Ionomycin and calcium resulted in exocytosis of neutrophil granules as demonstrated with electron microscopy. However proteomic analysis using two dimensional gel electrophoresis, in-gel trypsin digestion followed by nano liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) failed to identify any anticipated granule proteins in the reaction supernatants. Subsequent use of differential centrifugation and lysis followed by the application of the same proteomic analysis approach resulted in the isolation and confident identification of 39 proteins, many of which are known to be present in the granules of neutrophils of eutherian mammals or play a role in degranulation. These proteins notably consisted of the known antimicrobials, myeloperoxidase (MPO), serine proteinase, dermcidin, lysozyme and alkaline phosphatase. A number of important known antimicrobials, however, were not detected and these include defensins and cathelicidins. This is the first report of the neutrophil granule proteins of any marsupial and complements previous reports on the cytosolic proteins.     

Ultrastructure of the placenta of the tammar wallaby,Macropus eugenii: comparison with the grey short-tailed opossum,Monodelphis domestica

The ultrastructure of the tammar placenta was studied throughout pregnancy. The uterine epithelium grows from a columnar to an enlarged, undulating epithelium between early gestation and mid-gestation when the shell coat that surrounds the marsupial conceptus ruptures. Trophectoderm and uterine epithelium do not form syncytia, nor does invasion of the endometrium occur at any stage of pregnancy. Uterine secretion is provided to both the bilaminar and the trilaminar side of the yolk sac placenta up to birth. Fenestrations, abundant vesicles and lumenal processes of maternal capillaries, as well as deep basal folds of the uterine epithelium, suggest that there is transfer of hemotrophes adjacent to both parts of the yolk sac. In contrast, in the grey short-tailed opossum, these structures are lacking. The yolk sacs of adjacent embryos fuse to form a common yolk sac cavity, thus losing most of the bilaminar yolk sac. The bilaminar and trilaminar components of the yolk sac placenta of the tammar are less different in structure and function than those of the grey short-tailed opossum, but both types are fully functional placentas. The extended secretory phase of the tammar uterus and the maternal recognition of early pregnancy appear to be derived characters of macropodid marsupials.     

Immunohistochemistry of the lymphoid tissues of the tammar wallaby,Macropus eugenii

The lymphoid tissues of the metatherian mammal, the adult tammar wallaby, Macropus eugenii, were investigated using immunohistochemical techniques. Five cross-reactive antibodies previously shown to recognize surface markers in marsupial tissues and five previously untested antibodies were used. The distribution of T-cells in the tissue beds of spleen, lymph node, thymus, gut-associated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT) was documented using antibodies to CD3 and CD5. Similarly, B-cells were identified in the same tissues using anti-CD79b. Antibodies to CD8, CD31, CD79a and CD68 failed to recognize cells in these tissue beds. In general the pattern of cellular distribution identified using these antibodies was similar to that observed in other marsupial and eutherian lymphoid tissues. This study provides further information on the commonality of lymphoid tissue structure in the two major groups of extant mammals, metatherians and eutherians.     

Uterine morphology during diapause and early pregnancy in the tammar wallaby (Macropus eugenii)

In mammals, embryonic diapause, or suspension of embryonic development, occurs when embryos at the blastocyst stage are arrested in growth and metabolism. In the tammar wallaby (Macropus eugenii), there are two separate uteri, only one of which becomes gravid with the single conceptus at a post‐partum oestrus, so changes during pregnancy can be compared between the gravid and non‐gravid uterus within the same individual. Maintenance of the viable blastocyst and inhibition of further conceptus growth during diapause in the tammar is completely dependent on the uterine environment. Although the specific endocrine and seasonal signals are well established, much less is known about the cellular changes required to create this environment. Here we present the first detailed study of uterine morphology during diapause and early pregnancy of the tammar wallaby. We combined transmission electron microscopy and light microscopy to describe the histological and ultrastructural changes to luminal and glandular epithelial cells. At entry into diapause after the post‐partum oestrus and formation of the new conceptus, there was an increase in abundance of organelles associated with respiration in the endometrial cells of the newly gravid uterus, particularly in the endoplasmic reticulum and mitochondria, as well as an increase in secretory activity. Organelle changes and active secretion then ceased in these cells as they became quiescent and remained so for the duration of diapause. In contrast, cells of the non‐gravid, post‐partum, contralateral uterus underwent sloughing and remodelling during this time and some organelle changes in glandular epithelial cells continued throughout diapause, suggesting these cells are not completely quiescent during diapause, although no active secretion occurred. These findings demonstrate that diapause, like pregnancy, is under unilateral endocrine control in the tammar, and that preparation for and maintenance of diapause requires substantial changes to uterine endometrial cell ultrastructure and activity.     

Molecular characterisation of the tammar wallaby (Macropus eugenii) CD3 epsilon chain cDNA.

