RNA interference (RNAi) is a sequence-specific gene regulatory mechanism conserved among diverse eukaryotes. The sequence specificity in RNAi is determined by a family of 18-to 30-nucleotide (nt) regulatory small RNAs (for review, see Aravin and Tuschl 2005). Two major classes of endogenous small RNAs have been characterized: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs-the best-characterized endogenous small RNAs in eukaryotes-have been identified in diverse plants and animals, and are mainly involved in development and differentiation. miRNAs are processed from miRNA precursors (pre-miRNAs) with a stem-loop structure and regulate gene expression through translational repression or mRNA cleavage (for reviews, see Ambros 2004;Bartel 2004;He and Hannon 2004;Du and Zamore 2005). siRNAs are generated from long double-stranded RNA (dsRNA) and are mainly involved in defense against molecular parasites including viruses, transposons, and transgenes through RNAi (Sijen and Plasterk 2003;Shi et al. 2004). Endogenous siRNAs have been classified into at least three subclasses: repeat-associated siRNAs (rasiRNAs), trans-acting siRNAs (ta-siRNAs), and siRNAs derived from natural antisense transcripts (nat-siRNAs) (Lippman and Martienssen 2004;Peragine et al. 2004;Borsani et al. 2005). rasiRNAs corresponding to repetitive elements repress the repeat sequences at the transcriptional or post-transcriptional level and maintain a centromeric heterochromatic

The expression pattern and function of the murine endogenous retrovirus-like (MuERV-L) gene in mouse preimplantation embryos was investigated. MuERV-L was rapidly transcribed from the beginning of S phase (8 h after fertilization) in the first cell cycle. MuERV-L expression was completely repressed when transcription from the zygotic genome was inhibited by alpha-amanitin. These results reveal that MuERV-L is transcribed from the zygotic genome and that it is expressed earlier than any other genes previously reported. In addition, MuERV-L was expressed even when the first round of DNA synthesis was inhibited by aphidicolin, suggesting that its expression is controlled by the zygotic clock. The function of MuERV-L in the development of mouse embryos was also examined using antisense oligonucleotides. The developmental competence of embryos was markedly suppressed after the 4-cell stage when they were treated with antisense oligonucleotides. This result suggests that MuERV-L plays an important role in the development of mouse embryos at the early preimplantation stage.

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.

The effects of carbohydrates on meiotic maturation and ATP content of bovine oocytes under low oxygen tension (5%) were investigated. Furthermore, the developmental competence or intracellular H2O2 contents of the oocytes matured under 5% or 20% O2 was assessed. In vitro maturation of bovine cumulus–oocyte complexes was performed in synthetic oviduct fluid (SOF) containing 20 amino acids and hormones (SOFaa). The proportion of the oocytes that matured to the metaphase II stage in SOFaa containing 1.5 mM glucose, 0.33 mM pyruvate, and 3.3 mM lactate under 5% O2 was dramatically lower than that of oocytes matured under 20% O2 (P < 0.01). Similarly, the ATP content of the oocytes that matured under 5% O2 was much lower than that of oocytes matured under 20% O2 (P < 0.05). Under 5% O2 the proportion of metaphase II oocytes increased with increasing glucose concentration (0–20 mM) in SOFaa without pyruvate or lactate. In addition, the ATP content of oocytes cultured in 20 mM glucose was higher (P < 0.05) than that of oocytes cultured in 1.5 mM glucose. Two glucose metabolites (pyruvate and lactate) and a nonmetabolizable glucose analog (2‐deoxy‐glucose), however, had no noticeable effects on meiotic maturation under 5% O2. These results suggest that ATP production under 5% O2 is not dependent on the TCA cycle. Addition of iodoacetate, a glycolytic inhibitor, to SOFaa containing 20 mM glucose significantly reduced (P < 0.01) the proportion of metaphase II and ATP content. Moreover, the proportion of the development to the blastocyst stage of oocytes matured under 5% O2 was higher (P < 0.05) than that of oocytes matured under 20% O2. H2O2 contents of oocytes matured under 5% O2 was lower (P < 0.05) than that of oocytes matured under 20% O2. The results of the present study demonstrate that glucose plays important roles in supporting the completion of meiotic maturation in bovine cumulus–oocyte complexes under low oxygen tension and that low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine oocytes. Mol. Reprod. Dev. 57:353–360, 2000. © 2000 Wiley‐Liss, Inc.

Abstract. Zygotic gene activation (ZGA) is the first event of gene expression after fertilization. Following fertilization, ZGA occurs within a short time interval depending on the animal species. Until ZGA, maternal proteins and transcripts stored in oocytes control embryonic development, indicating the importance of maternal factors for development. Somatic cell cloning also proves the potential of oocyte to reprogram the differentiated cell nuclei to embryonic nuclei. Recent studies show that the epigenetic modifications of nuclei play important roles in controlling gene expression during ZGA. However, the mechanisms that control ZGA remain largely unknown. This review will cover the current understanding of ZGA. Specifically, it will focus on the maternal factors that control gene expression during early embryogenesis.