ZAR1
Zygote arrest protein 1 (Oocyte-specific maternal effect factor)
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The primary objective of this study was to compare expression of maternal transcripts in bovine oocyte populations with differential developmental competence: oocytes from prepubertal and pubertal animals; and oocytes from small (3-4 mm) and large (6-10 mm) follicles from pubertal animals. All transcripts were examined in oocytes prior to and after in vitro maturation (IVM). Genes were selected based on their known maternal effect in mouse (ZAR1, STELLA, HSF1, MATER/NLRP5 and its paralogue NLRP9), or their identification as markers of oocyte maturation, either involved in redox metabolism (PRDX1, PRDX2) or meiotic progression (AURKA). Total or polyadenylated forms of the transcripts were followed by reverse transcription coupled to real-time PCR. Six polyadenylated transcripts were found significantly reduced after maturation irrespective of donor age or follicle diameter (p<0.05). Within these six polyadenylated transcripts, ZAR1, NLRP9, HSF1, PRDX1 and PRDX2 were significantly reduced in oocytes from prepubertal animals compared to adult animals (p<0.05). A younger age was also associated with lower abundance (total form) of PRDX2/PRDX1 irrespective of maturation. Total HSF1, PRDX1 and polyadenylated NLRP9 showed a tendency (p values from 0.053 to 0.08) for a higher detection in oocytes from small follicles, thus encouraging further investigation of the follicle diameter model. However, at the present time, follicle size did not significantly affect expression of transcripts examined. In conclusion, this study demonstrates differences in the maternal store of RNA and its regulation during IVM which is dependent on donor age.
MeSH Terms
- Aging
- Animals
- Cattle
- Female
- Gene Expression
- Gene Expression Profiling
- Meiosis
- Oocytes
- Ovarian Follicle
- Peroxidases
- Peroxiredoxins
- Polymerase Chain Reaction
- RNA, Messenger
- Sexual Maturation
Improving human nuclear transfer (NT) efficiencies is paramount for the development of patient-specific stem cell lines, although the opportunities remain limited owing to difficulties in obtaining fresh mature oocytes. Therefore, the developmental competence of aged, failed-to-fertilize human oocytes as an alternate cytoplasmic source for NT was assessed and compared with use of fresh, ovulation-induced oocytes. To further characterize the developmental potential of aged oocytes, parthenogenetic activation, immunocytochemical analysis of essential microtubule proteins involved in meiotic and mitotic division, and RT-PCR in single oocytes (n = 6) was performed to determine expression of oocyte-specific genes [oocyte-specific histone 1 (H1FOO), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zygote arrest 1 (ZAR1)] and microtubule markers [nuclear mitotic arrest (NuMA), minus-end directed motor protein HSET and the microtubule kinesin motor protein EG5]. For NT, enucleation and fusion rates of aged oocytes were significantly lower compared with fresh oocytes (P < 0.05). Cleavage rates and subsequent development were poor. In addition, parthenote cleavage was low. Immunocytochemical analysis revealed that many oocytes displayed aberrant expression of NuMA and EG5, had disrupted meiotic spindles and tetrapolar spindles. One of the six oocytes misexpressed GDF9, BMP15 and ZAR1. Two oocytes expressed EG5 messenger RNA (mRNA), and HSET and NuMA were not detectable. RT-PCR of mRNA for oocyte specific genes and microtubule markers in single aged oocytes. Thus, aneuploidy and spindle defects may contribute to poor parthenogenetic development and developmental outcomes following NT.
MeSH Terms
- Aging
- Antigens, Nuclear
- Cell Cycle Proteins
- Female
- Fertilization in Vitro
- Humans
- Kinesin
- Nuclear Matrix-Associated Proteins
- Nuclear Transfer Techniques
- Oocytes
- Parthenogenesis
- Reverse Transcriptase Polymerase Chain Reaction
- Sperm Injections, Intracytoplasmic
- Tubulin