Mammalian target of rapamycin/eukaryotic initiation factor 4F pathway regulates follicle growth and development of theca cells in mice

The aim of the present study was to clarify the roles of the mammalian target of rapamycin (mTOR) signalling pathway in follicular growth and development of thecal cells. Using in vivo-grown and in vitro-cultured ovaries, histological changes were evaluated using haematoxylin and eosin (HE) staining. Differentially expressed genes (DEGs) from 0 day post partum (d.p.p.) to 8 d.p.p. ovaries were screened by microarray and verified by quantitative real-time polymerase chain reaction. Forty-two DEGs related to cell proliferation and differentiation were screened out, with most DEGs being related to the to mTOR signalling pathway. Then, 3 d.p.p. ovaries were retrieved and used to verify the role of mTOR signalling in follicle and thecal cell development using its activators (Ras homologue enriched in brain (Rheb) and GTP) and inhibitor (rapamycin). The development of follicles and thecal cells was significantly impaired in ovaries cultured in vitro Day 3 to Day 8. In in vitro-cultured ovaries, Rheb and GTP (is 100 ng mL–1 Rheb and 500 ng mL–1 GTP for 48 h) significantly increased follicle diameter, the percentage of primary and secondary follicles and the umber of thecal cells, and upregulated expression of mTOR, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), eukaryotic initiation factor (eIF) 4F and cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1). Rapamycin (10 nM rapamycin for 24 h) had opposite effects to those of Rheb and GTP, and partly abrogated (significant) the effects of Rheb and GTP when added to the culture in combination with these drugs. Thus, mTOR signalling plays an important role in follicle growth and thecal cell development.

KIT receptor-positive cells in the bovine corpus luteum are primarily theca-derived small luteal cells

The tyrosine kinase KIT receptor, the protooncogene CD117, plays a key role in growth and maturation of oocytes and follicles. Relevant data are sparse for the corpus luteum (CL). We first confirmed the presence ofKITmRNA and KIT protein in bovine CL homogenates. We then localized KIT-positive (KIT+) cells in CL sections by immunohistochemistry. At the CL stage of early development, the former theca transforming into capsule/septa showed a strong band-like KIT+ immunoresponse. In addition, CD45+ leukocytes in septa included subpopulations of CD45+/KIT+ and CD14+/KIT+ leukocytes as validated by double immunofluorescence localization. At the early secretory stage, KIT+ cells appeared within the septa/capsule region and in the periphery of the CL parenchyma, there forming a complex network. This was separate from the capillary bed as determined by double staining for CD117 and FVIII-related endothelial cell antigen (FVIIIr). The KIT+ network coincided with cells positive for cytochrome P450 17α-hydroxylase, a thecal cell-specific enzyme. The late secretory stage was defined by an advanced manifestation of the KIT+ network in the CL periphery. At the stage of regression, the KIT+ network was absent. The CL of pregnancy expressed high levels ofKITmRNA and KIT protein uniformly throughout pregnancy. The KIT+ immunolocalization revealed small fibroblast-like cells, luteal cells with granules, and clusters of large luteal cells with staining of the cell membrane. We conclude that a majority of KIT+ cells in the bovine CL are primarily theca-derived small luteal cells, and that a minority represent KIT+ leukocytes, in some cases KIT+ monocytes.