Denuded Oocytes Research Articles

Effects of intracytoplasmic sperm injection timing and fertilization methods on the development of bovine spindle transferred embryos

Cytoplasmic replacement by spindle transfer (ST) technique can be applied to improve the developmental competence of poor qualitied or aged oocytes. In cattle, ST technology has not been well established for producing embryos and the calves successfully using intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). The objective of this study was to develop a novel procedure for producing bovine ST embryos, which could be fundamental to applying ST technology in other mammals. In the present study, the efficacies of performing ICSI before (ICSI-ST) or after (ST-ICSI) and IVF on the development of ST bovine embryos were investigated. Results indicated that the blastocyst rate of ST embryos produced by ICSI-ST (24.7%) was higher (P < 0.05) than that produced by ST-ICSI (5.9%). On the other hand, ST-IVF had the highest fertilization rate (97.3%), polyspermy rate (24.7%), and lowest blastocyst rate (22.7%) when compared to denuded oocytes (DO), zona cut oocytes (ZC), and cumulus-oocyte complexes (COCs)-IVF groups. Finally, the in vitro development rates of ICSI-ST (24.5%) and ST-IVF (25.2%) were not significantly different (P > 0.05). However, the pregnancy rate (46.7%) and birth rate (33.3%) of ST-IVF were higher (P < 0.05) than those of ICSI-ST (6.3% and 0%, respectively). The percentage of mitochondrial DNA (mtDNA) heteroplasmy derived from donor karyoplasts of the 5 claves was between 2% and 18%. Taken together, performing ICSI prior to ST can improve the embryonic development of ST bovine embryos. Moreover, using IVF, instead of ICSI, for ST oocyte fertilization dramatically increased the pregnancy rate and birth rate of ST calves, in which mtDNA heteroplasmy derived from donor karyoplasts exists.

P–435 LH preserves oocyte-granulosa cell communication in mouse ovaries exposed to chemotherapy with alkylating agents

Abstract Study question Does Luteinizing Hormone (LH) protect the follicular endowment and growth by improving oocyte-granulosa cell (GC) communication of follicles exposed to chemotherapy at the primordial stage? Summary answer LH treatment protects mouse primordial follicles against alkylating agents by preventing the chemotherapy-induced follicular depletion and the impairment of oocyte-GC communication during follicular growth. What is known already Impaired folliculogenesis is one of the most common deleterious side effects of alkylating agents in ovaries. Bidirectional communication between the oocyte and surrounding GCs is crucial for oocyte development. Therefore, defective gap junctions and reduced oocyte-derived factors compromise folliculogenesis, oocyte competence and meiotic maturation. Previous findings reported a significant LH protection of follicular viability and meiotic potential of MII oocytes exposed to chemotherapy at primordial stage. Therefore, we aimed to investigate the LH effects on cell junctions and communication between oocyte and GCs in growing follicles derived from quiescent oocytes exposed to alkylating chemotherapy. Study design, size, duration Adult 6-week-old CD1 female mice were allocated to control (n = 3), chemotherapy (ChT, n = 5) and ChT+LH (n = 5) groups. Chemotherapy (120 mg/Kg of cyclophosphamide and 12 mg/Kg of busulfan) was intraperitoneally administrated to ChT and ChT+LH mice. ChT+LH animals were pretreated with 1 IU of LH, followed by a second LH dose (1 IU) along with chemotherapy 24 hours later. Control mice only received vehicle (DMSO). Mice were euthanized 30 days later to collect ovaries. Participants/materials, setting, methods Follicles were mechanically isolated by puncture with 30-gauge needles from frozen-thawed half-ovaries. Isolated follicles measuring ≥100µm were selected to represent the secondary and later developmental stages. Part of them were kept intact while others were decumulated by using narrow pipettes to obtain denuded oocytes (DOs), and GCs. Follicles, ODs and GCs were analyzed by qRT-PCR to evaluate key factors in oocyte-GC junctions (Cx37, Cx43, Cdh1, Cdh2, Tjp1) and communication (Gdf9, Bmp15, Bmpr2, Alk4, Alk5, Alk6). Main results and the role of chance Chemotherapy induced a 2.1-fold reduction in the number of total isolated follicles (p = 0.036), reducing 2.7-fold the number of primordial and primary follicles (&amp;lt;100 µm; p = 0.034) and 1.9-fold the amount of growing follicles (≥100 µm; p = 0.036) compared to controls. LH-treated ovaries showed a 1.6-fold increase in the total follicle isolation yield when compared to ChT (p = 0.032), recovering control-like values (p=ns). This LH protection specially benefited the early-stage follicles (&amp;lt;100 µm), where a 1.9-fold increase in the number of isolated follicles was detected compared to ChT group (p = 0.016). Gene expression analysis of follicles (n = 168), DOs (n = 110) and GCs (from n = 153 follicles) revealed a global downregulation pattern in ChT samples for all genes, when compared to controls, with a significant fold change (FC) reduction for Gdf9 in follicles (FC: 0.36±0.16); Cx37, Cdh2 and Gdf9 in DOs (FC: 0.23±0.17, 0.09±0.03, and 0.17±0.07, respectively); and Cx37, Cx43, Gdf9 and Bmp15 in GCs (FC: 0.40±0.23, 0.17±0.07, 0.17±0.08, and 0.04±0.01, respectively). However, LH treated samples showed an overall improvement of gene expression pattern reaching control-like levels for all genes excepting for a downregulation of the Bmp15 expression in GC (FC: 0.28±0.24; p = 0.036). Limitations, reasons for caution Animal model study performed with a reduced sample size. Therefore, these findings should be validated in further studies with human tissue samples. Wider implications of the findings: Our findings suggest that LH treatment prevents the chemotherapy-induced follicle depletion. The LH protection of primordial population seems to preserve its ability to properly establish oocyte-GC interactions during growth and development, which is required to regulate follicular maturation and oocyte competence. Trial registration number Not applicable

