Effects of fetectomy on oxytocin receptors in the myometrium of the tammar wallaby

Mesotocin, an oxytocin-like peptide, stimulates uterine contractions during marsupial parturition. Female marsupials have two separate uteri, and in monovular species, the uterus with the conceptus is gravid, whereas the contralateral uterus is nongravid. Marsupials are unique because systemic and feto-placental factors in the regulation of uterine function can be differentiated. In pregnant tammar wallabies, a marked increase in myometrial mesotocin receptors (MTRs) occurs on Day 23 of the 26-day gestation, but only in the gravid uterus. The objective of this study was to investigate the effects of removing the conceptus on this MTR up-regulation. Complete fetectomy on Day 20 of gestation resulted in significantly lower MTR mRNA and receptor concentrations on Day 23 compared with sham-operated controls. In contrast, there was no significant difference in MTR expression between controls and partially fetectomized animals in which uterine distension was maintained in the absence of a conceptus. In a related study, we examined MTRs in the myometrium of animals that appeared to be pregnant with a large, distended uterus. However, these uteri contained an abnormally developed fetus and avascular placenta. In these animals, MTR levels were significantly higher in the distended uterus compared with the nondistended uterus, and did not differ from controls. These data demonstrate that uterine occupancy is essential for the marked increase in uterine MTRs observed on Day 23 gestation. It also appears that distension may be one of the key factors involved.     

Mesotocin receptors during pregnancy,parturition and lactation in the tammar wallaby

Mestocin receptor concentrations in membrane preparations from reproductive tissues of the tammar Macropus eugenii throughout gestation and lactation were assessed using [³H]-oxytocin as the ligand. There was a single binding site which bound both mesotocin and oxytocin with high and similar affinities. Mesotocin receptor concentrations in the myometrium were low (708±199 fmol mg⁻¹ protein) in early and middle gestation but increased significantly on day 23 of pregnancy of the 26-day gestation period to 1921±552 fmol mg⁻¹ protein. Myometrial receptors reached a peak of 2483±575 fmol mg⁻¹ protein on days 25 and 26 of gestation, but returned to basal levels about an hour after birth. Receptor concentrations in the contralateral non-gravid uterus were much lower (605±75 fmol mg⁻¹) and did not significantly increase throughout the period of gestation but dropped one day before birth. Mesotocin receptors were undetectable in the endometrium, the yolk sac placenta and the lateral, median and anterior vagina of all animals tested. In the lactating mammary gland after birth mesotocin receptors were initially high (588±38 fmol mg⁻¹) but decreased after 200 days and by late lactation were 224±55 fmol mg⁻¹ protein on day 240, close to the time of weaning. Mesotocin receptors in the ipsilateral non-lactating gland were also high in early lactation (430±153 fmol mg⁻¹) and declined in late lactation (62±20 fmol mg⁻¹). The changing concentrations of mesotocin receptors in pregnancy and lactation demonstrate that they are specifically regulated in tammar reproductive tissues. The increase in mesotocin receptors in gravid, but not in the non-gravid myometrium three days before birth may make the uterus responsive to the surge of mesotocin at birth. Since this rise is unilateral and only occurs in the gravid myometrium it must be due to local effects from the ipsilateral ovary or the feto-placental unit. Likewise, the down-regulation of mesotocin receptors in the contralateral, non-gravid myometrium may be due to its proximity to the developing follicle. The changing concentrations in the lactating and the adjacent, non-lactating mammary gland also reflect a differential regulation of mesotocin receptors, probably mediated via the sucking stimulus. Thus, local influences appear to be of primary importance in the regulation of mesotocin receptors during reproduction in this marsupial.     

Estrogen-induced gonadal sex reversal in the tammar wallaby

Estrogens have a feminizing effect on gonadal differentiation in fish, amphibians, reptiles, and birds. However, the role of estrogen during gonadal differentiation in mammals is less clear. We investigated the effect of estrogen on gonadal differentiation of male tammar wallabies. Male pouch young were treated orally with estradiol benzoate or oil from the day of birth, before seminiferous cords develop, to Day 25 postpartum and were killed at Day 50 postpartum. In all estrogen-treated neonates, a decrease in gonadal volume, volume of the seminiferous cords, thickness of the tunica albuginea, and number of germ cells was found. The stage of treatment affected the magnitude of the response. Two of three male young born prematurely after 25 days of gestation and treated subsequently with estradiol had ovary-like gonads, with well-developed cortical and medullary regions and primordial follicle formation. Furthermore, at Day 50 postpartum, many (21%) of the germ cells in these sex-reversed ovaries were in the leptotene and zygotene stages of meiosis, similar to female germ cells at the same stage of development. In the other males born on Day 26 of gestation or later, estradiol treatment from the day of birth caused development of dysgenetic testes, with abnormal Sertoli cells, atrophy of the seminiferous tubules and tunica albuginea, and absence of meiotic germ cells. In this marsupial, therefore, estradiol can induce either partial or complete transformation of the male gonads into an ovary with meiotic germ cells. These results confirm that estrogen can inhibit early testicular development, and that testis determination occurs during a narrow window of time.     

