The structure of the TH/INS locus and the parental allele expressed are not conserved between mammals.

Parent-of-origin-specific expression of imprinted genes is critical for successful mammalian growth and development. Insulin, coded by the INS gene, is an important growth factor expressed from the paternal allele in the yolk sac placenta of therian mammals. The tyrosine hydroxylase gene TH encodes an enzyme involved in dopamine synthesis. TH and INS are closely associated in most vertebrates, but the mouse orthologues, Th and Ins2, are separated by repeated DNA. In mice, Th is expressed from the maternal allele, but the parental origin of expression is not known for any other mammal so it is unclear whether the maternal expression observed in the mouse represents an evolutionary divergence or an ancestral condition. We compared the length of the DNA segment between TH and INS across species and show that separation of these genes occurred in the rodent lineage with an accumulation of repeated DNA. We found that the region containing TH and INS in the tammar wallaby produces at least five distinct RNA transcripts: TH, TH-INS1, TH-INS2, lncINS and INS. Using allele-specific expression analysis, we show that the TH/INS locus is expressed from the paternal allele in pre- and postnatal tammar wallaby tissues. Determining the imprinting pattern of TH/INS in other mammals might clarify if paternal expression is the ancestral condition which has been flipped to maternal expression in rodents by the accumulation of repeat sequences.

Recent advances in reproductive research in Australia and New Zealand: highlights from the Annual Meeting of the Society for Reproductive Biology, 2022.

In 2022, the Society for Reproductive Biology came together in Christchurch New Zealand (NZ), for its first face-to-face meeting since the global COVID-19 pandemic. The meeting showcased recent advancements in reproductive research across a diverse range of themes relevant to human health and fertility, exotic species conservation, and agricultural breeding practices. Here, we highlight the key advances presented across the main themes of the meeting, including advances in addressing opportunities and challenges in reproductive health related to First Nations people in Australia and NZ; increasing conservation success of exotic species, including ethical management of invasive species; improvements in our understanding of developmental biology, specifically seminal fluid signalling, ovarian development and effects of environmental impacts such as endocrine-disrupting chemicals; and leveraging scientific breakthroughs in reproductive engineering to drive solutions for fertility, including in assisted reproductive technologies in humans and agricultural industries, and for regenerative medicine.

Marsupials have monoallelic MEST expression with a conserved antisense lncRNA but MEST is not imprinted.

The imprinted isoform of the Mest gene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. The imprinted isoform has a distinct differentially methylated region (DMR) at its promoter in eutherian mammals but in marsupials, there are no differentially methylated CpG islands between the parental alleles. Here, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how imprinting of this gene evolved in mammals. By confirming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform evolved at the MEST gene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identified in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might be important in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study shows that MEST is not imprinted in marsupials. MEST imprinting in eutherians, therefore must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the differentially methylated CpG islands between the parental alleles.

Diffusion and distal linkages govern interchromosomal dynamics during meiotic prophase.

SignificanceEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process are the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent "pairing" of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study provides observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.

Riboceine Rescues Auranofin-Induced Craniofacial Defects in Zebrafish.

Craniofacial abnormalities are a common group of congenital developmental disorders that can require intensive oral surgery as part of their treatment. Neural crest cells (NCCs) contribute to the facial structures; however, they are extremely sensitive to high levels of oxidative stress, which result in craniofacial abnormalities under perturbed developmental environments. The oxidative stress-inducing compound auranofin (AFN) disrupts craniofacial development in wildtype zebrafish embryos. Here, we tested whether the antioxidant Riboceine (RBC) rescues craniofacial defects arising from exposure to AFN. RBC rescued AFN-induced cellular apoptosis and distinct defects of the cranial cartilage in zebrafish larvae. Zebrafish embryos exposed to AFN have higher expression of antioxidant genes gstp1 and prxd1, with RBC treatment partially rescuing these gene expression profiles. Our data suggest that antioxidants may have utility in preventing defects in the craniofacial cartilage owing to environmental or genetic risk, perhaps by enhancing cell survival.

Chlorogenic Acid Supplementation Benefits Zebrafish Embryos Exposed to Auranofin.

Antioxidant supplementation may potentially be beneficial for embryonic development to reduce complications associated with increased levels of oxidative stress. Chlorogenic acid, one of the key polyphenolic antioxidants in S. oleraceus, was evaluated for potential protective effects during embryonic development of zebrafish exposed to the teratogen auranofin. Zebrafish embryos were transiently exposed to auranofin to induce developmental abnormalities. Phenotypic abnormalities were scored based on their severity at day 5 post-fertilization. The embryos supplemented with 250 µM chlorogenic acid showed a significantly lower score in phenotypic abnormalities compared to non-supplemented embryos after auranofin exposure. Therefore, supplementation with a low dose of chlorogenic acid showed a protective effect from auranofin-induced deformities and encouraged normal growth in zebrafish embryos. This study provides further support for the potential of using antioxidant supplementation during embryonic development for protection against malformation.

Antioxidant treatment ameliorates phenotypic features of SMC1A-mutated Cornelia de Lange syndrome in vitro and in vivo.

