Postnatal developmental trajectory of sex-biased gene expression in the mouse pituitary gland

Abstract

Background The pituitary gland regulates essential physiological processes such as growth, pubertal onset, stress response, metabolism, reproduction, and lactation. While sex biases in these functions and hormone production have been described, the underlying identity, temporal deployment, and cell-type specificity of sex-biased pituitary gene regulatory networks are not fully understood. Methods To capture sex differences in pituitary gene regulation dynamics during postnatal development, we performed 3’ untranslated region sequencing and small RNA sequencing to ascertain gene and microRNA expression, respectively, across five postnatal ages (postnatal days 12, 22, 27, 32, 37) that span the pubertal transition in female and male C57BL/6J mouse pituitaries (n = 5–6 biological replicates for each sex at each age). Results We observed over 900 instances of sex-biased gene expression and 17 sex-biased microRNAs, with the majority of sex differences occurring with puberty. Using miRNA–gene target interaction databases, we identified 18 sex-biased genes that were putative targets of 5 sex-biased microRNAs. In addition, by combining our bulk RNA-seq with publicly available male and female mouse pituitary single-nuclei RNA-seq data, we obtained evidence that cell-type proportion sex differences exist prior to puberty and persist post-puberty for three major hormone-producing cell types: somatotropes, lactotropes, and gonadotropes. Finally, we identified sex-biased genes in these three pituitary cell types after accounting for cell-type proportion differences between sexes. Conclusion Our study reveals the identity and postnatal developmental trajectory of sex-biased gene expression in the mouse pituitary. This work also highlights the importance of considering sex biases in cell-type composition when understanding sex differences in the processes regulated by the pituitary gland.

Age, sex, and cell type-resolved hypothalamic gene expression across the pubertal transition in mice

“…Firstly, while puberty is initiated in the hypothalamus, it’s crosstalk between other organs, particularly the pituitary gland and gonads, are key to understanding pubertal regulation overall. We previously investigated sexual dimorphisms across the pubertal transition within the same mice in the pituitary gland and found more evidence of sex-biased gene expression in the pituitary than the hypothalamus, suggesting that the pituitary may have key role in sex differences in puberty [19]. Integrating our current RNA-seq data with that from the pituitary gland and gene expression patterns in the gonads may elucidate regulatory crosstalk between organs [131].…”

“…RNA-seq libraries were prepared using an automated - QuantSeq 3’mRNA-seq (Lexogen GmbH, Vienna) and Agilent NGS Workstation (Agilent Technologies, Santa Clara) at The Centre for Applied Genomics (TCAG) (Toronto, Canada) as per the manufacturer’s protocol (UTRSeq). The automated QuantSeq 3’mRNA-seq library construction was described in detail in Hou et al, 2022 [19]. Briefly, 250 ng of total RNA spiked-in with ERCC Spike-In Control Mix 1 (Ambion) as per the manufacturer’s protocol was used to generate cDNA.…”

“…Fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to examine the quality of sequenced reads. A customized script to trim both the polyAs and adapters at the end of the reads [19] was used. The script implemented a “back search” strategy to account for cases where a mixture of adapters and polyAs were seen at the end of the reads.…”

“…Only a few studies have characterized hypothalamic gene expression across the pubertal transition, and fewer have incorporated both age and sex into their design [10, 19]. Our lab previously utilized multiplexed qPCR to measure the expression of 178 candidate puberty GWAS and disease-related genes at PD12, 22, 27, 32, and 37 in many tissues, including the hypothalamus [10].…”

“…There, we found that most age-biased expression in the hypothalamus occurred before puberty, perhaps reflecting the cellular development occurring before puberty [2, 16, 20]. Our lab has also performed RNA-seq through 3’UTR profiling of these same mice in the pituitary gland, where we discovered cell-type specific expression and cellular trajectories that became increasingly sexually dimorphic through the pubertal transition [19]. Recently, Han et al interrogated the premammillary nucleus and arcuate nucleus transcriptomes of female mice between postnatal day (PD) 20 and diestrus females (PD50-PD60), as well as a leptin-inducible model of puberty in adult mice, highlighting the importance of neuropil and somatodendritic organization [20].…”