The origin and evolution of genomic imprinting and viviparity in mammals

Genomic imprinting is widespread in eutherian mammals. Marsupial mammals also have genomic imprinting, but in fewer loci. It has long been thought that genomic imprinting is somehow related to placentation and/or viviparity in mammals, although neither is restricted to mammals. Most imprinted genes are expressed in the placenta. There is no evidence for genomic imprinting in the egg-laying monotreme mammals, despite their short-lived placenta that transfers nutrients from mother to embryo. Post natal genomic imprinting also occurs, especially in the brain. However, little attention has been paid to the primary source of nutrition in the neonate in all mammals, the mammary gland. Differentially methylated regions (DMRs) play an important role as imprinting control centres in each imprinted region which usually comprises both paternally and maternally expressed genes (PEGs and MEGs). The DMR is established in the male or female germline (the gDMR). Comprehensive comparative genome studies demonstrated that two imprinted regions, PEG10 and IGF2-H19, are conserved in both marsupials and eutherians and that PEG10 and H19 DMRs emerged in the therian ancestor at least 160 Ma, indicating the ancestral origin of genomic imprinting during therian mammal evolution. Importantly, these regions are known to be deeply involved in placental and embryonic growth. It appears that most maternal gDMRs are always associated with imprinting in eutherian mammals, but emerged at differing times during mammalian evolution. Thus, genomic imprinting could evolve from a defence mechanism against transposable elements that depended on DNA methylation established in germ cells

Postnatal epigenetic reprogramming in the germline of a marsupial, the tammar wallaby

Background: Epigenetic reprogramming is essential to restore totipotency and to reset genomic imprints during mammalian germ cell development and gamete formation. The dynamic DNA methylation change at DMRs (differentially methylated regions) within imprinted domains and of retrotransposons is characteristic of this process. Both marsupials and eutherian mammals have genomic imprinting but these two subgroups have been evolving separately for up to 160 million years. Marsupials have a unique reproductive strategy and deliver tiny, altricial young that complete their development within their mother's pouch. Germ cell proliferation in the genital ridge continues after birth in the tammar wallaby (Macropus eugenii), and it is only after 25 days postpartum that female germ cells begin to enter meiosis and male germ cells begin to enter mitotic arrest. At least two marsupial imprinted loci (PEG10 and H19) also have DMRs. To investigate the evolution of epigenetic reprogramming in the marsupial germline, here we collected germ cells from male pouch young of the tammar wallaby and analysed the methylation status of PEG10 and H19 DMR, an LTR (long terminal repeat) and a non-LTR retrotransposons. Results: Demethylation of the H19 DMR was almost completed by 14 days postpartum and de-novo methylation started from 34 days postpartum. These stages correspond to 14 days after the completion of primordial germ cell migration into genital ridge (demethylation) and 9 days after the first detection of mitotic arrest (re-methylation) in the male germ cells. Interestingly, the PEG10 DMR was already unmethylated at 7 days postpartum, suggesting that the timing of epigenetic reprogramming is not the same at all genomic loci. Retrotransposon methylation was not completely removed after the demethylation event in the germ cells, similar to the situation in the mouse. Conclusions: Thus, despite the postnatal occurrence of epigenetic reprogramming and the persistence of genome-wide undermethylation for 20 days in the postnatal tammar, the relative timing and mechanism of germ cell reprogramming are conserved between marsupials and eutherians. We suggest that the basic mechanism of epigenetic reprogramming had already been established before the marsupial-eutherian split and has been faithfully maintained for at least 160 million years and may reflect the timing of the onset of mitotic arrest in the male germline.ArticleEPIGENETICS & CHROMATIN. 6:14 (2013)journal articl

Placental expression of pituitary hormones is an ancestral feature of therian mammals

