A comparative proteomic analysis reveals important proteins for the fertilization and early embryonic development of the oyster Crassostrea gigas

Molluscan development involves important features that are important to understanding not only molluscan ontogeny but also animal evolution. To gain insight into the gamete proteome and protein function in fertilization and early development, we analyzed the proteomes of unfertilized oocytes and early embryos (2/4‐cell stage) of the Pacific oyster, Crassostrea gigas. An oocyte reference map containing 116 protein spots, of which 69 were identified, revealed a high abundance of vitellogenin‐derived protein spots. The differentially regulated protein spots during fertilization were screened using comparative proteomic approaches. In total, 18 differentially regulated protein spots were screened, and 15 of these were identified and divided into three groups. The proteins belonging to the first group function in energy supply and antioxidation and are proposed to ensure successful fertilization by regulating the levels of adenosine triphosphate, resisting oxidative stress, and preventing polyspermy. The proteins of the second group are associated with protein synthesis and modification, reflecting active protein synthesis after fertilization. The three proteins belonging to the final group are hypothesized to function in the regulation of embryonic development through the establishment of cell polarity and modulation of methylation reactions in nuclei. These results will enhance our knowledge of molluscan fertilization and development.     

Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns

It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.     

Aberrant protein expression is associated with decreased developmental potential in porcine cumulus–oocyte complexes

Oocyte developmental competence is progressively obtained during pubertal development in females. Poor developmental potential in oocytes derived from prepubertal females suggests that essential processes required for oocyte development have not been fulfilled. The objective of this experiment was to analyze the protein profiles of porcine cumulus–oocyte complexes (COC) derived from cyclic and prepubertal females to identify alterations in protein abundance that correlate with developmental potential. COC complexes, aspirated from prepubertal and cyclic ovaries, were pooled into three replicates of 400 COCs each per treatment in ～100 µl SOF‐HEPES medium. Protein samples were extracted and analyzed by two‐dimensional differential in gel electrophoresis (2D‐DIGE). Over 1,600 proteins were resolved on each of the three replicate gels. Sixteen protein spots were identified by mass spectrometry, representing 14 unique, differentially expressed proteins (volume ratio greater than 1.3). Glutathione‐S‐transferase and pyruvate kinase 3 were more abundant in COCs derived from cyclic females, whereas soluble epoxide hydrolase and transferrin were more abundant in prepubertal derived COCs. Abundance of several glycolytic enzymes (enolase 1, pyruvate kinase 3, and phosphoglycerate kinase) was increased in COCs derived from cyclic females, suggesting glucose metabolism is decreased in prepubertal derived COCs. We conclude that the abundance of proteins involved in metabolism and oxidative stress regulation is significantly altered in prepubertal derived COCs and may play a role in the mechanisms resulting in developmental competence. Mol. Reprod. Dev. 77: 51–58, 2010. © 2009 Wiley‐Liss, Inc.     

Protein phosphorylation changes reveal new candidates in the regulation of egg activation and early embryogenesis in D. melanogaster

Egg activation is the series of events that must occur for a mature oocyte to become capable of supporting embryogenesis. These events include changes to the egg's outer coverings, the resumption and completion of meiosis, the translation of new proteins, and the degradation of specific maternal mRNAs. While we know some of the molecules that direct the initial events of egg activation, it remains unclear how multiple pathways are coordinated to change the cellular state from mature oocyte to activated egg. Using a proteomic approach we have identified new candidates for the regulation and progression of egg activation. Reasoning that phosphorylation can simultaneously and rapidly modulate the activity of many proteins, we identified proteins that are post-translationally modified during the transition from oocyte to activated egg in Drosophila melanogaster. We find that at least 311 proteins change in phosphorylation state between mature oocytes and activated eggs. These proteins fall into various functional classes related to the events of egg activation including calcium binding, proteolysis, and protein translation. Our set of candidates includes genes already associated with egg activation, as well as many genes not previously studied during this developmental period. RNAi knockdown of a subset of these genes revealed a new gene, mrityu, necessary for embryonic development past the first mitosis. Thus, by identifying phospho-modulated proteins we have produced a focused candidate set for future genetic studies to test their roles in egg activation and the initiation of embryogenesis.     

