Markers Of Oocyte Maturation Research Articles

Mitofusin 1 as Marker of Oocyte Maturation in Relevance to ICSI Outcome in Infertile Females

Pro-fusion proteins as mitofusins-1 are required for controlling mitochondrial shape which determined by a dynamic balance between organelle fusion and fission and also supports oocyte development. When compare with somatic cell, mitochondria of oocyte are tiny and circular in presence. The aim is to study the mitofusin-1 in the follicular fluid as a marker for evaluating of oocyte maturation in women undergoing ICSI cycles. Fifty infertile females were included in cross-section study was undergoing ICSI procedure with age 20 to 42 years. After retrieval of oocyte and the number of oocytes was recorded by embryologist and follicular fluid Samples were used for the measurement of Mitofusin 1. Mitofusin 1 levels by ELISA kit (Mybiosource /USA).The results showed considerable higher mitofusin1levels in patients with good oocytes quality (3.71 ± 1.35 vs. 2.45 ± 1.12 & p=0.001). There were much positive correlations between follicular fluids mitofusin-1 with both total oocytes count (r= 0.374 & p= 0.007) and MII oocytes (r=0.383 & p=0.006); and there was no much correlation between mitofusin-1 levels with MI oocyte (r= - 0.100 & p= 0.490), germinal vesical oocyte (r=0.103 & p= 0.475) and fertilization rate (r= 0.054 & p= 0.711). High follicular fluid levels of Mitofusin 1 may positively impact oocyte development and pregnancy rate.

The expression levels of NOS2, HMOX1, and VEGFC in cumulus cells are markers of oocyte maturation and fertilization rate.

Throughout the reproductive life of women, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment, and act as a gatekeeper for oocyte developmental potential. One such mechanism relies on the hypoxia-tolerance response, which, with age, decreases systematically, including in the ovary. We aimed to evaluate the association between gene expression related to hypoxia and aging in CC and reproductive results in in vitro fertilization cycles. We recruited 94 women undergoing controlled ovarian stimulation. Total RNA was extracted from pooled CCs collected after oocyte pick-up (OPU) and reverse-transcribed to complementary DNA using random hexamers to test 14 genes related to hypoxia response via HIF1α activation, oxidative stress, and angiogenic responses. The expression of CLU, NOS2, and TXNIP had a positive correlation with age (rs = 0.25, rs = 0.24, and rs = 0.35, respectively). Additionally, NOS2 and HMOX1 expression correlated positively with the retrieval of immature oocytes (rs = 0.22 and rs = 0.40, respectively). Moreover, VEGFC levels decreased overall with increasing fertilization rate, independently of age (rs = -0.29). We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia with age, pointing at NOS2, HMOX1, and VEGFC expression as markers for oocyte maturation and fertilization success.

