Deviation of the living system from the stationary state during oogenesis

Summary The changes in respiration and glycolysis of whole oocytes and homogenates of oocytes during oogenesis have been studied.The respiration rate of whole oocytes increases during oocyte growth and decreases during oocyte maturation. The respiration rate of homogenates also increases during oocyte growth and does not change during egg maturation. At all oogenesis stages the respiration rate of homogenates is higher than the respiration rate of whole oocytes.Respiration intensity increases during the small growth stage and decreases during the following stages of oogenesis. Respiration intensity of homogenates under optimal conditions changes in a similar way. Respiration intensity under physiological conditions diminishes during oogenesis from 70% at the small growth stage to 42% in unfertilised eggs.The rate of glycolysis in whole oocytes and homogenates of oocytes increases during the growth period of oocytes but does not change during egg maturation.Glycolysis intensity of the whole oocytes increases at the large growth stage—stage of cytoplasmic vacuolisation—and becomes less during the following stages. Glycolysis intensity in homogenates under optimal conditions is much higher than the glycolysis intensity of whole oocytes and it decreases slightly during oogenesis. The efficiency of glycolysis in oocytes under physiological conditions is very low. It increases from the stage of cytoplasmic vacuolisation (3.6%) to the stage at which vitellogenesis starts (20%) and diminishes at the following stages.The data obtained are considered in the light of the Prigogine and Wiame interpretation of a thermodynamic theory of development.     

Persistence of nonribosome bound 5 S RNA in full-grown oocytes of Xenopus laevis

The 4 and 5 S RNA containing 42 S ribonucleoprotein (RNP) particles characteristic of previtellogenic and white oocytes cannot be detected in full-grown oocytes. When full-grown oocyte RNPs are separated on sucrose gradients 4 and 5 S RNA cannot be detected in the 42 S region. However, not all of the 5 S RNA stored during early oogenesis is incorporated into ribosomes at later stages. A substantial pool (20% of the total) of 5 S RNA remains in a non-ribosome-bound fraction sedimenting at about 7 S in full-grown oocytes.     

Translational control by cytoplasmic polyadenylation during Xenopus oocyte maturation: characterization of cis and trans elements and regulation by cyclin/MPF.

The expression of certain maternal mRNAs during oocyte maturation is regulated by cytoplasmic polyadenylation. To understand this process, we have focused on a maternal mRNA from Xenopus termed G10. This mRNA is stored in the cytoplasm of stage 6 oocytes until maturation when the process of poly(A) elongation stimulates its translation. Deletion analysis of the 3' untranslated region of G10 RNA has revealed that two sequence elements, UUUUUUAU and AAUAAA were both necessary and sufficient for polyadenylation and polysomal recruitment. In this communication, we have defined the U-rich region that is optimal for polyadenylation as UUUUUUAUAAAG, henceforth referred to as the cytoplasmic polyadenylation element (CPE). We have also identified unique sequence requirements in the 3' terminus of the RNA that can modulate polyadenylation even in the presence of wild-type cis elements. A time course of cytoplasmic polyadenylation in vivo shows that it is an early event of maturation and that it requires protein synthesis within the first 15 min of exposure to progesterone. MPF and cyclin can both induce polyadenylation but, at least with respect to MPF, cannot obviate the requirement for protein synthesis. To identify factors that may be responsible for maturation-specific polyadenylation, we employed extracts from oocytes and unfertilized eggs, the latter of which correctly polyadenylates exogenously added RNA. UV crosslinking demonstrated that an 82 kd protein binds to the U-rich CPE in egg, but not oocyte, extracts. The data suggest that progesterone, either in addition to or through MPF/cyclin, induces the synthesis of a factor during very early maturation that stimulates polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

