Final Stages Of Oocyte Maturation Research Articles

P-171 Mitochondrial complex I, a key component of the mitochondrial respiratory chain, is necessary for maintaining chromosomal stability during the final stages of oocyte maturation

Abstract Study question What is the role of mitochondrial complex I during the final stages of human oocyte maturation? Summary answer Mitochondrial complex I is necessary for the cytoplasmic maturation of germinal vesicle (GV) oocytes and the subsequent organization of the meiotic spindle. What is known already Mitochondria play a pivotal role in oogenesis. Primordial human oocytes are characterized by an abundance of immature mitochondria, which exhibit low activity and a diminished capacity to produce adenosine triphosphate (ATP). During oocyte recruitment, mitochondrial fusion and fission play an essential role, contributing to oocyte maturation and oocyte developmental competence. Furthermore, an increase in ATP levels during polar body extrusion has been associated with higher fertilization rates, suggesting a reliance on mitochondria as ATP suppliers during oocyte maturation. However, the precise role of mitochondria during the final stages of human oocyte maturation is yet to be understood. Study design, size, duration At total of 99 immature oocytes, including 56 GVs and 40 metaphase I (MI) oocytes, were included in the study. These were obtained from 40 oocyte donation cycles, performed at a single IVF centre between August 2023 and September 2023. Participants/materials, setting, methods Immature denuded oocytes were cultured in G2TM PLUS medium to reach nuclear maturation (rescue in vitro maturation; rIVM). rIVM was performed in the absence (0.1% DMSO, control) or presence of 0.5 µM Rotenone (an inhibitor of mitochondrial complex I) or 10 µM FCCP (an uncoupler of mitochondrial oxidative phosphorylation). To assess mitochondrial and nuclear status, we performed immunofluorescence analyses for TOMM20 and the meiotic spindle (DNA and TUBA). Main results and the role of chance After 40 hours of culture, 64.3% of control GV oocytes progressed to the MII stage, 7.1% reached MI, while 21.4% arrested, and 7.1% degenerated. Control MI oocytes showed a 90% rIVM rate after 24 hours, with none arresting and 10% degenerating. Different mitochondrial inhibitors had varied effects on GV and MI oocytes. FCCP resulted in the degeneration of all treated oocytes. When treated with rotenone, 87.5% of GVs advanced to the MI stage, after which they arrested, but no signs of degeneration were observed. Notably, 100% of MIs and MIIs derived from GVs subjected to rotenone treatment presented with misaligned chromosomes, compared to 33.3% in the control group. When starting from MIs, the rIVM rate decreased to 41.7% following rotenone treatment, with no signs of degeneration. Here, misalignments were observed in 40% of the rIVM-MII oocytes and 43% of those that arrested, compared to 33.3% in controls. These results suggest that both GV and MI oocytes have adequate complex I activity, essential for proper nuclear maturation at the MII stage. We further highlight the importance of complex I activity, especially in GVs, for processes such as polar body extrusion during oocyte maturation. Limitations, reasons for caution Rescue in vitro maturation poses a challenge due to its limited effectiveness in achieving sufficient MII developmental competence. Consequently, mitochondrial inhibitors may have unforeseen effects on mitochondria and spindle formation. The specific role of complex I should be confirmed by employing other complex I inhibitors not related to rotenone. Wider implications of the findings Our results suggest that complex I activity, a key component of the mitochondrial respiratory chain, plays a crucial role in spindle organization during oocyte maturation. This relationship between mitochondrial function and chromosomal stability in oocytes suggests that interventions targeting the mitochondrial respiratory chain may be promising for enhancing oocyte quality. Trial registration number Not applicable

Transcriptome and Proteome Analysis Revealed Key Pathways Regulating Final Stage of Oocyte Maturation of the Turkey (Meleagris gallopavo).

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte—called the inner perivitelline layer—is involved in sperm–zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1–4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte–granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation.

Outcomes comparison of IVF/ICSI among different trigger methods for final oocyte maturation: A systematic review and meta-analysis.