Comparative proteomic analysis of proteins involved in oocyte meiotic maturation in mice

After birth, oocytes stay at the diplotene stage in prophase of meiosis I. Meiosis resumes about 1 day before ovulation, and arrests in metaphase II (MII) after ovulation. The mature, MII oocytes are then ready for fertilization and to provide materials for early embryonic development. Proteomic characterization of oocytes can help identify proteins that are important for female meiotic maturation and early embryonic development. In this study, we compared the proteomic profiles between the germinal vesicle and MII mouse oocytes by two-dimensional electrophoresis; 95 differentially expressed protein spots corresponding to 63 proteins were identified. Many of these proteins are known to be essential for oocyte meiosis and early embryonic development, such as adenylosuccinate synthetase, nucleoplasmin-2, and protein-arginine deiminase type-6. Of the 12 proteins that were identified and are highly expressed in oocytes, a novel protein, E330034G19Rik, was found to be oocyte-specific. According to analysis by bioinformatics, it may regulate chromosome segregation during meiosis or cleavage. An in-depth study of these proteins will help us better understand the mechanisms of oocyte meiotic maturation, fertilization, and early embryogenesis. It will also help us understand the mechanisms of diseases that stem from abnormal oocyte maturation, such as polycystic ovary syndrome and premature ovary failure.     

Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in In Vitro–Matured Oocytes

Oocyte maturation is pertinent to the success of in vitro maturation (IVM), which is used to overcome female infertility, and produced over 5000 live births worldwide. However, the quality of human IVM oocytes has not been investigated at single-cell proteome level. Here, we quantified 2094 proteins in human oocytes during in vitro and in vivo maturation (IVO) by single-cell proteomic analysis and identified 176 differential proteins between IVO and germinal vesicle oocytes and 45 between IVM and IVO oocytes including maternal effect proteins, with potential contribution to the clinically observed decreased fertilization, implantation, and birth rates using human IVM oocytes. IVM and IVO oocytes showed separate clusters in principal component analysis, with higher inter-cell variability among IVM oocytes, and have little correlation between mRNA and protein changes during maturation. The patients with the most aberrantly expressed proteins in IVM oocytes had the lowest level of estradiol per mature follicle on trigger day. Our data provide a rich resource to evaluate effect of IVM on oocyte quality and study mechanism of oocyte maturation.     

Maternal diabetes and oocyte quality

Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging evidence has implicated that these effects are associated with compromised oocyte competence. Several developmental defects during oocyte maturation in diabetic mice have been reported over past decades. Most recently, we further identified the structural, spatial and metabolic dysfunction of mitochondria in oocytes from diabetic mice, suggesting the impaired oocyte quality. These defects in the oocyte may be maternally transmitted to the embryo and then manifested later as developmental abnormalities in preimplantation embryo, congenital malformations, and even metabolic disease in the offspring. In this paper, we briefly review the effects of maternal diabetes on oocyte quality, with a particular emphasis on the mitochondrial dysfunction. The possible connection between dysfunctional oocyte mitochondria and reproductive failure of diabetic females, and the mechanism(s) by which maternal diabetes exerts its effects on the oocyte are also discussed.     

Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle

The present study sought to assess the combined effects of body composition and diet (level of feeding) on the postfertilization developmental potential of oocytes recovered from heifers using ultrasound-guided transvaginal follicular aspiration and to relate oocyte quality to the metabolic status of these animals. By collecting oocytes on repeated occasions spanning several weeks, it was possible to assess the cumulative effects of changes in nutritional status on oocyte quality over this period. Twenty-four heifers of low and moderate body condition were placed on one of two levels of feeding (equivalent to once or twice the maintenance requirements of these animals). Oocytes were recovered at two defined time points within each of three successive estrous cycles and were matured, fertilized, and cultured to the blastocyst stage in vitro. The results show that the effect of feeding level on oocyte quality is dependent on the body condition of the animal, with the high level of feeding being beneficial to oocytes from animals of low body condition but detrimental to oocytes from animals of moderately high body condition. Furthermore, the effects of high levels of feeding on oocyte quality were cumulative, with blastocyst yields for relatively fat heifers on twice the maintenance requirement deteriorating with time relative to yields for relatively thin heifers on the same level of feeding. Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinemic, and we show, to our knowledge for the first time in ruminants, that this condition is associated with impaired oocyte quality.     

Proteome Dynamics and Physiological Responses to Short-Term Salt Stress in Brassica napus Leaves

Salt stress limits plant growth and crop productivity and is an increasing threat to agriculture worldwide. In this study, proteomic and physiological responses of Brassica napus leaves under salt stress were investigated. Seedlings under salt treatment showed growth inhibition and photosynthesis reduction. A comparative proteomic analysis of seedling leaves exposed to 200 mM NaCl for 24 h, 48 h and 72 h was conducted. Forty-four protein spots were differentially accumulated upon NaCl treatment and 42 of them were identified, including several novel salt-responsive proteins. To determine the functional roles of these proteins in salt adaptation, their dynamic changes in abundance were analyzed. The results suggested that the up-accumulated proteins, which were associated with protein metabolism, damage repair and defense response, might contribute to the alleviation of the deleterious effect of salt stress on chlorophyll biosynthesis, photosynthesis, energy synthesis and respiration in Brassica napus leaves. This study will lead to a better understanding of the molecular basis of salt stress adaptation in Brassica napus and provides a basis for genetic engineering of plants with improved salt tolerance in the future.     