ANALYSIS OF PARAMETERS OF MORPHOFUNCTIONAL STATE OF PIG OOCYTE LIPIDOME IN CASE OF IN VITRO CULTIVATION

Identification of effective markers of oocyte maturation based on morphological and functional characteristics of intracytoplasmic lipid droplets will allow to increaseinformative value of the criteria for assessing the quality of donor gametes for improvement of the efficiency of using cellular reproductive technologies in animal husbandry, biomedicine, and veterinary medicine. The aim of the study is to analyze the parameters of morphofunctional state of the lipidome (the morphology of lipid droplets and the intensity of fluorescence of the Nile red/lipid droplets complex - Nile red IF) in Susscrofadomesticus oocytes before and after in vitro cultivation. Oocytes were matured using Sage Media Cleavage (CooperSurgical, USA) with 5% Serum Protein Substitute serum (CooperSurgical, USA), 10 IU human chorionic gonadotropin (Sigma, USA). As a result of ranking the population of donor oocytes according to morphology of lipid droplets (granules and clusters) and Nile red IF (low, medium, high), it was found that, the proportion of oocytes with an average Nile red IF and lipid droplets (LD) in the form of granules was 63% before cultivation, which was significantly higher than the proportion of gametes with an average IF of Nile red and LD of a mixed form of 41% (P<0.05). After cultivation, the level of oocytes with medium Nile red IF and LD in the form of granules was 45%, which exceeded the level of gametes with the same Nile red IF and mixed LD of 23% (P<0.05). The proportion of in vitro matured oocytes with high Nile red IF and mixed LD morphology (granules and clusters) was 33%, 14% were characterized by the above parameters before cultivation (P<0.05). Comparative morphological analysis of the relation between the parameters of morphofunctional activity (morphology and IF Nile red) of lipidome in Susscrofadomesticus oocytes was carried out for the first time. Features of functional activity of lipidome before and after cultivation were revealed. The usage of a comprehensive analysis of parameters of functional activity of lipidomeforassessment of the quality of the oocyte is substantiated. Pig oocytes which morphology of lipid droplets is represented by a granular form are promising for cultivation, while Nile red IF is assessed as low. With regard to the analysis of the obtained results, it is recommended to use the proposed comprehensive assessment of lipidome based on morphology and Nile red IF to assess the initial population of donor oocytes in cellular reproductive technologies.

The insulin-like growth factor 1 stimulates ovarian steroidogenesis and oocyte maturation in spotted steed Hemibarbus maculatus

The insulin-like growth factor 1 (IGF1) plays important roles in fish reproduction. To understand the role of IGF1 in steroidogenesis and oocyte maturation, we investigated expression pattern and functions of Igf1 during in vitro maturation of spotted steed (Hemibarbus maculatus) oocytes. Igf1 expression quantified by qPCR and western blotting revealed that both Igf1 mRNA and protein was significantly increased in mature follicles. The immunofluorescence analyses showed IGF1 protein signal was detected in the theca cells of ovarian follicles during vitellogenesis stage, and in both theca cells and granulosa cells of matured oocytes. Incubation of ovarian follicles with recombinant IGF1 (rIGF1) significantly enhanced the rate of spotted steed oocyte germinal vesicle breakdown (GVBD), a marker of oocyte maturation. qPCR analysis showed that the transcription of steroidogenesis-related genes (3β-hsd, 20β-hsd, Cyp17a and Cyp19a1a) were influenced after IGF1 treatment. In addition, the expression profiles of receptors of Igf1, Fsh and Lh indicated that a cross talk response to IGF1 induced oocyte maturation. Furthermore, IGF1 treatment did not affect ERK protein expression but significantly upregulated CyclinB and AKT protein expression. All together, these findings help us to understand the molecular mechanism of IGF1 in regulation ovarian steroidogenesis and oocyte maturation of spotted steed.

Intact cell MALDI-TOF mass spectrometry, a promising proteomic profiling method in farm animal clinical and reproduction research

The objective of this review is to provide new insights into the possible use of a proteomic method known as Intact Cell Matrix-Assisted Laser Desorption-ionization Time-Of-Flight Mass Spectrometry (ICM-MS) in animal clinical research. Here, we give an overview of the basics of this technique, its advantages and disadvantages compared with other proteomic approaches, past applications and future perspectives. A special emphasis on its implementation in animal reproduction science is given, including examples of the reliable use of ICM-MS on fertility screening. In mammals, the ICM-MS profiles from pig epididymal spermatozoa reflect the proteome changes that they undergo during epididymal maturation and could be associated with the acquisition of fertilizing ability. In chicken, using adequate pre-processing and bioinformatics analysis tools, sperm ICM-MS profiles showed characteristic spectral features that allowed their classification according to their actual fertilizing ability. The association of ICM-MS and Top-down proteomic strategies allowed the identification of chicken fertility biomarkers candidates such as protein vitelline membrane outer layer protein 1 (VMO-1) and avian beta-defensin 10 (AvBD10). In female reproduction, a similar approach on ovarian follicular cells allowed the identification of specific markers of oocyte maturation in the oocyte and surrounding cumulus cells. Altogether, these results indicate that ICM-MS profiling could be a suitable approach for molecular phenotyping of male and female gametes.

Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation

Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. Biological significanceIntact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases.

