Age-associated changes in gene expression of goat oocytes

doi:10.1016/j.theriogenology.2013.04.019

Theriogenology

Volume 80, Issue 4, 1 September 2013, Pages 328-336

https://doi.org/10.1016/j.theriogenology.2013.04.019 Get rights and content

Abstract

Oocyte aging severely decreases the quality of oocytes, which hampers fertilization and subsequent embryo development. In the present study, age-dependent molecular changes in goat oocytes were investigated. First, the quality of goat oocytes with various in vitro culture times (24, 30, 36, 48, and 60 hours) was evaluated on the basis of developmental rates of parthenogenetically activated embryos and apoptosis of cumulus cells (CCs). Second, relative gene expression of six genes (mitochondrial genes: PGC-1α and NRF-1; epigenetic modification genes: SNRPN and HAT1; mitotic spindle checkpoint protein: SMAD2; and hyaluronan synthase gene: HAS3) were analyzed during oocyte aging. Third, we further studied the changes of seven genes (PGC-1α and NRF-1; apoptotic-related genes: BAX and BCL2; hyaluronan synthase gene: HAS2; metabolism-related gene: STAR; and superoxide dismutase gene: SOD1) in CCs during oocyte aging. In these studies, the blastocyst rate gradually decreased and the number of apoptotic cells significantly increased as the culture time increased (P < 0.05). Moreover, relative gene expressions of PGC-1α, NRF-1 and SMAD2 significantly decreased from 24 to 36 hours (P < 0.05), whereas the levels of HAT1 and HAS3 slowly increased as culture was prolonged. Furthermore, the levels of PGC-1α, BCL2, HAS2 and SOD1 quickly reduced, and BAX significantly increased from 24 to 36 hours in aged CCs (P < 0.05). In conclusion, goat oocytes started to age at 30 hours in vitro culture, and gene expression patterns of oocytes and CCs significantly changed as the oocytes aged. Gene expression pattern changes in CCs may provide a convenient and effective way to detect oocyte aging without compromising oocyte integrity.

Introduction

Oocyte quality has a critical role in embryonic development after fertilization [1]. In most mammals, ovulated oocytes are arrested at metaphase II (MII) until fertilization [2]. If fertilization does not occur on time, oocytes will undergo an aging process [3]. As the window for optimal fertilization is narrow, oocytes may miss the optimal fertilization time, resulting in fertilization of aged oocytes. Unintentional prolonging the culture time of oocytes in many research studies and clinical applications would have negative effects on further development. Therefore, studying the mechanisms of oocyte aging is vital for reproductive health and embryo technologies.

Goat is an important domesticated animal with great agricultural applications. Improper fertilization of aged oocytes would hamper goat reproduction. Although oocyte aging has been extensively studied in many species, such as sheep [4], mouse [5], pig [3], and cattle [6], there are only a few reports in goats [7]. Furthermore, previous studies mostly focused on biochemical changes, methods, or factors that affected the aging process [8], [9], whereas the inner changes of gene expression patterns were not characterized. To study the relationship between gene expressions and goat oocyte aging, we selected mitochondrial genes: PGC-1α (peroxisome proliferator-activated receptor 1α) and NRF-1 (nuclear respiratory factor 1); epigenetic modification genes: HAT1 (histone acetylation 1) and SNRPN (small-nuclear ribonucleoprotein polypeptide N); hyaluronan synthase gene: HAS3 (hyaluronan synthase 3); and mitotic spindle checkpoint protein: SMAD2 (mothers against decapentaplegic homolog 2).

In most mammals, >99% ovarian follicles never ovulate; therefore, only a few mature oocytes can be used for breeding [10]. Cumulus cells (CCs) are easily obtained and have a close relationship with oocytes [11]. Most IVF clinics rely on CCs for noninvasive examination of developmental and morphological aspects of oocytes, embryo selection from in vitro culture, and prediction of pregnancy rate [12], [13]. Moreover, CCs have an important role in oocyte aging and subsequent developmental potential [14], [15]. Therefore, we speculated the gene expression in CCs may provide indirect assessment for oocyte aging without compromising oocyte integrity. Until now, apparently no systematic study of mRNA expression levels of genes related to oocyte aging in CCs has been done. The mRNA expression levels of genes that are selectively expressed in CCs were investigated to predict the goat oocyte aging process, that is, mitochondrial genes: PGC-1α and NRF-1; apoptotic-related genes: BCL2 (B-cell CLL/lymphoma 2) and BAX (BCL2-associated X protein); hyaluronan synthase gene: HAS2 (hyaluronan synthase 2); metabolism-related gene: STAR (steroidogenic acute regulatory); and superoxide dismutase gene: SOD1 (Cu/Zn superoxide dismutase).

Section snippets

Materials and methods

All trials were conducted in accordance with the Guidelines for the Care and Use of College of Animal Science and Technology, Nanjing Agricultural University.

Unless otherwise indicated, all chemicals used were purchased from Sigma-Aldrich Company (St. Louis, MO, USA) and the media from Gibco (Grand Island, NY, USA).

Determination of goat oocyte aging time point

Groups of oocytes were activated at various in vitro maturation times (24, 30, 36, 48, and 60 hours), and cleavage and blastocyst rates were assessed (Table 2). As the culture time was prolonged, the numbers of oocytes at MII stage did not change from those at 24 hours. Compared with the 24-hour group (64.26%), cleavage rates of groups at 36 (71.74%), 48 (73.68%), and 60 hours (75.02%) increased (P < 0.05 respectively). However, blastocyst rates decreased from 31.31% to 14.34% (P < 0.05). The

Discussion

Matured oocytes are always needed for IVF, nucleus transfer, and intracytoplasmic sperm injection. Because these techniques would be compromised by aged oocytes, studying the mechanisms of oocyte aging is important. The first step is to identify the oocyte aging time point and then find an effective way to predict oocyte aging. In the present study, we concluded that the goat oocytes started to age at 30 hours after in vitro culture. Along with goat oocyte aging, gene expression patterns of

Acknowledgments

This study was financially supported by the Ministry of Science and Technology Support Project (No. 2011BAD19B02), the Fundamental Research Funds for the Central Universities (No. KYZ201211), and the Open Research Fund of State Key Laboratory of Bioelectronics, Southeast University.

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They were transported to our laboratory in physiologic (0.9%) NaCl solution containing gentamycin sulfate (2 mg/mL) within 2 h after slaughter. The COCs were aspirated from medium-sized follicles by a mouth pipette as described previously [19]. Thirty-five COCs in each group were cultured in 75 μL IVM medium with GSK-LSD1 under liquid paraffin oil.
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Research articleAge-associated changes in gene expression of goat oocytes

Abstract

Introduction

Section snippets

Materials and methods

Determination of goat oocyte aging time point

Discussion

Acknowledgments

Reprod Fertil Dev

Theriogenology

Theriogenology

Theriogenology

Int Rev Cytol

J Gynecol Obstet Biol Reprod (Paris)

Theriogenology

Theriogenology

Biochim Biophys Acta

Cell

J Biol Chem

Biochem Biophys Res Commun

Theriogenology

Theriogenology

J Biol Chem

Free Radic Biol Med

Cryobiology

Res Vet Sci

J Biol Chem

Reduced expression of MAD2, BCL2, and MAP kinase activity in pig oocytes after in vitro aging are associated with defects in sister chromatid segregation during meiosis II and embryo fragmentation after activation

Biol Reprod

Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns

PLoS One

Research article
Age-associated changes in gene expression of goat oocytes