Caspase-12 compensates for lack of caspase-2 and caspase-3 in female germ cells

Previously, we analyzed mice lacking either caspase-2 or caspase-3 and documented a role for caspase-2 in developmental and chemotherapy-induced apoptosis of oocytes. Those data also revealed dispensability of caspase-3, although we found this caspase critical for ovarian granulosa cell death. Because of the mutual interdependence of germ cells and granulosa cells, herein we generated caspase-2 and -3 double-mutant (DKO) mice to evaluate how these two caspases functionally relate to each other in orchestrating oocyte apoptosis. No difference was observed in the rate of spontaneous oocyte apoptosis between DKO and wildtype (WT) females. In contrast, the oocytes from DKO females were more susceptible to apoptosis induced by DNA damaging agents, compared with oocytes from WT females. This increased sensitivity to death of DKO oocytes appears to be a specific response to DNA damage, and it was associated with a compensatory upregulation of caspase-12. Interestingly, DKO oocytes were more resistant to apoptosis induced by methotrexate (MTX) than WT oocytes. These results revealed that in female germ cells, insults that directly interfere with their metabolic status (e.g. MTX) require caspase-2 and caspase-3 as obligatory executioners of the ensuing cell death cascade. However, when DNA damage is involved, and in the absence of caspase-2 and -3, caspase-12 becomes upregulated and mediates apoptosis in oocytes. Takai and Matikainen contributed equally to this work.     

Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5′monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-l-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.     

In vitro development of individually matured bovine oocytes in relation to follicular wall atresia

Morphologically good-quality cumulus oocyte complexes (COCs) can originate from slightly atretic follicles. Biochemical and ultrastructural investigations reveal that a very high percentage of bovine antral follicles express some degree of atresia. The aim of the present study was to determine the developmental competence of good quality COCs in relation to their biochemically estimated follicular wall apoptosis. For experimental design a single oocyte maturation system was established, followed by group culture processing oocytes together according to their level of follicular wall atresia estimated by an ELISA for apoptotic cell death. Single oocyte culture during maturation reduced the developmental capacity of oocytes significantly (P < 0.01), with 5% blastocysts versus 25% after common group culture. Blastocyst formation for single oocyte maturation was found exclusively in oocytes isolated from luteal stage ovaries with low degree of apoptosis. The level of follicular wall apoptosis in luteal stage follicles (0.79 +/- 0.05 units/mg protein, n = 198) was lower than in follicular stage follicles (1.14 +/- 0.05 units/mg protein, n = 208). This was caused by significant higher levels in small (< 3.5 mm diameter) and large (> 5.5 mm diameter) follicles of the latter group. In conclusion, despite reduced developmental capacity after single oocyte maturation, we were able to reveal some functional relationship between oocyte origin and quality. It was shown that morphologically good quality COCs isolated from follicles with higher degree of apoptosis lose their developmental capacity.     

Mitochondrial DNA variations in ova and blastocyst: Implications in assisted reproduction

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.     

Comparative proteomic analysis of leaf tissue from tomato plants colonized with <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis>

A comparative proteomic approach was performed to analyze the differential accumulation of leaf proteins in response to the symbiosis between Solanum lycopersicum and the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis. Protein profiling was examined in leaves from tomato plants colonized with AMF (M), as well as non-colonized plants fertilized with low phosphate (20 μM P; NM-LP) and non-colonized plants fertilized with regular phosphate Hoagland’s solution (200 μM P; NM-RP). Comparisons were made between these groups, and 2D-SDS-PAGE revealed that 27 spots were differentially accumulated in M vs. NM-LP. Twenty-three out of the 27 spots were successfully identified by mass spectrometry. Two of these proteins, 2-methylene-furan-3-one reductase and auxin-binding protein ABP19a, were up-accumulated in M plants. The down-accumulated proteins in M plants were associated mainly with photosynthesis, redox, and other molecular functions. Superoxide dismutase, harpin binding protein, and thioredoxin peroxidase were down-accumulated in leaves of M tomato plants when compared to NM-LP and NM-RP, indicating that these proteins are responsive to AMF colonization independently of the phosphate regime under which they were grown. 14-3-3 protein was up-accumulated in NM-RP vs. NM-LP plants, whereas it was down-accumulated in M vs. NM-LP and M vs. NM-RP, regardless of their phosphate nutrition. This suggests a possible regulation by P nutrition and AMF colonization. Our results demonstrate AMF-induced systemic changes in the expression of tomato leaf proteins, including the down-accumulation of proteins related to photosynthesis and redox function.