TSG-6 protein and its role during maturation of ovarian follicles

TSG-6 is an ~35 kDa glycoprotein belonging to the hyaluronan binding family. Its expression is induced as a result of an inflammatory condition and during ovulation. TSG-6 is a crucial protein engaged in extracellular matrix synthesis and organization of cumulus-oophorus-complexes (COCs) in preovulatory ovarian follicles. TSG-6 catalyzes cross-linking via heavy chains of trypsin α inhibitor and hyaluronan. This reaction is essential for proper cumulus cell expansion. This process is correlated with purchasing competence by the oocyte. Disorders of the synthesis of TSG-6 cause irregularities in expansion of cumulus cells during ovarian follicle maturation. Therefore, TSG-6 is a potential molecular marker of oocyte maturation.

FTIR spectral signatures of mouse antral oocytes: Molecular markers of oocyte maturation and developmental competence

Mammalian antral oocytes with a Hoescht-positive DNA ring around the nucleolus (SN) are able to resume meiosis and to fully support the embryonic development, while oocytes with a non-surrounded nucleolus (NSN) cannot. Here, we applied FTIR microspectroscopy to characterize single SN and NSN mouse oocytes in order to try to elucidate some aspects of the mechanisms behind the different chromatin organization that impairs the full development of NSN oocyte-derived embryos. To this aim, oocytes were measured at three different stages of their maturation: just after isolation and classification as SN and NSN oocytes (time 0); after 10h of in vitro maturation, i.e. at the completion of the metaphase I (time 1); and after 20h of in vitro maturation, i.e. at the completion of the metaphase II (time 2). Significant spectral differences in the lipid (3050–2800cm−1) and protein (1700–1600cm−1) absorption regions were found between the two types of oocytes and among the different stages of maturation within the same oocyte type. Moreover, dramatic changes in nucleic acid content, concerning mainly the extent of transcription and polyadenylation, were detected in particular between 1000 and 800cm−1. The use of the multivariate principal component–linear discriminant analysis (PCA–LDA) enabled us to identify the maturation stage in which the separation between the two types of oocytes took place, finding as the most discriminating wavenumbers those associated to transcriptional activity and polyadenylation, in agreement with the visual analysis of the spectral data.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

BackgroundWithout intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis.Results8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation.ConclusionUsing sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

Expression of Maternal Transcripts During Bovine Oocyte In Vitro Maturation is Affected by Donor Age

The primary objective of this study was to compare expression of maternal transcripts in bovine oocyte populations with differential developmental competence: oocytes from prepubertal and pubertal animals; and oocytes from small (3-4 mm) and large (6-10 mm) follicles from pubertal animals. All transcripts were examined in oocytes prior to and after in vitro maturation (IVM). Genes were selected based on their known maternal effect in mouse (ZAR1, STELLA, HSF1, MATER/NLRP5 and its paralogue NLRP9), or their identification as markers of oocyte maturation, either involved in redox metabolism (PRDX1, PRDX2) or meiotic progression (AURKA). Total or polyadenylated forms of the transcripts were followed by reverse transcription coupled to real-time PCR. Six polyadenylated transcripts were found significantly reduced after maturation irrespective of donor age or follicle diameter (p<0.05). Within these six polyadenylated transcripts, ZAR1, NLRP9, HSF1, PRDX1 and PRDX2 were significantly reduced in oocytes from prepubertal animals compared to adult animals (p<0.05). A younger age was also associated with lower abundance (total form) of PRDX2/PRDX1 irrespective of maturation. Total HSF1, PRDX1 and polyadenylated NLRP9 showed a tendency (p values from 0.053 to 0.08) for a higher detection in oocytes from small follicles, thus encouraging further investigation of the follicle diameter model. However, at the present time, follicle size did not significantly affect expression of transcripts examined. In conclusion, this study demonstrates differences in the maternal store of RNA and its regulation during IVM which is dependent on donor age.

Can the vascular endothelial growth factor (VEGF) and total estradiol 17β be used as a marker of equine oocyte maturation?

