Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs

Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa beta'-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs.     

Identification of two forms of vitellogenin-derived phosvitin and elucidation of their fate and roles during oocyte maturation in the barfin flounder, Verasper moseri

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.     

Derivation of major yolk proteins from parental vitellogenins and alternative processing during oocyte maturation in Fundulus heteroclitus

Various Coomassie blue-staining yolk proteins (YPs) present in oocytes and eggs of Fundulus heteroclitus, a teleost that produces low hydrated, demersal eggs (benthophil species), were subjected to N-terminal microsequencing. Four YPs were N-terminally blocked, while five yielded sequence information. Of the latter, four corresponded to internal sequences of vitellogenin 1 (Vg1), whereas a fifth band corresponded to the N-terminal sequence of Vg2. Phosphorylated YPs (phosvitins and phosvettes) derived from the polyserine domain of Vg were not successfully sequenced. The major N-terminally blocked 122-and 103-kDa YPs both represented the lipovitellin heavy chain of Vg1 (LvH1), and thus most of the oocyte YPs were derived from Vg1. During oocyte maturation in vivo and in vitro, the LvH1 122 is degraded, concomitant with an increased enzymatic activity of cathepsin B, while the 45-kDa YP is converted to a 42-kDa YP. The LvH1 122 was found to contain a consensus site for proteolytic degradation (PEST) near its C-terminus, which is missing from its stable, but truncated twin sequence, LvH1 103. We suggest that this site becomes exposed to cathepsin B during the hydration process that accompanies oocyte maturation and renders the LvH1 122 susceptible to proteolysis. PEST sites are found in Vg sequences from other benthophil fish, whereas, interestingly, they are missing in marine teleosts that spawn highly hydrated, pelagic eggs (pelagophil species), displaying a different pattern of Vg incorporation into YPs and LvH1 and LvH2 processing to that found in F. heteroclitus. Thus, different models of Vg/YP precursor/product relationship and further processing during oocyte maturation and hydration are proposed for pelagophil and benthophil teleosts.     

Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.     

Deduced primary structure of vitellogenin in the giant freshwater prawn, Macrobrachium rosenbergii, and yolk processing during ovarian maturation

A cDNA encoding vitellogenin (Vg) in the giant freshwater prawn, Macrobrachium rosenbergii, was cloned based on the cDNA sequence of vitellin (Vn) fragments A-N and B-42 determined previously, and its amino acid sequence deduced. The open reading frame (ORF) encoded 2,537 amino acid residues and its deduced amino acid sequence possessed three consensus cleavage sites, R-X-R-R, similar to those reported in Vgs of insects. The deduced primary structure of Vg in M. rosenbergii was seen to be similar to that of Penaeus japonicus, especially in the N-terminal region. It is therefore likely that Vgs in crustacean species including prawns and other related decapods exhibit a similar structural pattern. Based on the deduced primary structure of Vg and analysis of the various Vg and Vn subunits found in the hemolymph and ovary during ovarian maturation, we demonstrated the post-translational processing of Vg in M. rosenbergii. This is the first time that Vg processing has been clearly demonstrated in a crustacean species. Vg, after being synthesized in the hepatopancreas, is considered to be cleaved by a subtilisin-like endoprotease to form two subunits, A and proB, which are then released into the hemolymph. In the hemolymph, proB is possibly cleaved by a processing enzyme of unknown identity to give rise to subunits B and C/D. The three processed subunits A, B, and C/D are sequestered by the ovary to give rise to three yolk proteins, Macr-VnA, VnB, and VnC/D.     

Preimplantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro

Of eggs ovulated in LT/Sv mice, 10–20% undergo spontaneous parthenogenetic activation, and 40–50% of the parthenotes develop to blastocysts when cultured in simple defined medium from the one-cell stage. Similar percentages of oocytes isolated from Graafian follicles undergo parthenogenetic activation after spontaneous maturation in simple defined medium, but embryonic development proceeds no further than the two-cell stage. The simple defined medium that supported preimplantation development of ovulated eggs and spontaneous maturation of extrafollicular oocytes contained no serum, free amino acids, or vitamins. The present experiments were conducted to determine what conditions during spontaneous maturation of extrafollicular oocytes could promote the ability of oocytes to develop to blastocysts after parthenogenetic activation and mimic the environment of preovulatory follicles. Cumulus-enclosed oocytes that were matured in simple medium supplemented with fetal bovine serum (FBS) developed to blastocysts after spontaneous parthenogenetic activation. Furthermore, minimum essential medium (MEM), a complex medium containing free amino acids and vitamins, could substitute completely for FBS for maturing oocytes from (C57BL/6J × LT/Sv)F₁ mice, and to a lesser extent for maturing LT/Sv oocytes. Therefore, even though germinal vesicle breakdown in mouse oocytes and preimplantation development of mouse eggs can occur in the absence of an exogenous supply of free amino acids and vitamins, a complete, or normal, mouse oocyte maturation cannot. These results also demonstrated that gonadotropins are not necessary during oocyte meiotic maturation for parthenogenetically activated eggs to develop through the preimplantation stages. Luteinizing hormone or 17β-estradiol in MEM during oocyte maturation had no effect on the subsequent development of parthenotes. In contrast, follicle stimulating hormone (FSH) and progesterone in the maturation medium decreased the number of ova that subsequently cleaved, and FSH decreased the number of cleaved eggs that developed to blastocysts.     