Cornelia de Lange syndrome (CdLS) is a rare disease characterized by cognitive impairment, multisystemic alterations and premature aging. Furthermore, CdLS cells display gene expression dysregulation and genomic instability. Here, we demonstrated that treatment with antioxidant drugs, such as ascorbic acid and riboceine, reduced the level of genomic instability and extended the in vitro lifespan of CdLS cell lines. We also found that antioxidant treatment partially rescued the phenotype of a zebrafish model of CdLS. Gene expression profiling showed that antioxidant drugs caused dysregulation of gene transcription; notably, a number of genes coding for the zinc finger (ZNF)-containing Krueppel-associated box (KRAB) protein domain (KRAB-ZNF) were found to be downregulated. Taken together, these data suggest that antioxidant drugs have the potential to ameliorate the developmental phenotype of CdLS.

The Nucleoporin Nup2 Contains a Meiotic-Autonomous Region that Promotes the Dynamic Chromosome Events of Meiosis.

Meiosis is a specialized cellular program required to create haploid gametes from diploid parent cells. Homologous chromosomes pair, synapse, and recombine in a dynamic environment that accommodates gross chromosome reorganization and significant chromosome motion, which are critical for normal chromosome segregation. In Saccharomyces cerevisiae, Ndj1 is a meiotic telomere-associated protein required for physically attaching telomeres to proteins embedded in the nuclear envelope. In this study, we identified additional proteins that act at the nuclear periphery from meiotic cell extracts, including Nup2, a nonessential nucleoporin with a known role in tethering interstitial chromosomal loci to the nuclear pore complex. We found that deleting NUP2 affects meiotic progression and spore viability, and gives increased levels of recombination intermediates and products. We identified a previously uncharacterized 125 aa region of Nup2 that is necessary and sufficient for its meiotic function, thus behaving as a meiotic autonomous region (MAR). Nup2-MAR forms distinct foci on spread meiotic chromosomes, with a subset overlapping with Ndj1 foci. Localization of Nup2-MAR to meiotic chromosomes does not require Ndj1, nor does Ndj1 localization require Nup2, suggesting these proteins function in different pathways, and their interaction is weak or indirect. Instead, several severe synthetic phenotypes are associated with the nup2Δ ndj1Δ double mutant, including delayed turnover of recombination joint molecules, and a failure to undergo nuclear divisions without also arresting the meiotic program. These data suggest Nup2 and Ndj1 support partially overlapping functions that promote two different levels of meiotic chromosome organization necessary to withstand a dynamic stage of the eukaryotic life cycle.

Dietary Intake Influences Adult Fertility and Offspring Fitness in Zebrafish.

The burden of malnutrition, including both over- and undernutrition, is a major public health concern. Here we used a zebrafish model of diet-induced obesity to analyze the impact of dietary intake on fertility and the phenotype of the next generation. Over an eight-week period, one group received 60 mg of food each day (60 mg arm), while another received 5 mg (5 mg arm). At the end of the diet, the body mass index of the 60 mg arm was 1.5 fold greater than the 5 mg arm. The intervention also had a marked impact on fertility; breeding success and egg production in the 60 mg arm were increased 2.1- and 6.2-fold compared to the 5 mg arm, respectively. Transcriptome analysis of eggs revealed that transcripts involved in metabolic biological processes differed according to dietary intake. The progeny from the differentially fed fish were more likely to survive when the parents had access to more food. An intergenerational crossover study revealed that while parental diet did not influence weight gain in the offspring, the progeny of well-fed parents had increased levels of physical activity when exposed again to high nutrient availability. We conclude that dietary intake has an important influence on fertility and the subsequent fitness of offspring, even prior to breeding.

Embryonic oxidative stress results in reproductive impairment for adult zebrafish.

Exposure to environmental stressors during embryo development can have long-term effects on the adult organism. This study used the thioredoxin reductase inhibitor auranofin to investigate the consequences of oxidative stress during zebrafish development. Auranofin at low doses triggered upregulation of the antioxidant genes gstp1 and prdx1. As the dose was increased, acute developmental abnormalities, including cerebral hemorrhaging and jaw malformation, were observed. To determine whether transient disruption of redox homeostasis during development could have long-term consequences, zebrafish embryos were exposed to a low dose of auranofin from 6-24 hours post fertilization, and then raised to adulthood. The adult fish were outwardly normal in their appearance with no gross physical differences compared to the control group. However, these adult fish had reduced odds of breeding and a lower incidence of egg fertilization. This study shows that a suboptimal early life environment can reduce the chances of reproductive success in adulthood.

A neural crest origin for cohesinopathy heart defects.

Mutations in subunits or regulators of cohesin cause a spectrum of disorders in humans known as the 'cohesinopathies'. Cohesinopathies, including the best known example Cornelia de Lange syndrome (CdLS), are characterized by broad spectrum, multifactorial developmental anomalies. Heart defects occur at high frequency and can reach up to 30% in CdLS. The mechanisms by which heart defects occur are enigmatic, but assumed to be developmental in origin. In this study, we depleted cohesin subunit Rad21 by 70-80% in a zebrafish cohesinopathy model. The hearts of Rad21-depleted animals were smaller, often failed to loop, and functioned less efficiently than size-matched controls. Functional deficiency was accompanied by valve defects and reduced ejection fraction. Interestingly, neural crest cells failed to populate the heart and instead exhibited a wandering behavior. Consequently, these cells also failed to condense correctly into pharyngeal arches. Transcriptome analysis revealed that Wnt pathway, chemokine and cadherin genes are dysregulated at the time of cardiac neural crest development. Our results give insight into the etiology of heart defects in the cohesinopathies, and raise the possibility that mild mutations in cohesin genes may be causative of a fraction of congenital heart disease in human populations.