During the in vitro fertilization treatment, human chorionic gonadotrophin (hCG) is routinely used as a substitute for the natural endogenous LH surge during the final stage of oocyte maturation. However, it does not provide the FSH surge observed in the mid-cycle of the natural cycle. To date, whether the FSH surge can improve oocyte quality and pregnancy outcomes remains unknown. Randomized controlled trials comparing the following four trigger methods to conventional hCG were examined: GnRH agonist (GnRHa), kisspeptin, GnRHa plus hCG (dual trigger), and FSH plus hCG (FSH co-trigger). The results showed that the use of dual triggers was associated with a significantly higher number of retrieved cumulus-oocyte complexes (COCs) (weighted mean difference [WMD] 1.625, 95% CI 0.684-2.565), retrieved mature oocytes (WMD 0.986, 95% CI 0.426-1.545) and fertilized (2PN) oocytes (WMD 0.792, 95% CI 0.083-1.501), compared with the use of hCG. However, there was no significant difference between the two groups in terms of pregnancy rate. The FSH co-trigger resulted in significantly higher rates of 2PN oocytes retrieved than the hCG trigger (WMD 0.077, 95% CI 0.028-0.126). Notably, the risk of OHSS did not differ among the three treatment groups compared to that of the hCG group. This review protocol was registered with PROSPERO (CRD 42020194201).

Gonadal recrudescence and annual reproductive hormone pattern of captive female Spotted Scats (Scatophagus argus)

The Spotted Scat (Scatophagus argus), an important euryhaline fish inhabiting mangrove and coastal regions of Indo-Pacific waters, is both an ornamental and food fish in India. Detailed insight into maturation of Spotted Scat when maintained in aquaculture systems, therefore, needs to be elucidated. Lack of information on annual maturation dynamics of female scat collected from their natural habitat and reared in earthen ponds is the basis of this study. Oocytes were classified into five developmental stages: pre-vitellogenic, vitellogenic, late-vitellogenic, ripe, and follicular atresia. Ovarian maturity stages were subsequently categorized as immature (Stage 1), vitellogenesis (Stage 2), maturing (Stage 3), mature (Stage 4), and spent (Stage 5). In oocytes in primary, secondary and tertiary yolk stages, there are greater concentrations of E2 in vitellogenic females between March and June. Significant increases of E2, T, and 17-OHP paralleled the increase of diameter of late–vitellogenic oocytes in maturing females during July. The completion of vitellogenesis and initiation of germinal vesicle migration in the cytoplasm were evident in mature females (Stage 4) with a decreasing trend of sex steroids in and subsequent to the month of August. There were 50 % of oocytes in the final oocyte maturation stage (FOM) (490–620 μm) until completion of Stage 4 in September. The results of this study indicate there is complete ovarian maturation in female scats captured in their natural habitat and maintained in an earthen pond, which may be important information for hatchery management for induction of spawning of Spotted Scat in aquaculture systems.

Reproductive physiology of bonefishes (Albula spp.) across the Northwest Bahamas.

Bonefishes (Albula spp.) are classified within the superorder Elopomorpha, which is comprised of over 1000 species that share a unique leptocephalus larval stage. Bonefishes have a circum-tropical distribution, inhabiting inshore shallow water flats and gathering in presumptive nearshore pre-spawn aggregations (PSA) during spawning months. These fishes support economically important recreational fisheries and subsistence fisheries throughout their ranges, yet little is known regarding their reproductive biology. Analysis of oocyte development and nutrient composition, and sex and gonadotrophic hormone levels, was conducted on females sampled in Grand Bahama, Central Andros, and South Andros, The Bahamas, to assess their reproductive state. Fish collected from the flats habitats along all three islands exhibited four major reproductive phases (immature, developing, spawning capable, and regressing). In contrast, all females captured at presumptive PSA sites had eggs in the final stage of oocyte maturation, significantly higher levels of all reproductive hormones (17β-estradiol, testosterone, and LH), larger vitellogenic oocytes, and oocytes exhibiting germinal vesicle migration and germinal vesicle breakdown. In addition, monthly variability in hormone levels and spawning readiness between Grand Bahama and Andros PSAs suggest that peak spawning times may differ among regions. Fatty acid and free amino acid composition and profiles, with high proportions of docosahexaenoic acid, histidine, and taurine, suggest that these nutrients are not only relevant as energy reserves, but also help achieve buoyancy and osmoregulation of oocytes. This study expands upon our understanding of fish reproductive and developmental physiology, and indicates potential factors influencing the survival and recruitment of bonefishes.