Characterization of vitellin from the ovaries of the banana shrimp Litopenaeus merguiensis

Vitellin (Vt) was purified from ovary extracts of mature females of the banana shrimp Litopenaeus merguiensis using DEAE-Sephacel and Superdex 200 columns. Native Vt had an apparent molecular mass of 398 kDa as determined by native PAGE and by gel filtration chromatography. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of two major subunits of 87 and 78 kDa, although some faint bands were also detected. The N-terminal 10 amino acids sequence of the 78 kDa subunit is identical to that of Litopenaeus vannamei Vt and very similar to that of Litopenaeus japonicus vitellogenin (Vg) as well as Litopenaeus semisulcatus Vt, with an identity of 89%. Anti-Vt polyclonal antibody raised against purified Vt shows a high specificity with only ovarian Vt and hemolymph Vg of vitellogenic shrimps in double immunodiffusion and Western blot assays. Vg and Vt concentrations in hemolymph, hepatopancreas and ovaries were measured by ELISA. Vg concentrations increased in the hemolymph in the early stages of ovarian development and declined in the maturation stages. As there were undetectable concentrations of Vg in the hepatopancreas while an elevation of Vg levels occurred in the hemolymph, during the time that Vt was accumulating in the ovaries during oogenesis, this would suggest that the contribution of Vg synthesized by the hepatopancreas only might be not sufficient for adequate development of the oocytes in the banana shrimp L. merguiensis during vitellogenesis.     

Goldsinny wrasse (Ctenolabrus rupestris) is an extreme vtgAa-type pelagophil teleost

During oocyte maturation in the goldsinny wrasse (Ctenolabrus rupestris) extensive proteolysis of yolk proteins generates a large pool of free amino acids that drive hydration of the pelagic egg. By cloning hepatic vitellogenins (vtg) and using mass spectrometry, N-terminal microsequencing, and Western-immunoblotting to identify the yolk proteins (Yp), we show that multiple forms of vitellogenin mRNAs (vtgAa, vtgAb, and vtgC) are expressed in the liver, but only a single major class of the Yps derived from vtgAa predominates in the oocytes. Some Yps derived from vtgAb and vtgC appear also to be incorporated in the oocytes and eggs, but only at background levels. During oocyte hydration the vtgAa-derived lipovitellin heavy chain (LvH-Aa) and its cleavage variants are completely degraded leaving only a processed lipovitellin light chain (LvL-Aa) fragment as the major yolk protein for embryonic development. The maturational cleavage site of the LvL-Aa is identified as two amino acids downstream from the conserved Tyr(1168) of VtgAa in Atlantic halibut. In addition, although a beta'-component (approximately 18 kDa) is present in the oocytes, it is not fully degraded during the hydration process.     

Effects of polyunsaturated fatty acids and prostaglandins on oocyte maturation in a marine teleost, the European sea bass (Dicentrarchus labrax)

