Transcriptome analysis of rhesus monkey failed-to-mature oocytes: deficiencies in transcriptional regulation and cytoplasmic maturation of the oocyte mRNA population

Abstract

Study question: Which different pathways and functions are altered in rhesus monkey oocytes that fail to mature after an ovulatory stimulus?

Summary answer: Failed to mature (FTM) oocytes complete a large portion of the transition in transcriptome composition associated with normal maturation, but also manifest numerous differences that indicate incomplete transcriptional repression and cytoplasmic maturation affecting multiple processes.

What is known already: Oocyte maturation defects contribute to unexplained female infertility. Failure of some oocytes to undergo germinal vesicle breakdown or progress to second meiotic metaphase in response to an ovulatory stimulus can limit the number of high quality oocytes available for ART.

Study design, size, duration: The transcriptome of rhesus monkey oocytes that failed to mature (FTM; n = 11, 5 donors) in response to an ovulatory stimulus in vivo was compared to those of normal germinal vesicle stage (GV, n = 7, 2 donors) and metaphase II stage (MII, n = 7, 5 donors) oocytes by RNA-sequencing (RNAseq).

Participants/materials, setting, methods: Female rhesus monkeys of normal breeding age (6-12 years old) and with regular menstrual cycles were used. Animals underwent a controlled ovarian stimulation protocol for the collection of oocytes by ultrasound-guided needle aspiration of follicles.

Main results and the role of chance: We obtained a high quality RNAseq dataset consisting of n = 7, n = 7, and n = 11 libraries for normal GV, normal MII and FTM oocytes, respectively. Total reads acquired were an average of 34 million for each GV sample, 41 million for each FTM sample and 59 million for each MII oocyte sample. Approximately 44% of the total reads were exonic reads that successfully aligned to the rhesus monkey genome as unique non-rRNA gene transcript sequences, providing high depth of coverage. Approximately 44% of the mRNAs that undergo changes in abundance during normal maturation display partial modulations to intermediate abundances, and 9.2% fail to diverge significantly from GV stage oocytes. Additionally, a small group of mRNAs are grossly mis-regulated in the FTM oocyte. Differential expression was seen for mRNAs associated with mitochondrial functions, fatty acid beta oxidation, lipid accumulation, meiosis, zona pellucida formation, Hippo pathway signaling, and maternal mRNA regulation. A deficiency DNA methyltransferase one mRNA expression indicates a potential defect in transcriptional silencing.

Large scale data: All RNAseq data are published in the Gene Expression Omnibus Database (GSE112536).

Limitations, reasons for caution: These results do not establish cause of maturation failure but reveal novel correlates of incompetence to mature. Transcriptome studies likely do not capture all post-transcriptional or post-translational events that inhibit maturation, but do reveal mRNA expression changes that lie downstream of such events or that are related to effects on upstream regulators. The use of an animal model allows the study of oocyte maturation failure independent of covariates and confounders, such as pre-existing conditions of the female, which is a significant concern in human studies. Depending on the legislation, it may not be possible to collect and study oocytes from healthy women; and using surplus oocytes from patients undergoing ART may introduce confounders that vary from case to case. FTM oocytes were at various stages of meiotic progression, so correlates of specific times of arrest are not revealed. All the FTM oocytes failed to respond appropriately to an ovulatory stimulus in vivo. Therefore, this analysis informs us about common transcriptome features associated with meiotic incompetence.

Wider implications of the findings: These results reveal that some diagnostic markers of oocyte quality may not reflect developmental competence because even meiotically incompetent oocytes display many normal gene expression features. The results also reveal potential mechanisms by which maternal and environmental factors may impact transcriptional repression and cytoplasmic maturation, and prevent oocyte maturation.

Study funding/competing interest(s): This work was supported by grants from the National Institutes of Health Office of Research Infrastructure Programs Division of Comparative Medicine Grants R24 [OD012221 to K.E.L., OD011107/RR00169 (California National Primate Research Center), and OD010967/RR025880 to C.A.V.]; the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under the award number T32HD087166; MSU AgBioResearch, Michigan State University. Authors have nothing to disclose.

Figure 1

Schematic figure showing transitions of mRNA expression of the failed to mature oocytes in comparison to normal maturation in the rhesus monkey (Categories A–G). Differentially expressed genes (DEGs) identified by Cuffdiff analysis and defined as those with a q-value (false discovery rate) <0.05 are shown. The normal maturation (NM) category represents the mRNAs that are differentially expressed between germinal vesicle (GV) and metaphase II (MII) oocytes. Category A represents mRNAs that changed during normal maturation, being differentially expressed between GV and MII oocytes (i.e. in NM group) and between GV and failed to mature (FTM), but not between MII and FTM oocytes. Category B represents mRNAs that changed partially in FTM oocytes, reaching an abundance between and significantly different from GV and MII oocytes. Category C represents mRNAs that failed to be modulated, being differentially expressed between MII and GV oocytes, but not acquiring a statistical difference in FTM oocytes compared to GV oocytes. Category D represents mRNAs that displayed excessive modulation in FTM oocytes beyond the change seen in NM. Category E represents mRNAs that are oppositely regulated in FTM compared to MII relative to initial GV level of expression. Categories F (increased expression) and G (decreased expression) represent mRNAs displaying significant differences between FTM and both GV and MII, but not in NM.

Figure 2

Principle component analyses and frequency distribution graphs were used to visualize the differences between FTM, GV and MII oocytes. A. Principle component (PC) analysis plots show sample relationships between GV, FTM and MII samples. There is a clear delineation between GV and MII oocytes with FTM falling in between the two. B–D. Frequency distributions of the log two-fold changes between comparisons are indicated. Note that the overall variance in mRNA expression patterns was no greater among FTM oocytes than GV or MII stage oocytes. B = GV to FTM; C = FTM to MII; D = GV to MII. All graphs were created in R package ggplot2.

Figure 3

Distribution of mRNA expression values for FTM oocytes amongst categories A–E in comparison to NM. For each category, the total number of genes and percentage of the NM gene set are shown. Category NM includes mRNAs that are differentially expressed between GV and MII oocytes. Category A includes mRNAs that are differentially expressed between GV and MII and between GV and FTM, but not between MII and FTM. Category B includes NM mRNAs that are modulated in abundance during NM but only partially modulated in FTM oocytes (FTM different from both GV and MII stage oocytes). Category C includes mRNAs that failed to be modulated in abundance in FTM oocytes. Categories D and E include mRNAs that displayed excess and opposite regulation in abundance, respectively, compared to NM.

Figure 4

Summary of the top biological functions, pathways and upstream regulators aberrantly regulated in FTM, displaying (B) partial modulation, (C) failure to be modulated, and (F + G) altered expression in FTM oocytes but not during NM. All data came from IPA® canonical pathways, biological functions and upstream regulators (UR) analyses for these different gene categories (B, C and F + G).