Effects of follicular fluid of preovulatory follicles of repeat breeder dairy cows with subclinical endometritis on oocyte developmental competence

The aims of the present study were to determine the concentrations of lipopolysaccharide (LPS), hormonal progesterone, estradiol-17β, insulin growth factor (IGF-1) and magnesium in the serum and the preovulatory follicle follicular fluid (FF) in repeat breeder (RB) cows without (nSCE) or with subclinical endometritis (SCE), and further to examine the effects of this FF on developmental competence of cattle oocytes. In Experiment 1, 13 of 23 clinically healthy Holstein RB cows were identified (uterine PMNs) to have SCE. The cows were estrous synchronized, and 6–12 h after detection of standing estrus, FF and blood of the preovulatory follicles were collected. The mean (±SD) LPS (862.3 ± 148.1 compared with 1063.4 ± 262.8 EU/ml, P = 0.04) and estradiol-17β (188.9 ± 15.8 compared with 162.0 ± 31.5 ng/ml, P = 0.02) concentrations of FF was different between nSCE and SCE cows. In Experiment 2, FF of RB cows with relatively lesser (nSCE, n = 4) and greater (SCE, n = 4) percentages of uterine PMNs was separately added to the oocyte maturation medium for in vitro embryo production. Addition of FF from SCE cows to the oocyte maturation medium resulted in a lesser rate of development to the blastocyst stage than that of the nSCE cows (21.9 ± 1.8 compared with 27.8 ± 2.5%, P < 0.05). Results of the present study indicate greater FF LPS concentration may result in a lesser quality microenvironment milieu for the final stages of oocyte maturation in RB dairy cows with subclinical endometritis. In addition, supplementation of oocyte maturation medium with FF of preovulatory follicles from RB cows with subclinical endometritis resulted in a lesser potential of in vitro oocyte developmental competence.

Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation.

SummaryThe establishment of DNA methylation patterns in oocytes is a highly dynamic process marking gene-regulatory events during fertilization, embryonic development, and adulthood. However, after epigenetic reprogramming in primordial germ cells, how and when DNA methylation is re-established in developing human oocytes remains to be characterized. Here, using single-cell whole-genome bisulfite sequencing, we describe DNA methylation patterns in three different maturation stages of human oocytes. We found that while broad-scale patterns of CpG methylation have been largely established by the immature germinal vesicle stage, localized changes continue into later development. Non-CpG methylation, on the other hand, undergoes a large-scale, generalized remodeling through the final stage of maturation, with the net overall result being the accumulation of methylation as oocytes mature. The role of the genome-wide, non-CpG methylation remodeling in the final stage of oocyte maturation deserves further investigation.

Ultrasonography to assist with timing of spawning in European eel

Ovulation in the European eel can be induced by injection of DHP (17α,20β-dihydroxy-4-pregnen-3-one). The timing of injection is based on the developmental stage of the oocytes. The oocyte stage is determined by invasive biopsy or through external indicators of the oocyte hydration response: body weight index (BWI) and body girth index (BGI). However, in European eel, BWI and BGI are inaccurate indicators because the individual hydration response is highly variable. The aim of this study was to identify indicators of oocyte maturation non-invasively by applying ultrasonography. Ultrasound parameters that were determined were the body wall thickness; diameter of the cloaca opening; grey scale median (GSM) of the ovary, and diameters (vertical and horizontal) and surface area of ovary, liver and swim bladder. Data of farmed and wild eels were combined for correlation analyses and principal component analyses (PCA). Ovary and liver parameters were not correlated with the average oocyte stage. Body wall thickness was negatively correlated to the average oocyte stage but variability was high. The swim bladder vertical diameter (Sbv) was negatively correlated with the average oocyte stage and with the oocyte diameter. Swim bladder area (Sba) was also negatively correlated to the average oocyte stage but less accurate. Results showed that during the final stages of oocyte maturation, the swim bladder becomes smaller. This is most probably due to the pressure that is caused by the increasing ovaries as a result of the oocyte hydration response. Females with an average oocyte stage between 4.5 and 7.0 and an average oocyte diameter higher than 800 μm, had Sbv values decreasing from 4.3 to 2.4 mm and Sba values from 52 down to 11 mm2. The correlation between Sbv and oocyte stage is more strict than for BWI or BGI which makes Sbv not only a parameter that can be determined non-invasively but also one that is more accurate in indicating the oocyte developmental stage. Therefore, ultrasonography, and in particular the ultrasound imaging of the swim bladder, represents a useful tool to assist with the timing of spawning as induced by injection of DHP.

