ABSTRACT
Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cumulus Cells/metabolism , Gene Expression Regulation, Developmental/drug effects , In Vitro Oocyte Maturation Techniques/methods , Oxygen/pharmacology , Adenovirus E1B Proteins/metabolism , Analysis of Variance , Animals , Blotting, Western , DNA Primers/genetics , Dose-Response Relationship, Drug , Gene Expression Regulation, Developmental/physiology , Glucose Transporter Type 1/metabolism , Green Fluorescent Proteins/metabolism , Isoenzymes/metabolism , L-Lactate Dehydrogenase/metabolism , Lactate Dehydrogenase 5 , Mice , Mice, TransgenicABSTRACT
The IVM of mammalian cumulus-oocyte complexes (COCs) yields reduced oocyte developmental competence compared with oocytes matured in vivo. Altered cumulus cell function during IVM is implicated as one cause for this difference. We have conducted a microarray analysis of cumulus cell mRNA following IVM or in vivo maturation (IVV). Mouse COCs were sourced from ovaries of 21-day-old CBAB6F1 mice 46h after equine chorionic gonadotrophin (5IU, i.p.) or from oviducts following treatment with 5IU eCG (61h) and 5IU human chorionic gonadotrophin (13h). IVM was performed in α-Minimal Essential Medium with 50 mIU FSH for 17h. Three independent RNA samples were assessed using the Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA, USA). In total, 1593 genes were differentially expressed, with 811 genes upregulated and 782 genes downregulated in IVM compared with IVV cumulus cells; selected genes were validated by real-time reverse transcription-polymerase chain reaction (RT-PCR). Surprisingly, haemoglobin α (Hba-a1) was highly expressed in IVV relative to IVM cumulus cells, which was verified by both RT-PCR and western blot analysis. Because haemoglobin regulates O2 and/or nitric oxide availability, we postulate that it may contribute to regulation of these gases during the ovulatory period in vivo. These data will provide a useful resource to determine differences in cumulus cell function that are possibly linked to oocyte competence.
Subject(s)
Cumulus Cells/chemistry , Cumulus Cells/physiology , Microarray Analysis/methods , Oocytes/physiology , RNA, Messenger/genetics , Animals , Blotting, Western , Chorionic Gonadotropin/pharmacology , Cumulus Cells/metabolism , Gene Expression Profiling , Hemoglobin A/metabolism , In Vitro Oocyte Maturation Techniques/methods , Mice , Nitric Oxide/metabolism , Oligonucleotide Array Sequence Analysis , Oocytes/metabolism , Oxygen/metabolism , RNA, Messenger/drug effects , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The application of in vitro maturation (IVM) of oocytes as a technology to assist animal production and clinical infertility treatment remains poor because of the reduced developmental competence of oocytes after IVM, despite several decades of research. Reduced meiotic maturation rates, fertilization rates, and blastocyst production reveal short-term developmental insufficiency of oocytes when compared with in vivo-matured counterparts. However, there is an increasing body of evidence that demonstrates the capacity of IVM efficiency to be improved, some of which is reviewed here. Of more concern is the role that IVM of oocytes may play in causing or accentuating long-term development and health of fetuses and neonates after in vitro production of embryos and embryo transfer. This is a largely unexplored area, yet the application of such techniques, especially the safety of clinical IVM, is significant and requires monitoring before acceptance as a routine procedure.