The effects of the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and prostaglandins (PGs) on oocyte maturation were investigated in a marine teleost, the sea bass (Dicentrarchus labrax). Follicle-enclosed postvitellogenic, preovulatory oocytes were cultured in vitro and maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown. Human chorionic gonadotropin was administered as the maturation-inducing gonadotropin (GTH) and was capable of inducing maturation in a time- and dose-dependent manner. Free AA induced maturation in a dose- and time-dependent manner and enhanced GTH-induced maturation, while EPA, DHA, and oleic acid were ineffective. Maturation induced by GTH was significantly suppressed by a phospholipase A(2) blocker, suggesting that mobilization of AA was involved in GTH-induced maturation. Moreover, EPA and DHA exhibited a significant, dose-dependent attenuation of GTH-induced maturation. Maturation induced by GTH was inhibited in the presence of a cyclooxygenase inhibitor, indomethacin, and this inhibition was reversed by addition of AA, PGE(2), or PGF(2alpha). PGE(2) and PGF(2alpha) alone were both effective stimulators of maturation, while PGE(1) and PGE(3) were ineffective. The effect of PUFAs on oocyte maturation in vitro were corroborated with studies in vivo. Oocytes were obtained from females fed a commercial, PUFA-enriched diet (RD) and maturational behavior was compared with oocytes from females fed a natural diet (ND) with a higher EPA content and n-3:n-6 ratio. Although no significant difference was observed in the rate of spontaneous oocyte maturation, a higher percentage of GTH-induced maturation and lower percentage of atresia were observed in RD oocytes. Moreover, while basal PGE production from oocytes from both groups was the same, RD oocytes produced significantly higher levels of PGE in the presence of hCG. The results from this study provide evidence for the participation of AA metabolism in GTH-induced oocyte maturation, and suggest that other PUFAs and PGs may play important roles in the induction of maturation in a marine teleost.     

Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A2 induce starfish oocyte maturation

In starfish oocyte maturation (meiosis reinitiation) is induced by the natural hormone 1-methyladenine (1-Me-Ade). This paper shows that arachidonic acid (AA) induces oocyte maturation at concentrations above 0.5 microM. This maturation shares many characteristics with 1-MeAde-induced maturation: same kinetics, same required contact time, same stimulations of protein phosphorylation and sodium influx. Although calcium facilitates the AA-induced but not the 1-MeAde-induced maturation, AA, like 1-MeAde, does not stimulate the uptake of calcium. Calcium does not facilitate the uptake of AA by oocytes. Out of 36 different fatty acids (saturated and unsaturated), only eicosatetraenoic (AA) and eicosapentaenoic acids were found to mimic 1-MeAde. Calcium-dependent phospholipases A2 from bee venom and Naja venom also induce maturation (0.1-1 unit/ml) when added externally to the oocytes. Phospholipase A2 inhibitors (quinacrine, bromophenacylbromide) block maturation; inhibition is reversed by increasing the 1-MeAde concentration and only occurs during the hormone-dependent period. AA is usually metabolized through oxidation by cyclooxygenase or lipoxygenase. Cyclooxygenase inhibitors (acetylsalicylic acid, indomethacin, tolazoline) do not block maturation; prostaglandins E2, D2, F2 alpha, I2, and thromboxane B2 do not induce meiosis reinitiation. On the other hand, lipoxygenase inhibitors (quercetin, butylated hydroxytoluene, and eicosatetraynoic acid) block 1-MeAde-induced maturation; although leukotrienes (A4, B4, C4, D4, E4) have no effects on oocytes, two other lipoxygenase products, 12- and 15-hydroxyeicosatetraenoic acids (and their corresponding hydroperoxy-) induce oocyte maturation (around 1 microM). The possible mode of action of the fatty acids inducing oocyte maturation is discussed.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.     

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

In order to gain further insight into IGF-1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF-1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant-negative form of Grb2 specifically inhibits insulin-induced resumption of meiosis. We finally show that Grb2 binds to p60(Shc) in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2-Sos are implicated in ras-dependent Xenopus oocyte maturation induced by insulin/IGF-1; they also indicate that inability of insulin/IGF-1 to activate the Ras-MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.     

Cytokeratin phosphorylation, cytokeratin filament severing and the solubilization of the maternal mRNA Vg1

During meiotic maturation, the cortical cytokeratin filament system of the Xenopus oocyte disappears (Klymkowsky, M. W., and L. A. Maynell. 1989. Dev. Biol. 134:479). Here we demonstrate that this disappearance results from the severing of cytokeratin filaments into a heterogenous population of oligomers, with S- values ranging from 12S and greater. Cytokeratin filament severing correlates with the hyperphosphorylation of the type II cytokeratin of the oocyte. Both the severing of cytokeratin filaments and cytokeratin hyperphosphorylation are reversed by treatment with cycloheximide. These data suggest that fragmentation of cytokeratin filaments is controlled, at least in part, by the phosphorylation of the type II cytokeratin, and that the cytokeratin kinase activity responsible is biosynthetically labile. Cytokeratin filaments have been suggested to anchor the maternal mRNA Vg1 to the vegetal cortex of the oocyte (Pondel, M., and M. L. King. 1988. Proc. Natl. Acad. Sci. USA. 85:7216). By injecting fractions containing active maturation promoting factor or a purified, mutant cyclin protein, we find that the bulk of the Vg1 mRNA in the oocyte can be solubilized under conditions that block the fragmentation of cytokeratin filaments, and that the fragmentation of cytokeratin filaments itself leads to the solubilization of only a minor fraction of the Vg1 mRNA. Thus, at best, cytokeratin filaments directly anchor only a minor fraction of the Vg1 mRNA in the oocyte. Moreover, factors distinct from maturation promoting factor appear to be required for the complete solubilization of Vg1 mRNA during oocyte maturation.     