Corticosteroids deeply depress the in vitro steroidogenic capacity of Eurasian perch ovary at the end of the reproductive cycle

Corticosteroids play positive or negative role in the reproductive mechanisms of many fish species but the physiological contexts relating to such biphasic actions are not well defined. In the present study we investigated to what extent corticosteroids (cortisol-Co, 11-deoxycorticosterone-DOC) hormones may interfere with the steroidogenic capacity of Eurasian perch ovarian tissues, and we tested whether the negative effects of corticosteroids may be mitigated by potential stimulating endocrine factors, namely insulin-like growth factor-1 (IGF), human chorionic gonadotropin (HCG) or thyroid hormones (Triidothyronine-T3, thyroxine-T4). Ovarian tissues from six maturing fish at late vitellogenesis developmental stage (LVO) or at the start of the final meiotic oocyte maturation (FMO) were incubated during 6h in Cortland medium containing various endocrine compounds. Both corticosteroids drastically suppressed aromatase activity (AA) and sex-steroid production, namely 17-β estradiol (E2), 17α-20β-dihydroxy-4-pregnen-3-one (DHP) and testosterone (T). HCG significantly prevented the suppression of both AA and sex-steroid production by low and high cortisol doses, but a lesser AA protection was observed in the case of DOC. The protection of DHP and T productions by HCG from the negative effects by the two corticosteroids was higher at FMO than at LVO stage. IGF or thyroid hormone treatments were lesser effective or ineffective in mitigating the suppression of AA or sex-steroid production by cortisol. The results suggest that an increase in cortisol or DOC such as after mild or high stress intensity may inhibit drastically the ovarian steroidogenic capacity whatever the final oocyte maturation stage in percid fish by hampering AA and sex-steroid production. That inhibition may be partly mitigated by gonadotropins but not IGF nor thyroid hormones, especially at final meiotic oocyte maturation stage.

The effect of four C21-steroids on oocyte maturation in Siberian sturgeon (Acipenser baeri Brandt)

The effect of four C21-steroids, progesterone (P4), 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), 17,20β,21-trihydroxy-4-pregnen-3-one (20βS) and 11-desoxycortisol (S), on in vitro oocyte maturation in Siberian sturgeon (Acipenser baeri Brandt) females was demonstrated using short-term (5 and 30 min) exposures of ovarian follicles to steroid solutions followed by incubation in steroid-free medium. The study aimed to find out which of the four candidates for a maturationinducing steroid (P4, 17,20βP, 20βS or S) induces a fastest germinal vesicle breakdown (GVBD) in oocytes of Siberian sturgeon. Dissolution of the oocyte nucleus or GVBD was taken as a criterion of oocyte maturation. Dose-response profiles of hormone activities as well as effects of the hormones under short-term exposures of follicles to their equal doses were compared. P4 was found to be a most active GVBD inducer compared to other C21-steroids, S was the second in its activity, whereas 17,20βP and 20βS were less efficient. A comparison of the present and previously obtained data on the dynamics of C21-steroids in vivo and their effect on ovarian follicles in vitro indicates an important role of the above hormones, particularly P4 and 20βS, in the regulation of the final stage of oocyte maturation in sturgeons.

Utilization of laserpuncture induction as spawning stimulation in catfish (Clarias spp.) crossbreeding toward egg quality

The induction of laserpuncture on the reproductive acupoint of catfish can accelerate gonadotropin hormone formation from the pituitary especially gonadotropin II (GTH-II) which has a role in the final stage of oocyte maturation, ovulation and spawning stimulation. The purpose of this study is to evaluate the effects of laserpuncture induction toward the egg quality from crossbreeding catfish male var. Paiton and female var. Sangkuriang. The egg quality was measured by the following parameters: fertilization rate (FR), egg hatching rate (HR), and larvae survival rate (SR). The research treatments were conducted using two levels along with eight repetitions. The results show that the crossbreed catfish using laserpuncture induction affected the parameters by increasing the mean value of fertilization rate, egg hatching rate and larvae survival rate significantly (P<0.05) compared with mean value of fish without induction. This study concluded that laserpuncture induction on the crossbreeding between broodstock of male catfish var. Paiton and female var. Sangkuriang will increase FR, HR and SR.