Developmental changes in ventricular myosin isoenzymes of the tammar wallaby

Ventricular myosin in eutherian mammals undergoes a perinatal change in response to a sharp rise in thyroid hormone levels during development. In this investigation, changes in ventricular myosin heavy chains (MyHCs) of the tammar wallaby (Macropus eugenii) from early pouch life to adulthood were analysed using native gel electrophoresis, SDS-PAGE and western blotting. Adult wallaby ventricle showed three myosin isoenzymes, V₁, V₂ and V₃; western blots using specific anti-α-MyHC and anti-β-MyHC antibodies showed their MyHC compositions to be αα, αβ and ββ, respectively. Ventricular muscle in early pouch joeys expressed predominantly β-MyHC. Up to 200 days, the time of initial pouch exit, α-MyHC content was around 5%. Thereafter, there was a sharp increase of α-MyHC expression to 35% by 242 days of age, eventually falling back to 23% in the adult. These changes correlate with known surges in plasma levels of thyroid hormones around pouch exit. The results suggest that ventricular myosins in a marsupial mammal also undergo a developmental change, and that marsupial ventricular myosins are thyroid responsive as in eutherians. The increased α-MyHC expression empowers the heart to meet the enhanced cardiovascular demands of out-of-pouch activity and the thermogenic action of thyroid hormones.     

Phosphoglycerate kinase pseudogenes in the tammar wallaby and other macropodid marsupials

Phosphoglycerate kinase (EC 2.7.2.3; PGK) exists in two forms in marsupials. PGK1 is an X-linked house-keeping enzyme, and PGK2 is a mainly testis-specific enzyme under autosomal control. We have used PGK1 probes derived from two closely related species of macropodid marsupials (kangaroos and wallabies) to demonstrate the existence of a large family of pseudogenes in the tammar wallaby (Macropus eugenii). Over 30 fragments are detectable after Taq digestion. We estimate that there are 25–30 copies per genome. Most are autosomally inherited and are apparently not closely linked. Only two restriction fragments that appeared to be sex linked could be detected. Varying degrees of hybridization of fragments to the probes suggest different levels of homology, and hence different ages of origin. The existence of two PGK1 homologous restriction fragments from the X and a large number from the autosomes was also demonstrated by somatic cell hybridization for two other macropodid species, the wallaroo (M. robustus) and the red kangaroo (M. rufus). These results are compared with those from human and mouse, and it is suggested that the propensity of PGK1 to form pseudogenes is an ancient (130 MYR BP) characteristic of mammals. The high level of polymorphism detected in the tammar makes these PGK1 probes potentially useful for measuring genetic variability in this species and other macropodids.     

Expression of estrogen receptors alpha and beta in term human myometrium

We used immunohistochemistry to compare the expression of estrogen receptors (ERalpha and ERbeta) in term myometria of 32 pregnant women divided in two groups. Group I comprised of 16 women in labour and group II included 16 non-laboring gravidas. We observed cytoplasmatic localization of both ER isoforms and no differences in the ER expression between the two groups of patients. The abundance and specific localization of ERs in human term myometrium seems to be independent of its contractile activity which may point to the specific role of those receptors in late pregnancy myometrium.     

Dichromatic colour vision in an Australian marsupial, the tammar wallaby

Despite earlier assertions that most mammals are colour blind, colour vision has in recent years been demonstrated in a variety of eutherian mammals from a wide range of different orders. This paper presents the first behavioural evidence from colour discrimination experiments, that an Australian marsupial, the tammar wallaby (Macropus eugenii), has dichromatic colour vision. In addition, the experiments show that the wallabies readily learn the relationship between the presented colours rather than the absolute hues. This provides a sensitive method to measure the location of the neutral-point, which is the wavelength of monochromatic light that is indistinguishable from white. This point is a diagnostic feature for dichromats. The spectral sensitivity of the wallabies' middle-wavelength-sensitive photoreceptor is known (peak: 539 nm) and the behavioural results imply that the sensitivity of the short-wavelength-sensitive receptor must be near 420 nm. These spectral sensitivities are similar to those found in eutherian mammals, supporting the view that the earliest mammals had dichromatic colour vision. Accepted: 18 July 1999     

Class I genes have split from the MHC in the tammar wallaby

Genes within the Major Histocompatibility Complex (MHC) are critical to the immune response and immunoregulation. Comparative studies have revealed that the MHC has undergone many changes throughout evolution yet in tetrapods the three different classes of MHC genes have maintained linkage, suggesting that there may be some functional advantage obtained by maintaining this clustering of MHC genes. Here we present data showing that class II and III genes, the antigen processing gene TAP2, and MHC framework genes are found together in the tammar wallaby on chromosome 2. Surprisingly class I loci were not found on chromosome 2 but were mapped to ten different locations spread across six chromosomes. This distribution of class I loci in the wallaby on nearly all autosomes is not a characteristic of all marsupials and may be a relatively recent phenomenon. It highlights the need for the inclusion of more than one marsupial species in comparative studies and raises questions regarding the functional significance of the clustering of MHC genes.     