The aim of this study was to quantitate vascular endothelial growth factor (VEGF) and estradiol-17\beta in follicular fluid (FF) and serum and to correlate the levels of these substances with oocyte maturation. Transvaginal ultrasound-guided follicular aspirations were performed with the purpose of oocyte collection when the follicle was >35 mm in diameter. The mares were divided into 2 groups according to age, and the changes in VEGF and estradiol-17\beta in follicular fluid and serum were examined. Levels of estradiol-17\beta in the follicular fluid and serum of young mares (4-10 years old) did not differ from those in old mares (20-24 years old) (P > 0.05). The follicular fluid and serum concentrations of VEGF in old mares were lower (P < 0.001) than those in young mares. The VEGF levels in young mares did not differ in cases when either mature or intermediate oocytes were collected, whereas its levels in old mares were higher (P < 0.01) when mature oocytes were collected than when intermediate and immature oocytes were collected. Our data suggest that VEGF may play an important role in follicular growth and development, that VEGF levels appear to be age-dependent, and that VEGF levels might be valuable biochemical markers of oocyte maturation.

263 REGULATION OF SEVERAL TRANSCRIPTS DURING IN VITRO MATURATION IN PORCINE OOCYTES COLLECTED FROM DIFFERENT SIZE FOLLICLES

Oocyte-specific transcripts play important roles in oocyte maturation, fertilization, and early embryonic development. Currently, oocytes from medium-size follicles have been used for different assisted reproductive techniques after in vitro maturation (IVM). The aim of this study was to compare the mRNA expression level in porcine oocytes collected from medium (3–6 mm) and small (&lt;2 mm) size follicles. Genes were selected based on their described maternal effect (NALP9, HSF1), their identification as markers of oocyte maturation (AURK-A, AURK-B, MOS, and C-mos), their involvement in fertilization (ZP3, ZP4), and anti-apoptotic effect (Bcl-2). All transcripts were studied in oocytes just after collection [germinal vesicle (GV) stage] and after in vitro maturation (IVM; metaphase II stage). To ensure nuclear stage of immature oocytes, oocytes were mechanically denuded just after collection, centrifuged (10 000 rpm, 5 min, RT), and observed under the microscope (60×). Those oocytes with clear nucleolus and evident nuclear membrane were selected and stored (n = 10) until study. For metaphase II oocytes, only those exhibiting the extrusion of first polar body after IVM (n = 10) were selected. Total RNA was extracted from the pool of 10 immature and mature oocytes. One picogram of luciferase mRNA per oocyte was added as an exogenous standard. Total RNA was extracted from oocytes and cDNA was obtained and used as a template for quantitative PCR to analyse the level of different transcripts. The whole process was replicated 4 times. Data were normalized to the luciferase RNA and analysed by one-way ANOVA with maturational stage (GV or metaphase II) and follicle size (small or medium) as fixed factors. Results show that all transcripts were significantly decreased during IVM (P &lt; 0.05). Therefore, after IVM, NALP9, AURK-A, MOS, C-mos, ZP3, ZP4, and Bcl-2 transcripts were significantly reduced in matured oocytes compared with immature ones irrespective of follicle diameter. Transcripts of AURKAB and HSF1 decreased after IVM in oocytes from medium follicles or small follicles, respectively. A significant effect of follicular size was only detected in MOS transcripts in GV-stage oocytes because those collected from middle follicles had a higher amount than the ones from small follicles (Table 1). These results suggest that the variations in the maternal store of RNA during IVM are not related with follicle diameter for the studied genes. Further investigations are necessary to determinate the developmental competence of oocytes that came from different types of follicles (small and medium follicles). Table 1.Variation of transcripts during in vitro maturation in porcine oocytes collected from small and medium follicles This study was supported by Okayama University. R. Romar was given a grant by JSPS (Ref. S-09210).