Delaying Effects of Prolactin and Growth Hormone on Aging Processes in Bovine Oocytes Matured In Vitro.

Aging processes accelerate dramatically in oocytes that have reached the metaphase-II (M-II) stage. The present work aimed to study the patterns and intracellular pathways of actions of prolactin (PRL) and growth hormone (GH) on age-associated changes in bovine M-II oocytes aging in vitro. To this end, we analyzed spontaneous parthenogenetic activation (cytogenetic assay), apoptosis (TUNEL assay), and the developmental capacity (IVF/IVC) of in vitro-matured oocytes after prolonged culturing. Both PRL and GH reduced the activation rate of aging cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs), and their respective hormone receptors were revealed in the ova. The inhibitor of Src-family tyrosine kinases PP2 eliminated the effects of PRL and GH on meiotic arrest in DOs, whereas the MEK inhibitor U0126 only abolished the PRL effect. Furthermore, PRL was able to maintain the apoptosis resistance and developmental competence of aging CEOs. The protein kinase C inhibitor calphostin C suppressed both the actions of PRL. Thus, PRL and GH can directly support meiotic arrest in aging M-II oocytes by activating MAP kinases and/or Src-family kinases. The effect of PRL in maintaining the developmental capacity of aging oocytes is cumulus-dependent and related to the pro-survival action of the protein kinase C-mediated signal pathway.

Maturation conditions, post-ovulatory age, medium pH, and ER stress affect [Ca2+]i oscillation patterns in mouse oocytes.

Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca2+ concentration ([Ca2+]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca2+]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca2+]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca2+]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca2+]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca2+]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca2+]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca2+]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.

Reestablishment of transzonal projections and growth of bovine oocytes in vitro.

Transzonal projections (TZPs) that maintain bidirectional communication between oocytes and granulosa cells or cumulus cells are important structures for oocyte growth. However, whether TZPs develop between TZP-free oocytes and granulosa cells, and whether reestablished TZPs support oocyte growth, is unknown. We first examined changes in TZPs after denudation of bovine oocytes collected from early antral follicles (0.5–0.7 mm). Twenty-four hours after denudation, almost all the TZPs disappeared. We also examined the reestablishment of TZPs by coculturing TZP-free denuded oocytes (DOs) with mural granulosa cells (MGCs) collected from early antral follicles. In addition, to confirm if the reestablished TZPs were functional, the reconstructed complexes (DO+MGCs) were subjected to in vitro growth culture and found that the MGCs adhered to TZP-free DOs and TZPs were reestablished. During in vitro growth culture, DO+MGCs developed and formed antrum-like structures. After culture, the number of TZPs in DO+MGCs increased, and the oocytes grew fully and acquired meiotic competence. These results suggest that reestablished TZPs are able to support oocyte growth.