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.     

Uterine and embryonic metabolism after diapause in the tammar wallaby, Macropus eugenii

Pouch young were removed from lactating tammars to terminate embryonic diapause. Uterine metabolism was assessed at periods afterwards by incubating endometrial explants with [3H]leucine, and measuring the incorporation into acid-soluble material. Blastocysts were incubated with [3H]uridine to assess uptake and incorporation into acid-soluble material. Uterine reactivation, shown by an increase in the rate of leucine incorporation into secreted protein, was evident by Day 4 after removal of pouch young and was significantly more in both secreted and tissue protein by Day 6. Both continued to increase in gravid and non-gravid uteri up to Day 12. By the end of pregnancy (Day 26) uterine metabolism in the gravid uterus produced 2-3 times more secreted protein than in the non-gravid uterus, demonstrating a local feto-placental influence on the uterus. Tissue incorporation had declined in endometrium of gravid and non-gravid uteri by Day 26. Day 5 embryos were metabolically more active than in quiescence, although expansion of the embryos was not seen until Day 9. The early reactivation of the uterus and embryo from diapause suggests that it is not triggered by the previously described peaks of progesterone and oestradiol in plasma at Day 5, although there may be an earlier, increased sensitivity to these steroids which allows uterine reactivation to precede changes in peripheral plasma concentration.     

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.     

An ovarian independent population of uterine estrogen receptors

Estrogen receptor levels in uteri from ovariectomized mice varied markedly with time after surgery; these changes were not due to nuclear translocation. Receptor cyclicity was abolished in ovariectomized-adrenalectomized mice, and receptor levels remained low. This indicates that the adrenals significantly influenced uterine estrogen receptor levels. However, these adrenal-mediated changes did not alter uterine responsiveness to estrogen stimulation.     

The influence of photoperiod on embryonic diapause in the tammar wallaby,macropus eugenii

After inoculation of mycoplasmas via ductus deferens in male dogs with a vas deferens fistula the influence on gonads and general state of health was investigated using clinical, andrological, spermatological, microbiological and histological methods.On day 23 and 29 respectively after inoculation two animals showed local reactions within the fistulated gonads which were identified as orchitis and epididymitis. Clinical abnormalities correlated well with the decrease of sperm motility and significant increase (p < 0,05) of aberrant spermatozoa.The microbiological and histological findings are discussed in the light of the incidence of clinical symptoms and the influence of a mycoplasmal infection.     

Lysophosphatidylcholine disrupts the acrosome of tammar wallaby (macropus eugenii) spermatozoa

The acrosomal status of wallaby spermatozoa was evaluated by light and electron microscopy after incubation in 1–100 μM lysophosphatidylcholine (LPC) for up to 120 min. Treatment with 1 and 10 μM LPC for 120 min did not lead to acrosomal loss, or detectable alteration to the acrosome, as detected by Bryan's staining and light microscopy. Incubation with 25 μM LPC had little effect on acrosomal loss, however statistically significant changes (P < 0.05) in the acrosomal matrix (altered) were detected after 10-min incubation by light microscopy. Around 50% of acrosomes were altered after 20-min incubation in 50 μM LPC (P < 0.001), and 40% of spermatozoa had lost their acrosome after 60-min incubation (P < 0.001). Treatment with 75 and 100 μM LPC led to rapid acrosomal loss from around 50% of spermatozoa within 10 min (P < 0.001), and by 60 min acrosomal loss was 70–80%. LPC, like the diacylglycerol DiC₈ (1,2-di-octanoyl-sn-glycerol), is thus an effective agent to induce loss of the relatively stable wallaby sperm acrosome, and it also induces changes within the acrosomal matrix. Ultrastructure of the LPC-treated spermatozoa revealed that the plasma membrane and the acrosomal membranes were disrupted in a manner similar to that seen after detergent treatment (Triton X-100). There was no evidence of point fusion between the plasma membrane overlying the acrosome and the outer acrosomal membrane. The plasma membrane was the first structure to disappear from the spermatozoa. The acrosomal membranes and matrix showed increasing disruption with time and LPC concentration. Wallaby spermatozoa incubated with LPC at concentrations that induced significant acrosomal loss also underwent a rapid decline in motility that suggested that acrosomal loss may be due to cell damage, rather than a physiological AR. This study concluded that LPC-induced acrosomal loss from tammar wallaby spermatozoa is due to its action as a natural detergent and not as a phosphoinositide pathway intermediate. The study further demonstrates the unusual stability of the marsupial acrosomal membranes. © 1993 Wiley-Liss, Inc.