Oocyte secretion is regulated by somatic cells during oocyte maturation and mediates cumulus cell function

De novo mRNA synthesis ceases during the final stages of oocyte maturation and previously synthesized mRNAs are translated according to a well-orchestrated program of recruitment to the polysomes. The aim of this study is to investigate the regulation of oocyte mRNA translation and protein secretion during maturation, using interleukin (IL)-7 as the prototypic secreted factor, and to determine the role of this oocyte secreted factor in cumulus cell (CC) function.

MicroRNA-34 family expression in bovine gametes and preimplantation embryos.

BackgroundOocyte fertilization and successful embryo implantation are key events marking the onset of pregnancy. In sexually reproducing organisms, embryogenesis begins with the fusion of two haploid gametes, each of which has undergone progressive stages of maturation. In the final stages of oocyte maturation, minimal transcriptional activity is present and regulation of gene expression occurs primarily at the post-transcriptional level. MicroRNAs (miRNA) are potent effectors of post-transcriptional gene silencing and recent evidence demonstrates that the miR-34 family of miRNA are involved in both spermatogenesis and early events of embryogenesis.MethodsThe profile of miR-34 miRNAs has not been characterized in gametes or embryos of Bos taurus. We therefore used quantitative reverse transcription PCR (qRT-PCR) to examine this family of miRNAs: miR-34a, -34b and -34c as well as their precursors in bovine gametes and in vitro produced embryos. Oocytes were aspirated from antral follicles of bovine ovaries, and sperm cells were isolated from semen samples of 10 bulls with unknown fertility status. Immature and in vitro matured oocytes, as well as cleaved embryos, were collected in pools. Gametes, embryos and ovarian and testis tissues were purified for RNA.ResultsAll members of the miR-34 family are present in bovine spermatozoa, while only miR-34a and -34c are present in oocytes and cleaved (2-cell) embryos. Mir-34c demonstrates variation among different bulls and is consistently expressed throughout oocyte maturation and in the embryo. The primary transcript of the miR-34b/c bicistron is abundant in the testes and present in ovarian tissue but undetectable in oocytes and in mature spermatozoa.ConclusionsThe combination of these findings suggest that miR-34 miRNAs may be required in developing bovine gametes of both sexes, as well as in embryos, and that primary miR-34b/c processing takes place before the completion of gametogenesis. Individual variation in sperm miR-34 family abundance may offer potential as a biomarker of male bovine fertility.

Special research presentation: bmpr signaling in the mammalian oocyte improves fertilization and influences early embryo development

Much data supports the concept that there is little to no detectable new transcription during the final stages of oocyte maturation and the initial stages of early embryo development. Therefore most of the critical events at the time of fertilization and very early embryo development rely on an orchestrated program of translation of already existing mRNAs. BMPR (Bone morphogenetic protein receptor) mRNA is increased in polysome binding during oocyte maturation. Thus, BMP signaling may be important during oocyte maturation and early embryo development, since these events are dependent on the oocyte translational program of pre-formed maternal mRNAs.

Participation of PI3-kinase/Akt signalling in insulin stimulation of p34cdc2 activation in zebrafish oocyte: Phosphodiesterase 3 as a potential downstream target

Exposure of fully grown oocytes to growth factors (insulin/IGFs) initiates various signalling cascades that culminate to final stages of oocyte maturation. Regulation of signalling pathways during growth factor-induced meiosis resumption in fish is not well characterized. Here we studied the participation of PI3K/Akt signalling pathway during recombinant human insulin (rh-insulin)-induced meiotic maturation in zebrafish (Danio rerio) oocytes. Priming of defolliculated oocytes in vitro with rh-insulin promotes germinal vesicle breakdown (GVBD) in a dose- and time-dependent manner, an effect sensitive to translation but not transcription inhibition. More than 80% of the oocytes underwent GVBD due to 0.8IU/ml rh-insulin within 10h of incubation and the kinetics of p34cdc2 kinase activation corresponded well with GVBD data. PI3K inhibitors, wortmannin and LY294002 blocked insulin, but not 17α, 20β-DHP-induced GVBD. Immunoblot analyses of oocyte extract revealed that phospho-PI3K (p85α) was up regulated within 30–60min of insulin stimulation followed by phospho-Akt (Ser473) at 60–120min. Though PI3K/Akt phosphorylation was largely unaffected, pre-incubation with phosphodiesterase (PDE) inhibitors, IBMX and cilostamide, but not rolipram completely blocked rh-insulin-induced p34cdc2 activation and GVBD. These results suggest that PDE3 may be one potential downstream target to PI3K/Akt signalling necessary for rh-insulin-induced GVBD in zebrafish.