烟粉虱MEAM1隐种卵黄原蛋白受体基因cDNA的克隆、 序列分析及在不同发育时期的表达

卵黄原蛋白受体（vitellogenin receptor, VgR）是卵黄原蛋白被卵母细胞摄取的关键因子, 在卵黄发生和卵母细胞发育等生理过程中发挥着重要作用。为探讨烟粉虱Bemisia tabaci VgR的功能, 我们采用RT-PCR和RACE等技术扩增了烟粉虱MEAM1隐种B. tabaci Middle East-Asia Minor 1 (MAEM1) 的VgR基因cDNA 全长序列。生物信息学分析表明, 烟粉虱MEAM1隐种的VgR基因cDNA全长5 774 bp, 编码1 919个氨基酸, 推测分子量约201 kDa, N-端前31个氨基酸为信号肽。烟粉虱MEAM1隐种的VgR属于低密度脂蛋白受体（low density lipoprotein receptor, LDLR）家族, 蛋白质三维结构预测分析表明, 该受体具有LDLR家族基因典型的保守功能结构域。通过实时荧光定量PCR技术研究了烟粉虱MEAM1隐种VgR基因不同发育时期的表达, 结果表明VgR基因在伪蛹期开始表达, 并在羽化后1 d达到高峰, 此后逐渐降低, 3 d后又逐渐升高, 直至羽化后7 d达到峰值。研究结果丰富了卵黄原蛋白受体家族基因的数据库, 为今后深入研究并揭示烟粉虱卵黄发生的调控机制奠定了基础。     

Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development

The objective of this study was to determine the intrafollicular concentrations of free amino acids in pigs and to examine the effect of amino acids in a chemically defined maturation medium on oocyte maturation, in vitro fertilization (IVF), and embryo development in vitro. Pooled follicular fluid aspirated separately from small (<3mm in diameter), medium (3-8mm), and large follicles (>8mm) in three pairs of ovaries was analyzed for amino acid concentration. In addition, oocyte maturation, fertilization, and embryo development were examined after in vitro maturation (IVM) of oocytes in a defined maturation medium supplemented individually with glutamate (GLU), glutamine (GLN), glycine (GLY), aspartate (ASP), asparagine (ASN), arginine (ARG), alanine (ALA), leucine (LEU), lysine (LYS), proline (PRO), and valine (VAL). Irrespective of follicle size, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pig follicular fluid (pFF). Sperm penetration was not altered by amino acid treatment during IVM, but monospermic fertilization was increased (P<0.05) by GLN, ASP, and VAL. All amino acids except ASP and ASN stimulated (P<0.05) male pronuclear formation after IVF. ARG and ALA treatment during IVM improved (P<0.05) blastocyst formation. In conclusion, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pFF and amino acids in a defined medium improved porcine monospermic fertilization, male pronuclear formation, and preimplantation development.     

Bafilomycin A1 inhibits proteolytic cleavage and hydration but not yolk crystal disassembly or meiosis during maturation of sea bass oocytes

