Differentially Expressed miRNAs after GnRH Treatment and Their Potential Roles in FSH Regulation in Porcine Anterior Pituitary Cell

Abstract

Hypothalamic gonadotropin-releasing hormone (GnRH) is a major regulator of follicle-stimulating hormone (FSH) secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs) are small RNA molecules that control gene expression by imperfect binding to the 3′-untranslated region (3′-UTR) of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA) at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHβ mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR). Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHβ mRNA 3′-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially expressed miRNAs resolved in this study provide the first guide for post-transcriptional regulation of pituitary gonadotrope FSH secretion in pig, as well as in other mammals.

Follicular fluid and mural granulosa cells microRNA profiles vary in in vitro fertilization patients depending on their age and oocyte maturation stage

“…In our in silico prediction, we observed that several miRNAs were assigned to gene ontology processes termed as ''GnRH signaling pathway'' (among others). Previous studies have proven the effect of miRNA in GnRH signaling and also the effect of GnRH signaling in miRNA expression patterns (55)(56)(57)(58). The potential gene targets related to this process included kinases and calcium and MAPK signaling pathways, which are involved in FSH and LH production, which in turn could be affecting proper timing for oocyte meiosis and maturation.…”

Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis

“…The real-time PCR thermal profile was as follows: 5 min at 95 °C, 40 cycles of 30 s at 94 °C, 30 s at the corresponding annealing temperature (Tm) and 72 °C for 30 s, followed by 72 °C at 10 min, and 5S ribosomal RNA was used as an internal control for the PCR [17, 34]. The mRNAs primers were designed with Primer 5.0 (Table 1).…”

Rapid Communication: A MicroRNA-132/212 Pathway Mediates GnRH Activation of FSH Expression

“…Both miRNAs were demonstrated to target Zeb1, a repressor of Lhb gene transcription (15). FSH secretion has also been shown to be altered by overexpressing or inhibiting miR-361-3p in porcine primary pituitary cells, a miRNA that appeared to be down-regulated by GnRH (16). In neither case was there any evidence that miRNAs played a direct role in GnRH-induced gonadotropin synthesis.…”