Rosemary F. Bachvarova

Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse.

Previous work has shown that more than 50% or about 50 pg of polyadenylated RNA found in the full-grown mouse oocyte is deadenylated or degraded during meiotic maturation. Here we show that rRNA declines by 60 pg during this period, accounting for most of the 80-pg decline in total RNA and indicating that a significant amount of mRNA is deadenylated but not degraded during maturation. Actin mRNA is deadenylated at about 7 hr of in vitro maturation, following the decline in its translation. The poly(A) tail on hypoxanthine phosphoribosyltransferase (HPRT) mRNA is elongated at 7 hr of maturation, preceding an increase in HPRT activity. Actin mRNA is partially degraded in the one-cell embryo and falls to near the limit of detection in the late two-cell stage, while HPRT mRNA shows no change in early two-cell embryos, but is deadenylated and declines greatly during the two-cell stage. In aging unfertilized eggs, most of these changes occur on a delayed schedule. The various species of alpha-tubulin mRNA are largely deadenylated and more than half are degraded during maturation. Taken together with other published results, we conclude that each mRNA has its own pattern of changes in the length of the poly(A) tail (correlated with translation) and degradation during the period of maternal control of protein synthesis, and, for those examined, the maternal mRNAs remaining in the early two-cell embryo are degraded to low levels by the late two-cell stage.

Expression of c-kit encoded at the W locus of mice in developing embryonic germ cells and presumptive melanoblasts☆

The W locus of mice encodes the c-kit tyrosine kinase receptor. In embryos homozygous for severe W mutations, the number of germ cells does not increase after 8 days of development, melanocytes do not appear, and production of erythrocytes and mast cells is deficient. To gain some insight into the role of the c-kit receptor, we have used in situ hybridization to explore the time period of expression of c-kit transcripts in early germ cells and melanoblasts. At 6 1/2 days of development, expression was not seen in the embryonic cylinder, but did appear in parietal endoderm. Germ cells displayed a low level of c-kit transcripts from their first appearance in the 7 1/2 -day embryo, continuing through early proliferation and migration to the gonad. During migration, surrounding tissues also expressed c-kit. Expression increased in gonia and then ceased as they became nonproliferative. Expression in presumptive melanoblasts was first seen in the cervical region of 10-day embryos and continued as they spread over the surface of the body, entered the epidermis, and differentiated in hair follicles after birth. The effects of mutations of c-kit on germ cells and melanoblasts can be interpreted as an absence of a proliferative signal shortly after their segregation from other cell types. This signal may be required throughout the proliferative phase of early germ cells [and also in postnatal stages of germ cell development (Manova et al. (1990). Development 110, 1057-1069]. In melanoblasts, c-kit may play a role during both proliferation and differentiation.

Polyadenylated RNA of mouse ova and loss of maternal RNA in early development.

Ovulated mouse ova containing RNA labeled during oocyte growth were analyzed for the distribution of label between poly(A)+ and poly(A)− RNA by chromatography on poly(U) Sepharose columns. A small correction was made for contamination of the poly(A)+ fraction by poly(A)− RNA. About 9.6% of the RNA label was found in the poly(A)+ fraction for RNA labeled at any point throughout the growth phase of the oocyte 19 to 7 days before ovulation. The labeled polyadenylated RNA displayed a heterogeneous profile from about 14 S to 30 S on sucrose gradients. The amount of maternal mRNA present in the mouse egg calculated from available evidence is 5–8% of the total egg RNA, sufficient to contribute substantially to early embryonic protein synthesis. Labeled maternal RNA followed during preimplantation development declined by 40% on the first day, was stable on the second day, and declined by an additional 30% on the third day of development to the blastocyst stage. The proportion of RNA label in poly(A)+ RNA remained approximately constant throughout. Degradation of specific maternal messages and ribosomes may accompany changes in the pattern of protein synthesis on the first day of development. In addition, a substantial proportion of maternal RNA is apparently used during formation of the blastocyst.

Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes

The total RNA content of mouse oocytes, as measured by ethidium bromide fluorescence, was found to decrease by 19% during meiotic maturation (ovulated eggs contain 19% less RNA than full-grown oocytes). Consistent with these results, prelabeled stable RNA of full-grown oocytes decreased by about 20% during in vitro maturation. Polyadenylated RNA represented 19% of total prelabeled RNA in full-grown oocytes and 10% in oocytes matured in vitro, confirming previous results on in vivo prepared material. To distinguish between deadenylation and degradation for one mRNA, the amount and state of adenylation of actin mRNA was examined using Northern blots of oocyte RNA probed with a nick-translated beta-actin cloned chicken cDNA. The results showed that the amount of actin mRNA remained similar during maturation, but its molecular weight decreased slightly. Experiments in which RNA was treated with oligo(dT) and RNase H demonstrated that the actin mRNA was deadenylated during maturation, when actin synthesis is known to decline. These results indicate that the previously defined loss of bulk RNA and changes in the state of adenylation of mRNA during the first 11/2 days of embryogenesis actually begin during the 12 hr of meiotic maturation preceding fertilization.