Luteal-phase Ovarian Stimulation Case Report: Three-Year Follow-up of a Twin Birth

This unique study shows favorable outcomes to embryos developed from eggs retrieved following luteal phase stimulation and the children born from these eggs. A 40-year-old woman with a 10-year history of primary infertility was given hMG 150IU and letrozole 2.5 mg from cycle day 3 onwards. Her cycle transitioned into the luteal phase beginning on cycle day 10 as indicated by high serum progesterone levels. Follicle growth continued with hMG stimulation. GnRH agonist was given for the final stage of oocyte maturation. During collection, four mature oocytes were obtained and two top-quality embryos were vitrified for cryopreservation. Two months later, the two embryos were thawed and transferred during a natural cycle, creating a twin pregnancy and a favorable delivery. Three-year follow up showed that physical and psychomotor development of the twin babies were in the normal range of children conceived naturally. This case report documents that embryos developed following luteal-phase ovarian stimulation reached viability, and child development was found to be normal up to three years following birth. This study opens the door to the possibility of successful outcomes from luteal phase stimulation. This has a great potential for patients who did not respond well during the follicular phase, but most importantly, for newly diagnosed cancer patients who need immediate fertility preservation, i.e., to quickly produce viable oocytes.

Disruption of fibroblast growth factor receptor signaling in bovine cumulus-oocyte complexes during in vitro maturation reduces subsequent embryonic development

Several fibroblast growth factors (FGF) mediate folliculogenesis and oogenesis in cattle but it is unclear whether FGFs are required during the final stages of oocyte maturation. The objectives of this work were to determine whether blocking FGF receptor (FGFR) activity during in vitro maturation (IVM) affects oocyte fertilization and embryo development; examine changes in FGFR transcript profiles in cumulus cells and oocytes during IVM; and evaluate whether gonadotropins modulate FGFR transcript abundance during IVM. In the first set of studies, bovine cumulus-oocyte complexes (COCs) were matured in the presence of one of two FGFR kinase inhibitors (SU5402 or PD173074). After maturation, COCs were washed and cultured without inhibitors. Inhibitors did not affect cleavage rates but the percentage of ≥ 8-cell embryos at d 3 and blastocysts at d 7 and d 8 postfertilization were decreased when COCs were matured with either inhibitor. Profiles of FGFR mRNA variants were examined in cumulus cells and oocytes separated either immediately before (0 h) or at 6 or 21 h after beginning IVM. In cumulus cells, increases in R1b, R2b, and R2c abundance were detected when cultured in the absence of follicle-stimulating hormone (FSH). Supplementing FSH (1 or 25 μM) increased the abundance of R1b, R1c, R2b, and R2c. In oocytes, no time- or FSH-dependent changes in FGFR transcript abundance were detected. These observations implicate FGFs as crucial components of bovine oocyte competency and indicate that FSH augments FGFR mRNA abundance in cumulus cells during the final stages of oocyte maturation.

Recombinant or Urinary Human Chorionic Gonadotropin in Ovulation Induction?

Recombinant or Urinary Human Chorionic Gonadotropin in Ovulation Induction?

Disruption of Fibroblast Growth Factor Receptor Signaling in Bovine Cumulus-Oocyte Complexes During In Vitro Maturation Impacts Subsequent Embryonic Development.