30 The importance of cumulus cells for the survival and timing of meiotic resumption of porcine oocytes vitrified at the immature stage

Previous research revealed that vitrification at the immature (the germinal vesicle, GV) stage triggers premature meiotic resumption in cumulus-enclosed porcine oocytes and causes a damage in gap junctions (Appeltant et al. 2017 Reprod. Fertil. Dev. 29, 2419-2429). However, the correlation between the two phenomena was not investigated yet. The present research was conducted to clarify whether premature meiotic resumption is caused by gap junction disruption and to assess the importance of cumulus cells for the survival of porcine oocytes vitrified at the GV stage. Cumulus–oocyte complexes (COCs) were collected from 3- to 6-mm antral follicles of slaughtered gilts. Immediately after collection, approximately half of them were denuded mechanically (DOs). In each replicate, groups of COCs and DOs were processed without vitrification (control groups). Treatment groups of COCs and DOs were vitrified on Cryotop sheets in a combination of 17.5% propylene glycol and 17.5% ethylene glycol and warmed in 0.4M sucrose. The oocytes were then cultured for 22h in a chemically defined porcine oocyte medium (POM) supplemented with 10ngmL−1 epidermal growth factor, 10IUmL−1 equine chorionic gonadotrophin, 10IUmL−1 human chorionic gonadotrophin, and 1mM dibutyryl cAMP. After culture, COCs were denuded and oocyte survival was assessed by morphological evaluation of membrane integrity under a stereo microscope. Then, live oocytes were fixed and stained with 1% orcein and nuclear status was evaluated under a phase-contrast microscope. The experiment was replicated 5 times. Data were analysed by ANOVA followed by Tukey’s multiple comparisons test. After vitrification and culture, the survival rate in the COC group was higher (P&amp;lt;0.05) than that of the DO group (160/191=84.7±3.4% vs. 153/237=65.0±6.2%, respectively) but reduced (P&amp;lt;0.05) compared with those in the control COC and DO groups (138/143=96.6±1.0% and 152/153=99.3±0.6%, respectively). The majority of the control COCs and DOs were at the GV stage with similar percentages (95.6±2.2% and 94.0±2.2%, respectively). In contrast, the percentages of oocytes at the GV stage in the vitrified COC and DO groups were reduced (71.6±9.4% and 45.7±10.5%, respectively; P&amp;lt;0.05) compared with the control groups, which were associated with increased frequencies of diakinesis and MI stages. Percentages of oocytes at the GV stage in the vitrified COC and DO groups were not significantly different (P=0.23). In conclusion, cumulus cells can prevent vitrification-related membrane damage of oocytes. Furthermore, vitrification induced premature meiosis both in the cumulus-enclosed and denuded oocytes even in the presence of the meiotic inhibitor, dibutyryl cAMP. Nevertheless, cumulus removal without vitrification did not induce premature meiosis in the oocytes. Therefore, disruption in communication with cumulus cells might not be the primary reason for premature meiosis in vitrified oocytes.

FGF2/FGFR signaling promotes cumulus-oocyte complex maturation in vitro.

Fibroblast growth factor 2 (FGF2), a member of FGF family, binds with FGF receptors (FGFR) to initiate biological functions in various somatic cells. However, little is known regarding the role of FGF2/FGFR on oocyte meiosis. In this study, we investigated expression patterns and functions of FGF2/FGFR during in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs). Among four FGFRs, Ffgr1 was the most abundant in COCs. The transcripts for Fgf2 and Ffgr1 in COCs increased during IVM. Ffgr1 was present in oocytes and cumulus cells, while Fgf2 was present in only cumulus cells. Treatment of COCs with the selective FGFR inhibitor SU5402 blocked oocyte meiotic progression and downregulated expression of Bmp15 and Gdf9. In contrast, supplement of FGF2 promoted oocyte meiotic progression and upregulated Bmp15 and Gdf9 expression. Inhibition of FGFR with SU5402 reduced cumulus expansion and expressions of Ptx3, Has2 and Tnfaip6. Treatment with FGF2 increased Ptx3 and Has2 expression. Inhibition of FGFR had no effect on meiotic progression of denuded oocytes (DOs). However, co-culture of DOs with COCs or supplementation with FGF2 promoted meiotic progression of DOs. Inhibition of FGF2/FGFR signaling also downregulated Ffgr1 expression, while supplemental FGF2 upregulated Fgfr1 expression. Furthermore, inhibition of FGFR in COCs interrupted the c-Mos/MAPK pathway and maturation-promoting factor (MPF), as indicated by downregulation of oocyte c-mos and Ccnb1 transcripts, respectively. Overall, this study suggests that FGF2 produced by cumulus cells, activates a FGF2/FGFR autocrine/paracrine loop within COCs to regulate cumulus expansion and oocyte meiosis. These findings reveal a novel role for FGF2/FGFR signaling during in vitro maturation of COCs.

