Functions of maternal mRNA in early development

In this review, the types of mRNAs found in oocytes and eggs of several animal species, particularly Drosophila, marine invertebrates, frogs, and mice, are described. The roles that proteins derived from these mRNAs play in early development are discussed, and connections between maternally inherited information and embryonic pattern are sought. Comparisons between genetically identified maternally expressed genes in Drosophila and maternal mRNAs biochemically characterized in other species are made when possible. Regulation of the meiotic and early embryonic cell cycles is reviewed, and translational control of maternal mRNA following maturation and/or fertilization is discussed with regard to specific mRNAs.     

Maternal and embryonic sources of tyrosine hydroxylase during Drosophila embryogenesis

Tyrosine hydroxylase (TH), the enzyme which catalyzes the conversion of tyrosine to L-DOPA and is rate limiting in catecholamine biosynthesis, is biochemically expressed in late stage wild-type Drosophila oocytes as well as in early embryogenesis. Null mutant alleles of TH (pale) are embryonic lethals with death occurring in the late embryonic or early larval periods of development. Staging of embryos demonstrated that inhibition of the enzymatic activity of TH by alpha-methyl-p-tyrosine (alphaMT) retards the progression of embryos primarily during the organogenesis stages of embryonic development, with lesser effects on earlier and later stages. On the other hand, time of gene action studies with a conditional temperature sensitive pale mutant (ple(ts1)) at its restrictive temperature (29 degrees C) indicate an onset of tyrosine hydroxylase gene action beginning in the oocyte stage of development. Thus, maternal as well as embryonic effects on the secretion and/or functionality of this enzyme may play roles in the early developmental program of the organism.     

Injection of frozen-thawed porcine first polar bodies into enucleated oocytes results in fertilization and embryonic development

The objective was to determine whether enucleated oocytes injected with frozen porcine first polar bodies (pPB1s) could be fertilized and developed into viable embryos in vitro. Metaphase II (MII) oocytes with pPB1s were frozen (vitrified) and stored for 2 mo. The pPB1s were isolated from thawed MII oocytes and injected into enucleated recipient oocytes by micromanipulation. All recipients injected with thawed pPB1s were fertilized by intracytoplasmic sperm injection (ICSI), and the resulting recombinant zygotes were incubated to assess their developmental competence in vitro. Furthermore, double-antibody immunohistochemistry was used to verify that the nucleus of the pPB1 participated in fertilization and supported embryonic development. Porcine embryos (2- to 8-cell stage) were obtained from the recombinants. The average in vitro cleavage rate of 2-, 4-, and 8-cell stage recombinant embryos was 25.3, 17.7, and 9.3% (P < 0.05), respectively. Chromosomes in the labeled pPB1 participated in the formation of the two blastomere nuclei of 2-cell stage embryos derived from recombinant oocytes. In conclusion, nuclear materials of frozen-thawed pPB1 supported oocyte fertilization and subsequent embryonic development, thereby providing a new way to use frozen PB1s for preservation and reproduction of mammals.     

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10–11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.     

Expression of Maternally and Embryonically Derived Hypoxanthine Phosphoribosyl Transferase (Hprt) Activity in Mouse Eggs and Early Embryos

X-chromosome activity in early mouse development has been studied by a gene dosage method that involves measuring the activity level of the X-linked enzyme hypoxanthine phosphoribosyl transferase (HPRT) in single eggs and embryos from XO females and from females heterozygous for In(X)1H, a paracentric inversion of the X chromosome. The HPRT activity in oocytes increased threefold over a 24-hr period beginning after ovulation. Afterward, the activity plateaued in unfertilized eggs but continued to increase for at least 66 hr in presumed OY embryos. Both before and after ovulation, the level of activity in unfertilized eggs from In(X)/X females was twice that from XO females, and the distributions of activity in eggs for both sets of females remained unimodal. Beginning with the two-cell stage, distributions of activity for embryos from In(X)/X females were trimodal, which is evidence for embryonic activity. It is proposed that activation of a maternal mRNA or proenzyme is responsible for the HPRT activity increase in oocytes and early embryos and is supplemented by dosage-dependent activity of the embryonic Hprt gene as early as the two-cell stage.     

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.     

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development.     

端粒酶催化亚基基因在小鼠 胚胎发育早期的转录活性

用 RT-PCR 引物分别扩增成年昆明 (KM) 小鼠睾丸、脾脏、肾脏、肝脏和胸腺组织的总 RNA 发现,端粒酶催化亚基基因 tert 在这些组织中都有转录,目标产物正确组装到 PMD 18-T 载体后测序,结果与已知 cDNA 序列一致 . PMSG/hCG 超数排卵方法获得 KM 小鼠成熟卵母细胞和 CZB 溶液体外培养的胚胎 (KM ♀× KM ♂ ) ,用酸性 Tyrode's 溶液消化透明带后,采用巢式 RT-PCR ,同时分析 tert 基因和持家基因 hprt 的转录发现,对于单个样品来说 , 全部卵母细胞 (15 h-post hCG , 10/10) 都存在 hprt 转录本,其中,只有 40% (4/10) 还同时存在 tert 转录本 . 原核形成初期 (20 h-post hCG , 6/6) 和原核晚期 (30 h post-hCG , 8/8) 的受精卵,以及发育至 2-C 早期的胚胎 (35 h-post hCG , 7/7) 都不转录 tert 基因,只有 hprt mRNA 存在; 2-C 晚期 (50 h-post hCG) 时,两个基因同时转录 (4/8) 和一个基因单独转录 (4/8) 的胚胎各占 50% ;从 4-C 阶段 (65 h-post hCG , 4/4) 开始,包括 8-C 阶段 (75 h-post hCG , 4/4) ,桑椹胚阶段 (93 h-post hCG , 4/4) ,直至囊胚阶段 (118 h-post hCG , 4/4) ,所有的胚胎都同时转录 tert 和 hprt 基因,而且转录水平明显升高 . 以 20 枚胚胎量为模板进行 RT-PCR 发现,原核早期,原核晚期的胚胎中仍然没有 tert 基因转录,只有 hprt mRNA ,但是,在 2-C 早期胚胎中同时检测到了 hprt 和 tert 两种 mRNA. 结果表明,持家基因 hprt 在成熟卵母细胞受精前后,以及胚胎早期发育过程中均存在转录本 . 40% 卵母细胞中存在的 tert mRNA 在受精后很快降解,检测不到;胚胎基因组在 2-C 早期开始转录 tert mRNA ,转录水平逐渐上升 . 结果暗示,小鼠胚胎的基因组 DNA 在 2-C 早期开始启动,功能基因 tert 也在此时开始转录,可能与胚胎发育初期的染色体保护有关 .     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.