Several fibroblast growth factors (FGF) mediate folliculogenesis and oogenesis in cattle but it is unclear whether FGFs are required during the final stages of oocyte maturation. The objectives of this work were to determine whether blocking FGF receptor (FGFR) activity during in vitro maturation (IVM) affects oocyte fertilization and embryo development; examine changes in FGFR transcript profiles in cumulus cells and oocytes during IVM; and evaluate whether gonadotropins modulate FGFR transcript abundance during IVM. In the first set of studies, bovine cumulus-oocyte complexes (COCs) were matured in the presence of one of two FGFR kinase inhibitors (SU5402 or PD173074). After maturation, COCs were washed and cultured without inhibitors. Inhibitors did not affect cleavage rates but the percentage of 8-cell embryos at d 3 and blastocysts at d 7 and d 8 postfertilization were decreased when COCs were matured with either inhibitor. Profiles of FGFR mRNA variants were examined in cumulus cells and oocytes separated either immediately before (0 h) or at 6 or 21 h after beginning IVM. In cumulus cells, increases in R1b, R2b, and R2c abundance were detected when cultured in the absence of follicle-stimulating hormone (FSH). Supplementing FSH (1 or 25 M) increased the abundance of R1b, R1c, R2b, and R2c. In oocytes, no timeor SH-dependent changes in FGFR transcript abundance were detected. These observations implicate FGFs as crucial components f bovine oocyte competency and indicate that FSH augments FGFR mRNA abundance in cumulus cells during the final stages f oocyte maturation. 2012 Elsevier Inc. All rights reserved.

276 FIBROBLAST GROWTH FACTOR 2 PROMOTES BOVINE OOCYTE MEIOTIC MATURATION AND DEVELOPMENTAL COMPETENCE

Oocyte competency is acquired during the course of folliculogenesis and is controlled by various endocrine and paracrine signals. One of these is fibroblast growth factor 2 (FGF2). Its expression is up-regulated in theca and granulosa cells during final maturation of a bovine follicle, and its cognate receptors are expressed in cumulus cells and oocytes throughout the final stages of oocyte maturation. The overall goal of this work was to describe how supplementing FGF2 during oocyte maturation in vitro affects oocyte maturation and subsequent embryo development. Cumulus–oocyte complexes (COC) were collected from bovine ovaries obtained from a local abattoir and cultured in defined TCM-based oocyte maturation medium. Depending on the study, oocytes were examined either during (6 h) or after (21 h) maturation or were fertilized in vitro and examined throughout in vitro embryo development in modified SOFF. Data were analysed with least-squares ANOVA using GLM of SAS. Adding 0.5 to 50 ng mL–1 of FGF2 did not affect cleavage rate or the percentage of 8 to 16 cell embryos at day 3 post-IVF. However, the blastocyst rate at day 7 was greater when oocytes were exposed to 0.5 ng mL–1 of FGF2 during maturation [30.0 ± 1.9% (17/109) v. 16.0 ± 2.6% (23/77) for nontreatment control; 4 replicates; P &lt; 0.05], whereas higher doses of FGF2 did not affect blastocyst rates when compared with controls. Total cell number per blastocyst was not affected by FGF2 addition. The effects of FGF2 on oocyte maturation and cumulus expansion were examined to better understand how FGF2 improves oocyte competency. Adding 0.5 ng mL–1 of FGF2 did not affect the percentage of oocytes containing condensed chromatin after 6 h IVM or metaphase II (MII) rate after 21 h IVM, but 0.5 ng mL–1 of FGF2 treatment increased the cumulus expansion index score after 21 h IVM (P &lt; 0.05). Interestingly, adding 5 ng mL–1 but not 50 ng mL–1 of FGF2 increased MII rate [61.5 ± 4.3% (53/120) for 5 ng mL–1 of FGF2 v. 46.9 ± 5.9% (64/104) for nontreatment controls; 7 replicates; P &lt; 0.05], but neither FGF2 affected rates of chromatin condensation and cumulus expansion. Changes in the relative abundance for several putative oocyte competency markers and maternal genes (CTSB, Sprouty2, EGFR, FSHR, Has2, BMP15, GDF9, JY-1, Follistatin, H2A) were examined at 6 and 21 h after treatment with 0.5 ng mL–1 of FGF2 by quantitative RT-PCR. Relative amounts of 18S RNA was used as an internal control, and 2-ΔΔCT was used to quantify relative gene expression. The relative abundance of most of the transcripts examined was not affected by FGF2, but EGFR mRNA levels were greater after 6 h but not 21 h IVM in cumulus cells isolated from FGF2-supplemented COC (P = 0.057). In summary, improvements in blastocyst development were achieved by FGF2 treatment during oocyte maturation. The reason for the enhanced oocyte competency remains unclear, but it may occur in part because of improvements in cumulus expansion and production of EGFR. This project was supported by NRICGP number 2008-35203-19106 from the USDA-NIFA.