Production of sHLA-G molecules by in vitro matured cumulus-oocyte complex

Oocyte selection with the highest competence is a major goal in IVF. Several studies demonstrated that non-classical HLA class I HLA-G molecule modulation creates a tolerogenic microenvironment at the feto-maternal interface and is implicated in embryo implantation. This study investigated if soluble HLA-G molecules producted by the cumulus-oocyte complex (COC) are markers of oocyte maturation. sHLA-G molecule levels were analyzed using Bio-Plex assay in 152 COC supernatants obtained from 42 women and maturated by an 'in vitro maturation procedure'. The presence of sHLA-G molecules was confirmed by Western blotting technique. The results demonstrate detectable amounts of sHLA-G molecules ranging from 300 to 800 pg/ml in 14/73 (19%) COCs that generated mature oocytes and complete absence of detectable sHLA-G antigens in the supernatants of COCs that corresponded to immature oocytes. The detection of sHLA-G molecules in the COC culture supernatants corresponding to matured oocytes is proposed to be a marker to identify gametes with higher functionality. This non-invasive marker could be used, in addition to morphological approaches, to reduce the number of fertilized oocytes and transferred embryos.

Production of vascular endothelial growth factor and angiogenic factor in human follicular fluid

The aim of this study was to quantitate of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and angiogenin in follicular fluid (FF) and to correlate the levels of these substances with oocyte maturation. FF were aspirated from women undergoing in vitro fertilization and embryo transfer. Sera were collected from women with normal menstrual cycles. VEGF in FFs and sera were measured by enzyme linked immunosorbent assay. VEGF, HGF, and angiogenin mRNA expression aspirated folliculars cell was analyzed by reverse transcription and polymerase chain reaction (RT-PCR). The concentrations of VEGF, HGF, and angiogenin in FF were significantly higher than those in serum ( P<0.001). VEGF, HGF, and angiogenin mRNA in the aspirated follicles cell was detected by RT-PCR. HGF levels were higher in FF containing mature oocyte. The levels of VEGF in FF containing mature oocytes in women under 39 years of age were significantly lower than those in FF from women more than 40 years old ( P<0.01). Our data suggest that VEGF, HGF, and angiogenin may play an important role in follicular growth and development, that VEGF levels in FF appear to be age-dependent; and that VEGF and HGF levels might be valuable biochemical markers of oocyte maturation.

Synthesis of a 180 kDa protein is a molecular marker of Xenopus oocyte meiotic maturation

Xenopus oocyte maturation can be induced in vitro by a number of agents including hormones, proto-oncogenes and cyclins. Xenopus oocytes matured by a variety of treatments, including progesterone, insulin, testosterone and injection of Xenopus c- mos RNA, Xenopus cyclin B2 RNA and egg extracts, synthesize a predominant 180 kDa protein (p180). p180 synthesis begins during maturation prior to germinal vesicle breakdown (GVBD) but is prominent only in oocytes that have undergone GVBD. p180 synthesis is thus a useful, reliable molecular marker of oocyte maturation. p180 continues to be synthesized in eggs and embryos up to the gastrula stage of embryogenesis, but not in the neurula or tailbud embryo.

Study of the conversion of GDP-mannose into GDP-fucose in Nereids: a biochemical marker of oocyte maturation.

Homogenates of Perinereis cultrifera oocytes were found to transform GDP-D-mannose into another sugar nucleotide. Ultraviolet absorption spectra, chromatographic behaviour, gas-liquid chromatography coupled to mass spectrometry analysis revealed that GDP-D-mannose had been converted into GDP-L-fucose. This conversion is a multi-step reaction as proved by the involvement of two intermediates identified as GDP-4-oxo-6-deoxy-D-mannose and GDP-4-oxo-6-deoxy-L-galactose, this latter being reduced by NADPH to give GDP-L-fucose. It is shown that the enzymatic activities responsible for the conversion of GDP-D-mannose into GDP-L-fucose is recovered only in oocytes and is not present in the other coelomic cells (i.e. coelomocytes). More interesting is the fact that maximum activity is recovered at a well defined stage of the hormone-controlled oogenesis. Thus, this enzymatic system appears as a biochemical marker of the oocyte maturation in P. cultrifera.