SEBOX is essential for early embryogenesis at the two-cell stage in the mouse

Previously, we found high levels of skin-embryo-brain-oocyte homeobox (Sebox) gene expression in germinal vesicle (GV)-stage oocytes. The objective of the present study was to determine the role played by SEBOX in oocyte maturation and early embryogenesis using RNA interference (RNAi). Microinjection of Sebox double-stranded RNA into GV oocytes resulted in a marked decrease in Sebox mRNA and protein expression. However, Sebox RNAi affects neither oocyte maturation rate nor morphological characteristics, including spindle and chromosomal organization of metaphase II oocytes. In addition, Sebox RNAi had no discernible effect on the activities of M-phase promoting factor or mitogen-activated protein kinase. In contrast, microinjection of Sebox double-stranded RNA into pronuclear-stage embryos resulted in holding embryo development at the two-cell (84.9%) and the four- and eight-cell (15.1%) stages. We concluded that Sebox is a new addition to maternal effect genes that produced and stored in oocytes and function in preimplantation embryo development.     

Poly (A) polymerases in the nucleus and cytoplasm of frog oocytes: dynamic changes during oocyte maturation and early development.

Poly(A) can be added to mRNAs both in the nucleus and in the cytoplasm. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs provides a common mechanism of translational control. In this report, to begin to understand the regulation of polyadenylation activities during early development, we analyze poly (A) polymerases (PAPs) in oocytes and early embryos of the frog, Xenopus laevis. We have cloned and sequenced a PAP cDNA that corresponds to a maternal mRNA present in frog oocytes. This PAP is similar in size and sequence to mammalian nuclear PAPs. By immunoblotting using monoclonal antibodies raised against human PAP, we demonstrate that oocytes contain multiple forms of PAP that display different electrophoretic mobilities. The oocyte nucleus contains primarily the slower migrating forms of PAP, whereas the cytoplasm contains primarily the faster migrating species. The nuclear forms of PAP are phosphorylated, accounting for their retarded mobility. During oocyte maturation and early postfertilization development, preexisting PAPs undergo regulated phosphorylation and dephosphorylation events. Using the cloned PAP cDNA, we demonstrate that the complex changes in PAP forms seen during oocyte maturation may be due to modifications of a single polypeptide. These results demonstrate that the oocyte contains a cytoplasmic polymerase closely related to the nuclear enzyme and suggest models for how its activity may be regulated during early development.     

RNA SYNTHESIS IN THE MOUSE OOCYTE

RNA synthesis in the oocyte and granulosa cell nuclei of growing follicles has been studied in the mouse ovary. The RNA precursor [³H]uridine was administered intraperitoneally to adult mice and the amount of label incorporated into ovarian RNA was quantitated autoradiographically using grain-counting procedures. Uridine incorporation into the nucleus is low in oocytes of small, resting follicles but increases during follicle growth and reaches a peak prior to the beginning of antrum formation. Thereafter uptake rapidly declines and is very low in the oocytes of maturing follicles. Uridine incorporation into granulosa cell nuclei, in contrast to that found in the oocyte, increases gradually during most of the period of follicle growth. Qualitative studies of the activity of endogenous, DNA-dependent RNA polymerases have also been made in fixed oocytes isolated from follicles at different stages of growth. Polymerase activity is demonstrable in the nucleolus and nucleoplasm of oocytes from growing follicles, but is absent from maturing oocytes of large follicles.     