<p>Abstract</p> <p>Background</p> <p>The placenta is essential for supplying nutrients and gases to the developing mammalian young before birth. While all mammals have a functional placenta, only in therian mammals (marsupials and eutherians) does the placenta closely appose or invade the uterine endometrium. The eutherian placenta secretes hormones that are structurally and functionally similar to pituitary growth hormone (GH), prolactin (PRL) and luteinizing hormone (LH). Marsupial and eutherian mammals diverged from a common ancestor approximately 125 to 148 million years ago and developed distinct reproductive strategies. As in eutherians, marsupials rely on a short-lived but functional placenta for embryogenesis.</p> <p>Results</p> <p>We characterized pituitary GH, GH-R, IGF-2, PRL and LHβ in a macropodid marsupial, the tammar wallaby, <it>Macropus eugenii</it>. These genes were expressed in the tammar placenta during the last third of gestation when most fetal growth occurs and active organogenesis is initiated. The mRNA of key growth genes GH, GH-R, IGF-2 and PRL were expressed during late pregnancy. We found significant up-regulation of GH, GH-R and IGF-2 after the start of the rapid growth phase of organogenesis which suggests that the placental growth hormones regulate the rapid phase of fetal growth.</p> <p>Conclusions</p> <p>This is the first demonstration of the existence of pituitary hormones in the marsupial placenta. Placental expression of these pituitary hormones has clearly been conserved in marsupials as in eutherian mammals, suggesting an ancestral origin of the evolution of placental expression and a critical function of these hormones in growth and development of all therian mammals.</p

Effects of progesterone on parturition in the tammar, Macropus eugenii

Summary. Tammar wallabies were treated with progesterone injections or implants during late pregnancy to determine whether progesterone withdrawal was essential for parturition. Neither physiological (implanted group) nor pharmacological (injected group) levels of circulating progesterone prevented parturition occurring at about the expected time in about two-thirds of animals that were pregnant. The neonates of both groups were normal in size and weight, but about a third of treated pregnant animals retained their fetuses or aborted. The retained fetuses were retarded in development. Therefore, progesterone treatment had no influence on the duration of gestation, or parturition, in the tammar wallaby, but high progesterone concentrations may interfere with the normal course of development and birth in a proportion of treated animals

Comparative analysis of the mammalian WNT4 promoter

BACKGROUND: WNT4 is a critical signalling molecule in embryogenesis and homeostasis, but the elements that control its transcriptional regulation are largely unknown. This study uses comparative cross species sequence and functional analyses between humans and a marsupial (the tammar wallaby,Macropus eugenii) to refine the mammalian Wnt4 promoter. RESULTS: We have defined a highly conserved 89 bp minimal promoter region in human WNT4 by comparative analysis with the tammar wallaby. There are many conserved transcription factor binding sites in the proximal promoter region, including SP1, MyoD, NFkappaB and AP2, as well as highly conserved CpG islands within the human, mouse and marsupial promoters, suggesting that DNA methylation may play an important role in WNT4 transcriptional regulation. CONCLUSION: Using a marsupial model, we have been able to provide new information on the transcriptional regulators in the promoter of this essential mammalian developmental gene, WNT4. These transcription factor binding sites and CpG islands are highly conserved in two disparate mammals, and are likely key controlling elements in the regulation of this essential developmental gene

Expression and protein localisation of IGF2 in the marsupial placenta

<p>Abstract</p> <p>Background</p> <p>In eutherian mammals, genomic imprinting is critical for normal placentation and embryo survival. <it>Insulin-like growth factor 2 </it>(<it>IGF2</it>) is imprinted in the placenta of both eutherians and marsupials, but its function, or that of any imprinted gene, has not been investigated in any marsupial. This study examines the role of <it>IGF2 </it>in the yolk sac placenta of the tammar wallaby, <it>Macropus eugenii</it>.</p> <p>Results</p> <p><it>IGF2 </it>mRNA and protein were produced in the marsupial placenta. Both IGF2 receptors were present in the placenta, and presumably mediate IGF2 mitogenic actions. <it>IGF2 </it>mRNA levels were highest in the vascular region of the yolk sac placenta. IGF2 increased <it>vascular endothelial growth factor </it>expression in placental explant cultures, suggesting that IGF2 promotes vascularisation of the yolk sac.</p> <p>Conclusion</p> <p>This is the first demonstration of a physiological role for any imprinted gene in marsupial placentation. The conserved imprinting of <it>IGF2</it> in this marsupial and in all eutherian species so far investigated, but not in monotremes, suggests that imprinting of this gene may have originated in the placenta of the therian ancestor.</p

ATRX has a critical and conserved role in mammalian sexual differentiation

BACKGROUND X-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities. RESULTS We examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients. CONCLUSIONS There is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution.This study was supported by a National Health and Medical Research Council R D Wright Fellowship to AJP the Australian Research Council Centre of Excellence in Kangaroo Genomics and a Federation Fellowship to MBR