Oocytes of the black sea bass, Centropristes striata, were enlarged in volume more than three-fold over a 24-hr period during oocyte maturation, both in vivo and in vitro. At the same time, the opaque oocytes clarified while the crystalline yolk inclusions lost their ordered structure, fused with one another, and formed a continuous electron-lucent mass. The oocyte size increase was due almost entirely to water uptake, which was accompanied by the accumulation of Na+, K+, and free amino acids (FAAs). The absolute amounts of each of these small molecular weight osmotic effectors increased 2x, 4x, and over 10x, respectively, indicating that the generation of FAAs is the major cause of water uptake during maturation. Amino acid analyses indicated that the amounts of all amino acids except taurine increased, so that selective amino acids were not produced during maturation. The increase in FAAs was accompanied by the loss of certain high-molecular-weight yolk proteins and the generation of many smaller peptides. Oocytes stimulated to undergo maturation in the presence of bafilomycin A1, a specific inhibitor of the vacuolar ATPase-dependent proton pump, clarified and underwent maturation but did not increase significantly in size. Cytological examination revealed that yolk crystals fused and became homogeneous but maintained their electron density. No evidence of proteolysis was found in bafilomycin A1-treated oocytes and the generation of FAAs together with hydration was inhibited in a dose-dependent manner (I50 = 3 nM bafilomycin A1). Taken together, we postulate that the pronounced oocyte hydration in marine teleosts that spawn pelagic (floating) eggs is accomplished by a two-step process whereby (i) K+ influx promotes yolk crystal disassembly and yolk sphere fusion and (ii) acidification of the yolk spheres activates yolk proteolysis and concomitant hydration. Bafilomycin A1 inhibits only the second step so that many of the events of oocyte maturation, including germinal vesicle breakdown, occur in its presence but oocyte hydration is suppressed.     

Amino acids can induce nondisjunction during maturation of mammalian oocytes in vitro

Certain chromosomal disorders in mammalian embryos are traceable to meiotic errors during oocyte maturation. This report evaluates the influence of amino acids on meiotic maturation in vitro of oocytes from pigs, hamsters, and rats. The results indicate that maturing porcine oocytes respond not only to 1-glutamine (1-gln) but also to 1-isoleucine (1-ileu) in complex or chemically defined media by exhibiting significantly (P less than .05) increased incidences of nondisjunction when compared with oocytes in control medium. Nondisjunction was highly correlated (r = 0.981) with dose of 1-gln in porcine oocytes (incidences of maturing oocytes exhibiting nondisjunction were 19.3%, 39.7%, 41.5%, 66.2%, and 88.5% at 1-gln concentrations of 0 mM, 0.5 mM, 1.0 mM, 3.0 mM and 10.0 mM, respectively). Hamster oocytes also exhibited significantly (P less than .05) increased nondisjunction when cultured in medium containing 1-gln (52% of maturing cumulus-enclosed oocytes exhibited nondisjunction in medium with 3.0 mM 1-gln vs. 6.3% in control medium). Denuded hamster oocytes also responded to 1-gln (35% exhibited nondisjunction in 4.0 mM 1-gln, 8% in control medium). In contrast the incidence of nondisjunction in rat oocytes was not increased significantly by 1-gln over a concentration range of 0-12.0 mM. This study demonstrates that maturing oocytes respond to certain environmental conditions by undergoing chromosomally abnormal maturation. Specifically, amino acids can induce nondisjunction when present in elevated concentrations during oocyte maturation. The amino acid influence not only was dose dependent for porcine oocytes but also was species specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Essential role of ubiquitin C-terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1, and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.     

Enhanced glycolysis after maturation of bovine oocytes in vitro is associated with increased developmental competence

The effect of maturation in vitro on metabolism of individual bovine oocytes was examined. Three maturation media were used: standard, consisting of tissue culture medium 199 supplemented with serum and pyruvate, and a chemically defined medium supplemented with either amino acids or lactate. Development to blastocyst was significantly higher (P < 0.05) after maturation in standard medium (47%) than in defined medium with lactate (17%) but was not different than maturation in defined medium with amino acids (29%). Glucose metabolism through the Krebs cycle was not different after maturation in standard or defined medium with amino acids or lactate (0.48, 0.43, 0.38 pmol/oocyte/3 hr, respectively) but was affected by the removal of unlabeled pyruvate from the metabolic measurement medium (0.16, 0.21, 0.27 pmol/oocyte/3 hr, respectively). When physiological concentrations of glucose (0.52 mM) and pyruvate (0.5 mM) were used, oxidation of pyruvate was not different after maturation in standard or defined medium with amino acids or lactate (1.38, 1.13, 1.13 pmol/ oocyte/3 hr, respectively); however, glycolysis was significantly increased (P < 0.05) in treatments that supported higher blastocyst development (standard medium, 1.77 pmol/oocyte/3 hr; defined medium with amino acids, 1.58 pmol/oocyte/3 hr; defined medium with lactate, 1.32 pmol/oocyte/3 hr). Metabolism of glucose through the Krebs cycle was low in all media. In contrast, oxidation of pyruvate readily occurred after maturation in vitro. Metabolism of glucose through the Embden-Meyerhof pathway is important during oocyte maturation in vitro, and higher glycolytic rates in in vitro matured oocytes may reflect increased developmental competence.