OOCYTE SECRETED FACTORS REGULATE FSHR AND AMH mRNA LEVELS IN CUMULUS CELLS

Considering that (1) FSH activity decreases cumulus-oocyte communication via retraction of transzonal processes (TZP), (2) AMH inhibits FSH signalling by decreasing FSHR expression and adenylyl cyclase activation, and (3) the oocyte is an active player in the cumulus-oocyte complex (COC) regulating cumulus cell metabolism and gene expression via oocyte-secreted factors (OSF), we tested the hypothesis that the oocyte decreases mRNA abundance of FSHR while increasing that of AMH in cumulus cells through OSF, as part of an effort to preserve its communication with cumulus cells thus safeguarding its developmental competence. The relative mRNA abundance of FSHR and AMH in cumulus cell was compared among 3 treatment-groups: (1) Intact COC subjected to in vitro maturation (IVM); (2) oocytectomized COC (OOX) subjected to IVM and (3) OOX subjected to IVM with the addition of denuded oocytes (OOX+DO; 1 DO/μL). Four culture replicates including all three treatment-groups were performed. COC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by aspiration of the ooplasm with a micromanipulator while preserving the structure of the COC. COC and OOX underwent IVM in pools of 20 for 22 hours. IVM was performed in 100 μL of TCM199 with Earl’s salts supplemented with 1μg/mL FSH, 0.4% BSA, 22 μg/mL sodium pyruvate, 75 μg/mL of amikacin at 38,5°C and 5.5% CO2 in humid atmosphere. Abundance of mRNA encoding FSHR and AMH in cumulus cells was assessed by real-time RT-PCR using Power SybrGreen (LifeTech®) and RPL15 (ribosomal protein 15) as the reference gene. Effects of treatments were tested by ANOVA and groups were compared with the Tukey test. Statistically significant differences were those with p<0.05, while p values >0.05 and <0.1 were considered as tendency of difference. Oocyctectomy decreased AMH mRNA levels (p=0.005) while tended to increase FSHR mRNA abundance (p=0.077) in cumulus cells. The addition of denuded oocytes (DO) reversed the effects of oocytectomy on both genes. Relative AMH mRNA levels (AMH/RPL15 mRNA abundance, mean ± EPM) were 0.875 ± 0.1, 0.3675 ± 0.02 and 0.7825 ± 0.1 for COC, OOX and OOX+DO, respectively. For FSHR, the respective values (FSHR/RPL15 mRNA abundance) were 1.495 ± 0.54, 2.575 ± 0.52 and 0.875 ± 0.29. Oocyte secreted factors seem to reduce FSHR expression while enhancing AMH expression in cumulus cells. We speculate that this is part of an effort of the oocyte to restrain FSH activity in the COC in order to preserve its communication with cumulus cells thus favouring developmental competence.

