Research Resource: Preovulatory LH Surge Effects on Follicular Theca and Granulosa Transcriptomes

Abstract

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle.

Regulation of ovulatory genes in bovine granulosa cells: lessons from siRNA silencing of PTGS2

“…Another important observation is related with PTX3, which is considered as a bona fide ovulatory protein necessary for ECM stability during cumulus expansion (Varani et al 2002, Salustri et al 2004. PTX3, as shown before and also in this study, is induced in GCs by the LH surge in mice and bovine (Varani et al 2002, Christenson et al 2013. However, our study demonstrates that although GCs do express PTX3, it was not induced in isolated cell culture but rather inhibited by elevated cAMP levels.…”

“…Follicles were collected from Holstein-Friesian cows as described (Vanselow et al 2010, Christenson et al 2013. Large dominant follicles before LH surge were collected after carefully, monitoring a growing cohort of follicles in normally cycling cows by transrectal ultrasonography (Aloka SSD-500, Aloka GmbH, Meerbusch, Germany).…”

“…The fluid was centrifuged (2 min, 400 g) and the sediment cells were frozen in liquid nitrogen and stored at K80 8C for RNA preparation. RNA isolation, RT and qPCR were carried out as described previously (Christenson et al 2013). …”

Characterization of microRNAs differentially expressed during bovine follicle development

“…Each of these developmental steps involves sequential and profound changes in tissue structure and function, requiring finely tuned and coordinated changes in the expression of thousands of genes. Transcriptome analyses have identified many such genes during follicle selection, maturation, and the follicle-luteal transition (Gilbert et al 2011, Rao et al 2011, Christenson et al 2013, although relatively little is still known about the molecular mechanisms regulating their expression, particularly at the posttranscriptional level.…”

Toward building the cow folliculome

“…We do not have theca cell data but Lane Christenson has published a recent paper where the microarray data revealed that of the 24,016 probe sets which represent, 11,548 independent annotated bovine genes and 7091 unannotated expressed sequence tags, the majority of the probe sets, 10,770 were present in all 3 cell types (mural, antral and theca) before the LH surge, respectively and the theca cells only showed 1247 unique probes (Christenson et al, 2013). The cumulus on the other side is considered as a sub-population of granulosa where some differentiation events are prevented by the proximity of the oocyte (Eppig et al, 1998;Diaz et al, 2007).…”