A maternal tail of poly(a): The long and the short of it

Rosemary F. Bachvarova Department of Cell Biology Cornell University Medical College New York, New York 10021 Just as most cells transcribe only some of their many genes, so growing oocytes, maturing oocytes, and early embryos translate only some of the many messages pres- ent in the cytoplasm. A large fraction of the mRNA synthe- sized in growing oocytes is not used for immediate transla- tion, but is dormant or masked, and stored for future translation. Most stored messages are activated at various times during oocyte maturation and early embryonic de- velopment, when little or no mRNA is produced. In general, stored messages carry short poly(A) tails (15-90 A’s), and activation is accompanied by cytoplasmic polyadenylation increasing the tail to 150 A’s or more. Likewise, cessation of translation is accompanied by deadenylation of the mes- sage. The poly(A) tail is known to promote initiation of translation (reviewed in Jackson and Standart, 1990) and in yeast, thiseffect is mediated through the poly(A)-binding protein, whose gene is required for viability. Two classes of maternal message whose translation is regulated during meiotic maturation have been studied in frog and mouse oocytes (reviewed in Wickens, 1990; Richter, 1991). First is a set of stored messages whose translation commences during meiotic maturation (see tPA mRNA in Figure 1). The several members studied are all polyadenylated in a process that requires specific sequences in the 3’ untranslated region (UTR): the poly-

Gene Expression During Oogenesis and Oocyte Development in Mammals

Mouse oocytes progress through early meiotic prophase during fetal life and reach the diplotene stage by birth. During prepubertal and reproductive life, oocytes are continuously selected to grow from the pool of small primordial oocytes. Growing oocytes reach full size in 2 weeks, and full-grown oocytes are present in rapidly enlarging follicles for about 5 days before meiotic maturation and ovulation. RNA synthesis during early meiotic prophase, as estimated from [3H]uridine incorporation followed by autoradiography and from electron microscopic analysis of nuclear components, proceeds at a moderate rate throughout except for a brief period in early pachytene when synthesis is low or absent. RNA synthesis continues in primordial oocytes at a moderate rate. Incorporation studies, electron microscopic analyses, and particularly measurements of ongoing RNA polymerase activity (completion of initiated chains as analysed in tissue sections) indicate a distinctly increased rate of synthesis during oocyte growth over that of primordial oocytes, followed by a decline in full-grown oocytes. During growth, this rate increases severalfold. The absolute rate of synthesis of heterogeneous nuclear RNA (using rRNA as a standard) during mid-growth is very rapid, but nevertheless still much lower than that in typical lampbrush chromosomes. Most of the hnRNA turns over with a half-life of about 20 min, as is typical in somatic cells. Newly synthesized mRNA-like RNA enters the cytoplasm at about one-half the rate of rRNA, and about one-third of the ribosomes and one-fourth of the mRNA appear in polysomes. In full-grown oocytes, the rate of synthesis falls distinctly, but a significant level of synthesis continues until it essentially ceases at breakdown of the germinal vesicle. During meiotic prophase, chromosomes are most compact at pachytene and unfold lateral projections as RNA synthesis increases in late pachytene-early diplotene. In primordial oocytes, the diplotene state of chromosomes is obvious in most mammals, but in rodents the chromosomes are more evenly dispersed and are said to be in a dictyate state, although they are still presumably in a diplotene configuration. The chromosome core, which is present in leptotene through early diplotene stages, apparently disappears in the dictyate stage.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of activins and TGFβ1 and β2 RNAs in early postimplantation mouse embryos and uterine decidua

The expression of the mesoderm inducing factors, activins and TGF beta s, was characterized in 5 1/2-9 1/2 day mouse embryos and implantation sites by in situ hybridization. Activin beta A RNA was not detected within the embryo, but is expressed in nearby decidual cells from 5 to 7 days. Thus activin A could play a role within the embyro during gastrulation. Activin beta A is also expressed in more mesometrially located decidual cells from 6 to 9 1/2 days. Activin beta B and inhibin alpha RNAs were not detected, while a control tissue was highly positive. TGF beta 1 is expressed in the secondary decidual zone and in developing endothelial cells in the decidua and embryo. TGF beta 2 is expressed in the mesometrial decidua at 6 1/2 days and in the midline of the cranial neural plate.

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution

Summary The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.

Half-lives and relative amounts of stored and polysomal ribosomes and poly(A)+ RNA in mouse oocytes☆

Growing mouse oocytes were labeled in vitro with [3H]uridine and chased for 2 or for 7 days to estimate the relative amounts of RNA appearing in different fractions and to follow their turnover. Oocytes were lysed and thoroughly dispersed in the presence of 1% DOC, and centrifuged on sucrose gradients to separate polysomes from smaller components not engaged in translation. After the short chase, one-third of the labeled ribosomes appeared in EDTA-sensitive polysomes. The proportion of ribosomes in both fractions remained stable during the long chase, demonstrating no net flow from one fraction to the other. When gradient fractions were analyzed by poly(U) Sepharose chromatography, it was found that about 20% of the labeled poly(A)+ RNA appeared in polysomes after the short chase. The half-lives of stored and translated mRNA were followed relative to stable rRNA during the long chase. Stored mRNA was completely stable, but translated mRNA turned over with a t1/2 of about 6 days. Other methods for separating stored from translated components were not successful, including sedimentation of putative large complexes (fibrillar lattices) containing stored components, or chromatography of lysates on oligo(dT)-cellulose. Results presented here combined with our previous results demonstrate that, during meiotic maturation, the percent of labeled stable RNA which is polyadenylated declines from 19 to 10%, suggesting deadenylation or degradation of half of the accumulated maternal mRNA.

Incorporation of tritiated adenosine into mouse ovum RNA

Abstract The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng. The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-3H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation. The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-3H or uridine-5,6-3H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.