Ultrastructure of oogenesis in the bluefin tuna, Thunnus thynnus

Ovarian ultrastructure of the Atlantic bluefin tuna (Thunnus thynnus) was investigated during the reproductive season with the aim of improving our understanding of the reproductive biology in this species. The bluefin, like the other tunas, has an asynchronous mode of ovarian development; therefore, all developmental stages of the oocyte can be found in mature ovaries. The process of oocyte development can be divided into five distinct stages (formation of oocytes from oogonia, primary growth, lipid stage, vitellogenesis, and maturation). Although histological and ultrastructural features of most these stages are similar among all studied teleosts, the transitional period between primary growth and vitellogenesis exhibits interspecific morphological differences that depend on the egg physiology. Although the most remarkable feature of this stage in many teleosts is the occurrence of cortical alveoli, in the bluefin tuna, as is common in marine fishes, the predominant cytoplasmic inclusions are lipid droplets. Nests of early meiotic oocytes derive from the germinal epithelium that borders the ovarian lumen. Each oocyte in the nest becomes surrounded by extensions of prefollicle cells derived from somatic epithelial cells and these form the follicle that is located in the stromal tissue. The primary growth stage is characterized by intense RNA synthesis and the differentiation of the vitelline envelope. Secondary growth commences with the accumulation of lipid droplets in the oocyte cytoplasm (lipid stage), which is then followed by massive uptake and processing of proteins into yolk platelets (vitellogenic stage). During the maturation stage the lipid inclusions coalesce into a single oil droplet, and hydrolysis of the yolk platelets leads to the formation of a homogeneous mass of fluid yolk in mature eggs.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Possible involvement of Nemo-like kinase 1 in Xenopus oocyte maturation as a kinase responsible for Pumilio1, Pumilio2, and CPEB phosphorylation

Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.     

Responses of oocytes and embryos to the culture environment

Embryo development is strongly influenced by events occurring during oocyte maturation. Although many immature oocytes are capable of completing meiosis in vitro, only a small percentage of the original pool of immature oocytes is competent to continue development to the blastocyst stage and subsequently result in a pregnancy. This indicates that maturation of oocytes in vitro may not be occurring in an entirely normal manner. Cytoplasmic changes occurring during maturation, collectively termed cytoplasmic maturation, are essential for embryonic development. The cytoplasm of the oocyte may play a crucial role in assembling the correct metabolic machinery for production of sufficient energy for cellular functions during maturation, cleavage and blastocyst formation. A better understanding of the structural, functional and metabolic characteristics of the oocyte during maturation, and the consequence of changes in these parameters on developmental competence is needed. Understanding the role of cytoplasmic changes during oocyte maturation will help increase the efficiency of in vitro embryo production. Better embryo production strategies will facilitate basic research into the control of early development, improve implementation in endangered species, provide a source of high quality oocytes for nuclear transfer and transgenic technologies and benefit the commercial embryo transfer industry.     

Temporal pattern of synthesis of the mouse cortical granule protein, p75, during oocyte growth and maturation.

We previously demonstrated that a protein of M(r) 75,000 (p75) is localized to cortical granules (CGs) in mouse oocytes and eggs and is released upon activation or fertilization of eggs (K.E. Pierce, M. C. Siebert, G. S. Kopf, R. M. Schultz, and P. G. Calarco, 1990, Dev. Biol. 141, 381-392). To examine the temporal pattern of synthesis of p75 during the early stages of CG formation, growing oocytes, which were isolated from juvenile mice, were incubated for 4 hr in medium containing [35S]methionine, and radiolabeled proteins were immunoprecipitated using an antiserum that detects p75. Synthesis of p75 is detected at low levels in the smallest oocytes examined (less than 20 microns). Synthesis of p75 relative to total protein synthesis increases about 12-fold during oocyte growth from the 20-40 microns size and then remains constant throughout the remaining period of oocyte growth (40-70 microns). In the fully grown, germinal vesicle (GV)-intact oocyte (70-80 microns), immunoprecipitated p75 comprises approximately 1.5% of the total amount of radiolabeled protein. Three hours after the transfer of these oocytes to a medium that supports resumption of meiosis and GV breakdown in vitro, oocytes subjected to a 1-hr labeling pulse display a 35% decrease in the relative level of p75 synthesis. By 15 hr of maturation, p75 synthesis was reduced to 14% of that in the fully grown, GV-intact oocyte and this is similar to the level of p75 synthesis in ovulated eggs. The level of p75 synthesis following in vitro translation of total egg RNA is only 38% lower than that obtained from total oocyte RNA. In addition, synthesis of p75 is observed following in vitro translation of oocyte, but not egg, poly(A)+ RNA. These results are consistent with p75 synthesis during oocyte maturation being under translational control.     

