Transcriptomic diversification of developing cumulus and mural granulosa cells in mouse ovarian follicles

Abstract

Cumulus cells and mural granulosa cells (MGCs) have functionally distinct roles in antral follicles, and comparison of their transcriptomes at a global and systems level can propel future studies on mechanisms underlying their functional diversity. These cells were isolated from small and large antral follicles before and after stimulation of immature mice with gonadotropins, respectively. Both cell types underwent dramatic transcriptomic changes, and differences between them increased with follicular growth. Although cumulus cells of both stages of follicular development are competent to undergo expansion in vitro, they were otherwise remarkably dissimilar with transcriptomic changes quantitatively equivalent to those of MGCs. Gene ontology analysis revealed that cumulus cells of small follicles were enriched in transcripts generally associated with catalytic components of metabolic processes, while those from large follicles were involved in regulation of metabolism, cell differentiation, and adhesion. Contrast of cumulus cells versus MGCs revealed that cumulus cells were enriched in transcripts associated with metabolism and cell proliferation while MGCs were enriched for transcripts involved in cell signaling and differentiation. In vitro and in vivo models were used to test the hypothesis that higher levels of transcripts in cumulus cells versus MGCs is the result of stimulation by oocyte-derived paracrine factors (ODPFs). Surprisingly ∼48% of transcripts higher in cumulus cells than MGCs were not stimulated by ODPFs. Those stimulated by ODPFs were mainly associated with cell division, mRNA processing, or the catalytic pathways of metabolism, while those not stimulated by ODPFs were associated with regulatory processes such as signaling, transcription, phosphorylation, or the regulation of metabolism.

Keywords: cumulus cells; mouse; mural granulosa cells; oocyte; oocyte-derived paracrine factors; ovarian follicle; transcriptome.

FIG. 1

Photomicrographs of histological sections of (A) a small antral follicle (SAF) typical of those in the ovaries of 22-day-old B6SJLF1 mice not stimulated with eCG and (B) a large antral follicle (LAF) typical of those in the ovaries 44 h postinjection with 5 international units eCG showing mural granulosa cells (MGC), cumulus cells (CC), and oocyte (oo). C) The follicular remnants after puncturing an LAF with a needle to express (yellow arrow) MGC and the oocyte cumulus cell complex. Note that many MGCs most immediately adjacent to the follicular basal lamina remain unexpressed and therefore not included in the microarray analyses. Bar = 100 μm.

FIG. 2

Number of transcripts expressed at higher levels in SAFs of unstimulated ovaries (blue bars) versus those of LAFs 44 h post-eCG-stimulation (red bars) in cumulus cells or MGCs. Only transcripts that encode proteins with annotated biological functions are included.

FIG. 3

VLAD depiction of GO terms in the contrast of cumulus cells from SAFs versus LAFs. The top 25 GO terms, selected by VLAD on the basis of local maximum P value, are shown. A local maximum term is one whose significance (−log [p]) is greater than its immediate neighbors (children or parents). The size of the rectangle is proportional to the P value; the larger the rectangle the lower, and more significant, is the P value. The green portion of the rectangle shows the relative contribution of cumulus cell transcripts from SAFs to that GO term while the red portion shows the relative contribution of transcripts from LAFs. Supplemental Table S1 presents a complete list of transcripts expressed differently in SAFs and LAFs for each GO term.

FIG. 4

Number of transcripts expressed at levels higher in cumulus cells versus MGCs (blue bars) or higher in MGCs versus cumulus cells (red bars). Samples were taken from the SAFs of 22-day-old mice or the LAFs of 22-day-old mice 44 h poststimulation with eCG. Only transcripts that encode proteins with annotated biological functions are included.

FIG. 5

VLAD depiction of GO terms in the contrast of cumulus cells versus MGCs from SAFs. The top 25 GO terms, selected by VLAD on the basis of local maximum P value, are shown. The size of the rectangle is proportional to the P value; the larger the rectangle the lower, and more significant, is the P value. The green portion of the rectangle shows the relative contribution of cumulus cell transcripts from SAFs to that GO term while the red portion shows the relative contribution of MGC transcripts from SAFs. Supplemental Table S3 presents a complete list of transcripts expressed differently in SAFs and LAFs for each GO term.

FIG. 6

VLAD depiction of GO terms in the contrast of cumulus cells versus MGCs from LAFs. The top 25 GO terms, selected by VLAD on the basis of local maximum P value, are shown. The size of the rectangle is proportional to the P value; the larger the rectangle the lower, and more significant, is the P value. The green portion of the rectangle shows the relative contribution of cumulus cell transcripts from LAFs to that GO term while the red portion shows the relative contribution of transcripts from LAFs. Supplemental Table S4 presents a complete list of transcripts expressed differently in SAFs and LAFs for each GO term.

FIG. 7

VLAD depiction of GO terms in the contrast of consensus transcripts higher in cumulus cells than MGCs stimulated by ODPFs or not. The top 25 GO terms, selected by VLAD on the basis of local maximum P value, are shown. The size of the rectangle is proportional to the P value; the larger the rectangle the lower, and more significant, is the P value. The green portion of the rectangle shows the relative contribution of cumulus cell transcripts not stimulated by ODPFs to that GO term while the red portion shows the relative contribution of transcripts stimulated by ODPFs. Supplemental Table S8 presents complete list of transcripts expressed differently in SAFs and LAFs for each GO term.

FIG. 8

Working model depicting possible mechanisms regulating the expression of two groups of transcripts enriched in cumulus cells relative to MGCs. These groups of transcripts affect the diversity in functions of these cell types. Both groups are expressed at higher levels in cumulus cells than in MGCs. One group of transcripts is elevated in cumulus by oocyte-derived paracrine factors (ODPFs) and the other is not. The group whose expression is stimulated by ODPFs includes, among others, transcripts enriched in the GO terms associated with cell division and catalytic enzymes of metabolic pathways. The transcripts not affected by ODPFs include, among others, transcripts enriched in the GO terms associated with the regulation of these pathways and various aspects of gene expression (see Fig. 7 and Supplemental Table S8). The transcripts unaffected by ODPFs may be expressed at higher levels in cumulus cells than MGCs because expression is suppressed in the MGCs.