The cDNA encoding the epsilon chain of the tammar wallaby CD3 complex (CD3epsilon) was isolated by PCR. This is the first CD3 component to be cloned in a marsupial. The tammar wallaby cDNA coding region was 61.7 and 63.0% identical to the human and mouse cDNA coding sequences, respectively. Similarly, the predicted amino acid sequence was 56.5 and 52.9% identical to the human and mouse sequences. When compared with other known CD3epsilon peptide sequences, the most conserved region of the tammar wallaby CD3epsilon chain peptide was the cytoplasmic domain and the least conserved was the extracellular portion. Phylogenetic reconstruction based on the deduced amino acid sequence placed the tammar wallaby sequence in its expected position outside of all the eutherian mammals.     

Bistratified amacrine cells in the retina of the tammar wallaby — Macropus eugenii

Summary Cajal (1911) noted that bistratified amacrine cells were common in non mammalian species and extremely rare in the mammalian retina. An examination of the marsupial retina of the tammar wallaby, stained with a modified Golgi procedure, revealed that a particular type of bistratified amacrine was frequently impregnated with the silver stain. Flat mount and transverse sections showed that the morphology of this cell did not correspond with any of the species-dependent bistratified amacrines reproduced in Cajal's drawings. Instead, the cell appeared to be almost identical to the AII or rod amacrine that has been observed in a number of mammalian retinas. The relative frequency with which the cell appears in our material, and its confirmed rod input in other species, are both consistent with the grazing habits of the tammar wallaby which is a crepuscular animal that does most of its feeding at dusk and after dark.     

Development of the penis and clitoris in the tammar wallaby, Macropus eugenii

 The development of the phallus from the indifferent stage to sexual dimorphism has not been described in any marsupial. This study describes the morphological and histological changes occurring in the development of the phallus of the tammar wallaby. The development of the penis and clitoris in the tammar closely follow the most widely accepted model for the development of the same organs in eutherian mammals. The urogenital plate that is present in both sexes at birth hollows out to form a urogenital groove at approximately 70 days postpartum (p.p.). There is then greater growth of the phallus in males than in females, which results in sexual dimorphism in length approximately 100 days p.p. In males, the urogenital groove secondarily closes over at this time and fuses in the midline and by 128 days p.p. the penile urethra is fully formed. In females, the groove remains open. The clitoris changes little morphologically from the time of formation of the urogenital groove until adulthood. The pattern of development of the penis in the tammar is similar to that seen in eutherian mammals. There is strong evidence that penis development is androgen-dependent in the tammar, yet unusually it becomes sexually dimorphic at a time when androgen content of the developing testis is low. Accepted: 10 November 1998     

Postnatal development of the telencephalon of the tammar wallaby (Macropus eugenii). An accessible model of neocortical differentiation

Summary The sequence of development of cell layers in the neocortex of the tammar has been followed from 24 days gestation to 213 days postnatal. The tammar is born at 27 days gestation and the major period of its development occurs during the subsequent 250 days, most of this time being spent within the pouch. Although the pattern of differentiation of the cell layers appears to resemble that described for many Eutherian mammals, the neocortex is at an embryonic 2 layered stage at birth and a cortical plate is not present throughout the telencephalon until 10–15 days postnatal. A transient subplate zone, presenting a characteristic appearance with widely spaced rows of cells aligned parallel to the cortical surface, develops between 20 and 70 days postnatal, but no secondary proliferative region is seen in the subventricular zone of the dorso-lateral wall.Preliminary experiments with (³H)-thymidine injections indicate that the cortical plate follows the inside-out pattern of development described in many Eutherian mammals and that the oldest neurons are found in the parallel cell rows of the subplate zone. The importance of the late differentiation of the neocortex in relation to the time of birth and the resulting usefulness of the tammar as an experimental model of cortical development is discussed.     

CD15 immunoreactivity in the developing brain of a marsupial, the tammar wallaby ( Macropus eugenii)

We have studied the distribution of the CD15 epitope in the developing brain of an Australian diprotodontid metatherian mammal, the tammar wallaby (Macropus eugenii), using immunohistochemistry in conjunction with hematoxylin and eosin staining. At the time of birth (28 days after conception), CD15 immunoreactivity labeled somata in the primordial plexiform layer of the parietal cortex in a similar position to that seen in the early fetal eutherian brain. CD15 immunoreactivity in the brain of the developing pouch-young wallaby was found to be localized on the surface of radial glia at boundaries between developmentally significant forebrain compartments in a similar distribution to that seen in developing eutherian brain. These were best seen in the developing diencephalon, delineating epithalamus, ventral and dorsal thalamus and hypothalamic anlage, and in the striatum. Immunoreactivity for CD15 identified radial glia marking the lateral migratory stream at the striatopallial boundary, peaking in intensity at P19 to P25. From P37 to P54, CD15 immunoreactivity also demarcated patch compartments in the developing striatum. In contrast, CD15 immunoreactivity in hindbrain structures showed some differences from the temporospatial pattern seen in eutherian brain. These may reflect the relatively early brainstem maturation required for the newborn wallaby to be able to traverse the distance from the maternal genital tract to the pouch. The wallaby provides a convenient model for testing hypotheses concerning the role of CD15 in forebrain development because all events in which CD15 may play a critical role in forebrain morphogenesis occur during pouch life, when the young wallaby is accessible to experimental manipulation.     