Cytoplasmic remodelling and the acquisition of developmental competence in pig oocytes

The progression of oocyte meiosis is accompanied by major changes in the ooplasm that play a key role in the completion of a coordinate nuclear and cytoplasmic maturation. We review evidence from the literature and present data obtained in our laboratory on different aspects of pig oocyte cytoplasm compartmentalization during maturation and early embryo development. In particular, we will discuss the changes in adenosine triphosphate (ATP) concentration and distribution taking place during the maturation process and their possible significance for oocyte developmental competence. We describe two important aspects of cytoplasmic streaming: mitochondrial distribution patterns in oocytes and early embryos and the complex rearrangements of cytoplasmic microtubule networks, while discussing their possible correlations with ooplasm compartmentalization. Recent evidence indicates that the cytoskeleton is used to shuttle not only organelles but also mRNAs to specific sites within the oocyte cytoplasm. Localization is driven by specific molecular motors belonging to the kinesin superfamily and requires the involvement of the RNA targeting molecule Staufen. We present recent experimental evidence, obtained in our laboratory, on the pig orthologues for kinesin KIF5B and Staufen, describe their expression patterns and discuss their possible role in oocyte maturation.     

RNA synthesis in the ovary and oocytes of the starfish

Qualitative studies on the in vitro uptake and incorporation of tritiated uridine into RNA of the somatic and germinal elements of the starfish ovary were carried out prior to and during hormone-induced oocyte maturation and spawning.Autoradiography of nonhormone-treated ovaries indicated that the outer ovarian wall contained the highest concentration of label, with lesser amounts in the follicle cells and least in the oocytes. Oocytes and follicle cells localized at the periphery of the ovary were labeled first, and both cells became progressively labeled throughout the ovary with time; the label first appeared localized in the nucleolus of the oocyte.Sucrose gradient analysis of the separated cellular components of prelabeled hormone-treated ovaries indicated that RNA synthesis occurred in all segments of the ovary and that the spawned oocyte fraction was the least active. Synthesis of ribosomal RNA was detectable after a lag period of approximately 4 hr. Oocytes incubated in ³H-uridine during and subsequent to 1-methyladenine-induced spawning and maturation synthesized 15–19 S and low molecular weight RNA but not ribosomal RNA. Synthesis of the 15–19 S RNA was inhibited with ethidium bromide and to a limited extent by actinomycin D. Isolated mitochondrial fractions contained most of the labeled 15–19 S RNA. These data suggest the mitochondrial origin of most, if not all, of this intermediate-weight RNA. On the basis of these studies, it appears that starfish oocytes and follicle cells are metabolically active at the transitional period from growth to maturational stages in oocytes. Synthesis of RNA furthermore apparently continues in the cytoplasm subsequent to germinal vesicle breakdown and spawning.     

A conserved role of a DEAD box helicase in mRNA masking.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3' UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD--> DQAD and HRIGR-->HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.     

Behavior of individual maternal pA+ RNAs during embryogenesis of Xenopus laevis

Of the 10 Xenopus oocyte cDNA clones previously examined in this laboratory (L. Golden, U. Schafer, and M. Rosbash, 1980, Cell22, 835–844), 5 are complementary to RNAs which which decrease in abundance during early development. We have further examined the behavior during embryogenesis of these 5 sets of clone-complementary RNAs. The results indicate that for 3 of these 5 sets of RNAs there is an increase in the per embryo levels of RNA. Thus, 8 of the 10 clones originally examined are complementary to RNAs which increase in amount during early embryogenesis. One of the remaining two clones is complementary to (at least) 4 RNAs which vary somewhat in their levels during embryogenesis. The last clone (XOC 2–7) is complementary to an RNA species which is largely destroyed at late blastula or early gastrula. This RNA is therefore the only maternal sequence, of the ten clones examined, which unambiguously decreases in amount during embryogenesis. The data also show that XOC 2–7 RNA is largely adenylated at oocyte maturation and then deadenylated during subsequent embryogenesis while another clone, XOC 1–2, is largely dead-enylated at oocyte maturation. The results also suggest that a large fraction of oocyte RNAs are present in early embryos (and in liver) and are largely the same size as in oocytes.