Project description:Age of the donor animal has a significant influence on the oocyteM-bM-^@M-^Ys ability to complete maturation and acquire the mRNA and proteins required for normal embryonic development. It is well known that developmental competence is reduced in oocytes derived from juvenile animals when compared to that derived from adults. However, the molecular mechanisms associated with these differences are not well elucidated. The aim of this study was to analyze the transcriptome differences between adult versus prepubertal derived preimplantation embryos and to identify genes associated with oocyte developmental potential. We performed cDNA microarray analysis on populations of preimplantation embryos (8- to 16-cell and Day 7 blastocysts) derived from adult versus prepubertal Japanese Black cattle. Total RNA was extracted and amplified in a linear fashion and then subjected to the microarray hybridization using the Affymetrix GeneChip Bovine Genome Array. The Bovine Array contains 24 072 probe sets representing over 23000 transcripts and 19000 UniGene clusters. Following normalization of the microarray data, analysis revealed differences in gene expression abundance in 8- to 16-cell and blastocysts harvested from adult versus prepubertal heifers. Results indicated that a total of 591 and 490 genes were differentially expressed (M-bM-^IM-%2-fold) in 8- to 16-cell and blastocyst respectively between the two groups (p<0.01). In 8- to 16-cell stage, 373 genes were up-regulated and 218 gens down-regulated in adult when compared to the prepubertal heifer group. In case of blastocyst stage embryos, 242 genes were up-regulated and 248 genes were down-regulated in adult when compared to prepubertal group. The ovaries of adult cows (Japanese black cattle) were collected from local abattoir while ovaries of prepubertal Japanese black heifers (9 to 12 months old) were collected by spay device at several commercial farms. The ovaries were transported to the research laboratory in 0.67% (w/v) NaCl solution containing 100 mg/L kanamycin sulfate. COC were placed in 200 M-BM-5L drop of the maturation medium (TCM 199 + 10% FBS) and cultured at 38.5M-BM-0C in a humidified atmosphere of 5% CO2 in air for 20 to 22 h for in vitro maturation (IVM). After 20 to 22 h IVM, the COC were fertilized in vitro with frozenM-bM-^@M-^Sthawed semen of Japanese Black bull in BO medium. Six hours after IVF, oocytes were denuded of surrounding cumulus cells by gentle pipetting in a solution of 0.1% (w/v) hyaluronidase. The presumptive zygotes were cultured in CR1aa medium supplemented with fatty acid-free BSA (Sigma) for 2 days and cultured in CR1aa medium supplemented with 10% (v/v) fetal bovine serum (FBS) for next 5 days at 38.5 M-BM-0C in 5% CO2, 5% O2 and 90% N2 with high humidity (15 to 20 zygotes per 50 M-BM-5l microdrop). 8- to 16-cell refers to day 3, 70 to 72 hours post insemination( hpi) preimplantation embryo stage containing 8 to 16 cells, while blastocyst refer to pre-hatching day 7 expanded blastocysts. Pools of 8- to 16- cell embryos (50 in each sample) and blastocysts (10 in each sample) from prepubertal and adult groups were used for cDNA microarray.

Project description:Developmental competences of oocytes derived from prepubertal heifers are lower than those derived from adult counterparts. The objective of this study was to identify a range of genes associated with reduced oocyte competence that are differentially expressed between adult versus prepubertal donors. Microarray experiments were conducted using total RNA isolated from GV and MII stages oocytes collected from adult and prepubertal animals using Affymetrix GeneChip Bovine Genome Array containing 24,072 probe sets representing over 23,000 transcripts. A total of 549 and 333 genes were differentially expressed between prepubertal versus adult bovine MII and GV stages oocytes respectively. Out of these, 312 and 176 genes were up-regulated, while 237 and 157 were down-regulated in prepubertal when compared with adult MII and GV oocytes respectively. Ontological classification of the differentially expressed genes revealed that up-regulated genes in adult oocytes were involved in signal transduction, regulation of transcription DNA-dependent, and transport. Results from the present study indicated that significant number of genes were differentially expressed (>2-fold, p<0.01) between the two groups. Thus the decreased developmental competence of oocytes from prepubertal heifers may be induced due to difference in gene expression abundance as observed in our study. In conclusion, transcript abundance analyses of oocytes using microarray approach have been carried out in bovine and several other species. However, to our knowledge, this is the first study carried out to examine genes expression differential abundance in oocytes derived from perpubertal versus adult Japanese Black Cattle. Bovine 4b PP biological rep1, Bovine 78b PP biological rep2, Bovine 79 PP biological rep3 represents GV stage oocytes derived from Prepubertal (PP) heifer group, while Bovine 74b A biological rep1, Bovine 80b A biological rep2, Bovine 81 A biological rep3 represents GV stage oocytes derived from Adult (A) cow group. Bovine 7 PP biological rep1, Bovine 53 PP biological rep2, Bovine 57 PP biological rep3 represents MII stage oocytes derived from Prepubertal heifer group, while Bovine 59 A biological rep1, Bovine 70 A biological rep2, Bovine 71 A biological rep3 represents MII stage oocytes from Adult cow group. The ovaries of adult cows (Japanese black cattle) were collected from local abattoir while ovaries of prepubertal Japanese black heifers (9 to 12 months old) were collected by spay device at several commercial farms. The collected ovaries of both the adult cows and prepubertal heifers groups were transported to the research laboratory in 0.67% (w/v) NaCl solution containing 100 mg/L kanamycin sulfate (Meiji Seika, Tokyo, Japan). For both groups, cumulus oocyte complexes (COCs) from ovarian follicles 2 to 8M-BM- mm in diameter were aspirated by using an 18 gauge needle (Terumo co, Tokyo, Japan) attached to a 5 ml disposable syringe (Nipro, Osaka, Japan). After collection, the COCs were washed twice with Tyrode-lactate-pyruvate-polyvinylalcohol (Hepes-TLP-PVA) and TCM 199 (Invitrogen, Gibco, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (maturation medium). Only COCs with evenly granulated cytoplasm surrounded by multiple layers of compact cumulus cells were used in all the experiments. The COCs (70 to 80) were placed in 200 M-BM-5L drop of the maturation medium in petri dish (35x10mm, Becton Dickinson Labware, Oxnard, CA, USA) covered with paraffin liquid (Nacalai Tesque Inc, Kyoto, Japan) and cultured at 38.5M-BM-0C in a humidified atmosphere of 5% CO2 in air for 20 to 22 h for maturation.

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