A novel MSMB-related microprotein in the postovulatory egg coats of marsupials

<p>Abstract</p> <p>Background</p> <p>Early marsupial conceptuses differ markedly from those of eutherian mammals, especially during cleavage and early blastocyst stages of development. Additionally, in marsupials the zona pellucida is surrounded by two acellular layers, the mucoid coat and shell, which are formed from secretions from the reproductive tract.</p> <p>Results</p> <p>We report the identification of a novel postovulatory coat component in marsupials, which we call uterinesecreted microprotein (USM). USM belongs to a family of disulfide-rich microproteins of unconfirmed function that is found throughout deuterostomes and in some protostomes, and includes β-microseminoprotein (MSMB) and prostate-associated microseminoprotein (MSMP). We describe the evolution of this family in detail, including USM-related sequences in other vertebrates. The orthologue of <it>USM </it>in the tammar wallaby, <it>USM1</it>, is expressed by the endometrium with a dynamic temporal profile, possibly under the control of progesterone.</p> <p>Conclusions</p> <p>USM appears to have evolved in a mammalian ancestor specifically as a component of the postovulatory coats. By analogy with the known properties of MSMB, it may have roles in regulating sperm motility/survival or in the immune system. However, its C-terminal domain is greatly truncated compared with MSMB, suggesting a divergent function.</p

Differential roles of TGIF family genes in mammalian reproduction

<p>Abstract</p> <p>Background</p> <p>TG-interacting factors (TGIFs) belong to a family of TALE-homeodomain proteins including TGIF1, TGIF2 and TGIFLX/Y in human. Both TGIF1 and TGIF2 act as transcription factors repressing TGF-β signalling. Human <it>TGIFLX </it>and its orthologue, <it>Tex1 </it>in the mouse, are X-linked genes that are only expressed in the adult testis. <it>TGIF2 </it>arose from <it>TGIF1 </it>by duplication, whereas <it>TGIFLX </it>arose by retrotransposition to the X-chromosome. These genes have not been characterised in any non-eutherian mammals. We therefore studied the TGIF family in the tammar wallaby (a marsupial mammal) to investigate their roles in reproduction and how and when these genes may have evolved their functions and chromosomal locations.</p> <p>Results</p> <p>Both <it>TGIF1 </it>and <it>TGIF2 </it>were present in the tammar genome on autosomes but <it>TGIFLX </it>was absent. Tammar <it>TGIF1 </it>shared a similar expression pattern during embryogenesis, sexual differentiation and in adult tissues to that of <it>TGIF1 </it>in eutherian mammals, suggesting it has been functionally conserved. Tammar <it>TGIF2 </it>was ubiquitously expressed throughout early development as in the human and mouse, but in the adult, it was expressed only in the gonads and spleen, more like the expression pattern of human <it>TGIFLX </it>and mouse <it>Tex1</it>. Tammar <it>TGIF2 </it>mRNA was specifically detected in round and elongated spermatids. There was no mRNA detected in mature spermatozoa. TGIF2 protein was specifically located in the cytoplasm of spermatids, and in the residual body and the mid-piece of the mature sperm tail. These data suggest that tammar <it>TGIF2 </it>may participate in spermiogenesis, like <it>TGIFLX </it>does in eutherians. <it>TGIF2 </it>was detected for the first time in the ovary with mRNA produced in the granulosa and theca cells, suggesting it may also play a role in folliculogenesis.</p> <p>Conclusions</p> <p>The restricted and very similar expression of tammar <it>TGIF2 </it>to X-linked paralogues in eutherians suggests that the evolution of <it>TGIF1</it>, <it>TGIF2 </it>and <it>TGIFLX </it>in eutherians was accompanied by a change from ubiquitous to tissue-specific expression. The distribution and localization of TGIF2 in tammar adult gonads suggest that there has been an ultra-conserved function for the TGIF family in fertility and that <it>TGIF2 </it>already functioned in spermatogenesis and potentially folliculogenesis long before its retrotransposition to the X-chromosome of eutherian mammals. These results also provide further evidence that the eutherian X-chromosome has actively recruited sex and reproductive-related genes during mammalian evolution.</p