Project description:Morphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, with in vitro produced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. To investigate the transcriptional sex difference during early development, in vitro produced bovine blastocysts were collected and sexed by PCR. A significant male-biased development was observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, with 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such as SOX21 and PRDM14, and splicing factors FMR1 and HNRNPH2. Additionally, we revealed 1,555 significantly sex-biased differential alternative splicing (AS), predominantly skipped exons, mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, we identified 1,151 sex-biased differentially expressed isoforms (DEIs) associated with 1,017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage in DENND1B, DIS3L2, DOCK11, IL1RAPL2, and ZRSR2Y, indicating their sex-specific regulation in early embryo development. This study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis.

Project description:CLAMP transcription factor is a GA binding protein that recruits the male dosage compensation complex MSL to male X-chromosome, thus upregulating transcription level twice that of female X-chromosomes. Interestingly, CLAMP is essential for survival of both male and female individuals. However, what function it plays at the autosomes, especially in females is not known. It is well establised that GA-rich sequences to which CLAMP bind have evolved from polypyrimidine tracts that regulate splicing. Also, MALDI-MS data shows that CLAMP binds to RNA binding proteins involved in sex-specific splicing like, Squid. Furthermore, as part of MSL complex CLAMP interacts with MLE, a RNA helicase which is a known component of spliceosome complex conserved from flies to humans. Thus, we hypothesized that CLAMP plays a role in sex-spcific splicing. Given that CLAMP is a transcription factor, understanding mechanism of how it regulates splicing will add to our understanding about how regulators of gene expression shape the transcriptome differentially between males and females. We tested our hypothesis by identifying CLAMP dependent female and male sex-specific splicing events by analysing RNA-seq data from control as well as clampnull mutant male and female third instar larvae (L3) using suppa based time2splice pipeline (https://github.com/ashleymaeconard/time2splice). We identified 189 and 211 CLAMP dependent splicing events in female and male L3. Out of these 139 were female sex-specific splicing whereas 161 was male-sex-specific splicing. 50 splicing events identified as CLAMP dependent were non-specific to any particular sex, and were affect in both the sexes. Interestingly, we identified splicing of master regulator of sex-determination sxl affected by absence of CLAMP in females whereas splicing of one of the sex-determination pathway effectors, tra regulated by CLAMP in males. These findings indicate CLAMP plays important role in determining sex-specific transcript diversity in Drosophila.

Project description:Background: The placenta is vital for fetal development and its contributions to various developmental issues, such as pregnancy complications, fetal growth restriction, and maternal exposure, have been extensively studied in mice. Contrary to popular belief, the placenta forms mainly from fetal tissue; therefore, it has the same biological sex as the fetus it supports. However, while placental function is linked to increased risks of pregnancy complications and neurodevelopmental diseases in male offspring in particular, the sex-specific epigenetic (e.g., DNA methylation) and transcriptomic features of the late-gestation mouse placenta remain largely unknown.Methods: We collected male and female mouse placentas at late gestation (E18.5, n = 3/sex) and performed next-generation sequencing to identify genome-wide sex-specific differences in transcription and DNA methylation. Results: Our sex-specific analysis revealed 358 differentially expressed genes (DEGs) on autosomes, which were associated with signaling pathways involved in transmembrane transport and the responses to viruses and external stimuli. X chromosome DEGs (n = 39) were associated with different pathways, including those regulating chromatin modification and small GTPase-mediated signal transduction. Sex-specific differentially methylated regions (DMRs) were more common on the X chromosomes (n = 3756) than on autosomes (n = 1705). Interestingly, while most X chromosome DMRs had higher DNA methylation levels in female placentas and tended to be included in CpG dinucleotide-rich regions, 73% of autosomal DMRs had higher methylation levels in male placentas and were distant from CpG-rich regions. Several sex-specific DEGs were correlated with sex-specific DMRs. A subset of the sex-specific DMRs present in late-stage placentas were already established in mid-gestation (E10.5) placentas, while others were acquired later in placental development.Conclusion: Our study provides comprehensive lists of sex-specific DEGs and DMRs that collectively cause profound differences in the DNA methylation and gene expression profiles of late-gestation mouse placentas. Our results demonstrate the importance of incorporating sex-specific analyses into epigenetic and transcription studies to enhance the accuracy and comprehensiveness of their conclusions and help address the significant knowledge gap regarding how sex differences influence placental function.

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Sex-related differences in gene expression in early-stage bladder cancer revealed by whole-transcriptome sequencing

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