Sexual differentiation of the urogenital system of the fetal and neonatal tammar wallaby, Macropus eugenii

In male tammar wallabies, the scrotum is the first organ to become sexually differentiated, 4–5 days before birth (day 22 of gestation). This is followed by enlargement of the gubernaculum and processus vaginalis one day before birth. However the indifferent gonad does not show any signs of testicular cord formation or androgen production until later, at around the time of birth; this is more pronounced at 2 days post-partum (p.p.), when the testis takes on a characteristic rounded appearance. Primordial germ cells proliferate throughout the testis at this time, although the testis does not become significantly heavier than the ovary until around 80 days p.p.. In females, the appearance of the mammary glands is the first sign of sexual differentiation 4–5 days before birth. The indifferent gonad first shows signs of developing an ovarian cortex and medulla 7 days after birth. The migrating germ cells are confined to the cortex, and first start to enter meiosis about 25 days after birth. The Wolffian (mesonephric) ducts are patent to the urogenital sinus in fetuses at day 21 of gestation. In the female they have started to regress by 10 days p.p. and only rudiments remain by day 25 p.p.. The Müllerian (paramesonephric) ducts develop adjacent to the cranial pole of the mesonephros at about day 25 of gestation and grow caudally to meet the urogenital sinus between days 2 and 7 p.p.. The Müllerian duct of the female develops a prominent ostium abdominale by day 9 p.p., but this structure has completely regressed in males by day 13 p.p.. The testis and ovary both migrate caudally, together with the adjacent mesonephros, at about day 10 p.p.. The ovaries remain around the level of lumbar vertebra 4 after about day 7 p.p., while the testes continue to descend. The testes enter the internal inguinal ring at about day 25 p.p., about the time that prostatic buds first appear in the urogenital sinus, and are in the inguinal canal from days 25 to 36 p.p.. They enter the scrotum at around day 36 p.p., and testicular descent is complete by days 65–72 p.p.. Melanin develops in the tunica vaginalis 72 days after birth. The overall development of the urogenital system in this marsupial is similar to that of eutherians but the sequence of events differs, with some aspects of genital differentiation preceding gonadal differentiation, apparently because they are directly controlled by X-linked genes, rather than indirectly controlled by gonadal steroids.     

A longitudinal study of changes in blood leukocyte numbers in the tammar wallaby, Macropus eugenii

Changes in leukocyte numbers were monitored over a 3-year period in a small group of captive tammar wallabies, Macropus eugenii, maintained in the animal research facilities at Macquarie University (NSW, Australia). The neutrophil to lymphocyte ratio (N/L), a commonly used parameter in the assessment of health status in wildlife populations, was not useful when applied between animal populations but did reliably predict changes within individual animals and between animals within the study cohort. This study also demonstrated the importance of obtaining haematological values from animals on more than one occasion to ensure that differential cell counts from asymptomatic individuals do not unduly influence the determination of reference values.     

A longitudinal study of the protein components of marsupial milk from birth to weaning in the tammar wallaby (Macropus eugenii)

The major milk whey proteins of the tammar wallaby (Macropus eugenii) have been identified over the total period of lactation using proteomic analysis techniques comprising two-dimensional electrophoresis, comparative image analysis, matrix assisted laser desorption ionisation mass spectrometry (MALDI MS), de novo peptide sequencing and cross species protein matching. Samples were collected at the periods coinciding with major milestones of immunological development in the developing marsupial and in the four phases of milk production, specifically, Days 0, 5 (Phase 1); 27, 68 (Phase 2A); 137, 174 (Phase 2B) and 250 (Phase 3). Major changes in the protein content of marsupial milk whey correlated with the changing needs of the pouch young for stages in growth and development. We have shown that the levels of milk whey proteins vary with the developmental stage of the young animal, with a high number of proteins detected in early and late milk compared with the middle phases of lactation. Over 41 proteins were confidently identified, of which most had known roles in immunological protection. Proteins providing immunological protection across the lactation period included transferrin, beta2 microglobulin, haptoglobulin and a 78kDa glucose regulated protein. Immunoglobulin IgJ linker chain and a known antimicrobial cathelicidin, were only detected for the first 100-137 days, after which time Complement B factor was found to be present (Phase 2B). The changes which correlated with development and growth in the pouch young were reflected by the presence of proteins such as an alpha-fetoprotein like protein and clusterin found in early milk (Phase 1-2A) and two unknown proteins which were apparent in very early mammary gland secretions. This is the first comprehensive proteomic study of the major whey proteins of a marsupial across the entire period of lactation and provides fundamental data on proteins secreted by the mammary gland during key stages of immunological development of the young animal.