Effects of cyclic adenosine monophosphate modulators on maturation and quality of vitrified-warmed germinal vesicle stage mouse oocytes

BackgroundIt is still one of the unresolved issues if germinal vesicle stage (GV) oocytes can be successfully cryopreserved for fertility preservation and matured in vitro without damage after warming. Several studies have reported that the addition of cyclic adenosine monophosphate (cAMP) modulators to in vitro maturation (IVM) media improved the developmental potency of mature oocytes though vitrification itself provokes cAMP depletion. We evaluated whether the addition of cAMP modulators after GV oocytes retrieval before vitrification enhances maturation and developmental capability after warming of GV oocytes.MethodsRetrieved GV oocytes of mice were divided into cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). Then, GV oocytes were cultured with or without dibutyryl-cAMP (dbcAMP, cAMP analog) and 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) during the pre-vitrification period for 30 min.ResultsOne hour after warming, the ratio of oocytes that stayed in the intact GV stage was significantly higher in groups treated with cAMP modulators. After 18 h of IVM, the percentage of maturation was significantly higher in the COC group treated with dbcAMP. The expression of F-actin, which is involved in meiotic spindle migration and chromosomal translocation, is likewise increased in this group. However, there was no difference in chromosome and spindle organization integrity or developmental competence between the MII oocytes of all groups.ConclusionsIncreasing the intracellular cAMP level before vitrification of the GV oocytes maintained the cell cycle arrest, and this process may facilitate oocyte maturation after IVM by preventing cryodamage and synchronizing maturation between nuclear and cytoplasmic components. The role of cumulus cells seems to be essential for this mechanism.

Determination of 70 kDa Proteins for Different Temperatures in Cattle Oocytes Matured in vitro

This study was conducted to determine the effect of 36.5 (low) or 38.5 °C (conventional) culture temperatures during in vitro maturation (IVM) on 70 kDa proteins in cattle oocytes. In the study, cattle cumulus oocyte complexes (COC) were subjected to IVM in bicarbonate-buffered TCM–199 supplemented with 10% FCS for 22 hours with a humidified 5% CO2 in air at either 36.5 or 38.5 °C culture temperatures. Maturation of COCs was determined according to fully cumulus expansion at the end of IVM. The cumulus cells of matured COCs were removed by vortexing in TCM-199 with HEPES buffered TCM-199 containing 0.1% hyaluronidase enzyme. Following, denuded oocytes were washed in 9.6 % phosphate buffered saline (PBS) solution for protein isolation. Denuded oocytes (n = 100 for both groups) were centrifuged for 5 min at 300 rpm at 4 °C for PBS removed than the lysis buffer (100 μl) was added and shake 15 min on ice for 30 minutes. For protein isolation, the protein was centrifuged again at 13,000 rpm for 5 minutes and the protein samples were stored at -20 ° C. To increase the concentration of isolated protein samples, the samples were frozen at -80 °C for 24 hours and lyophilized at 0.140 hPa in a lyophilizer. Lyophilized protein samples were stored at -20 °C until SDS-PAGE analysis. The level of 70 kDa proteins in oocytes were determined by SDS-PAGE method. There were no significant differences between low (81.11%) or conventional (84.41%) incubation temperatures in IVM in terms of full cumulus expansion. The 70 kDa proteins band size in SDS-PAGE polyacrylamide gel image of cattle oocytes maturated at low culture temperature was weak compared to cattle oocytes maturated at conventional culture temperature. The results of this study may show that low incubation temperature during IVM decreases the amount of 70 kDa proteins in cattle oocytes.

Follicle-stimulating hormone administration affects amino acid metabolism in mammalian oocytes†.

Culture media used in assisted reproduction are commonly supplemented with gonadotropin hormones to support the nuclear and cytoplasmic maturation of in vitro matured oocytes. However, the effect of gonadotropins on protein synthesis in oocytes is yet to be fully understood. As published data have previously documented a positive in vitro effect of follicle-stimulating hormone (FSH) on cytoplasmic maturation, we exposed mouse denuded oocytes to FSH in order to evaluate the changes in global protein synthesis. We found that dose-dependent administration of FSH resulted in a decrease of methionine incorporation into de novo synthesized proteins in denuded mouse oocytes and oocytes cultured in cumulus-oocyte complexes. Similarly, FSH influenced methionine incorporation in additional mammalian species including human. Furthermore, we showed the expression of FSH-receptor protein in oocytes. We found that major translational regulators were not affected by FSH treatment; however, the amino acid uptake became impaired. We propose that the effect of FSH treatment on amino acid uptake is influenced by FSH receptor with the effect on oocyte metabolism and physiology.

Combination of L-Ascorbic Acid and 2- Mercaptoethanol Reduces the Damaging Effect of Hydrogen Peroxide During in Vitro Maturation of Nili Ravi Buffalo Denuded Oocytes

Combination of L-Ascorbic Acid and 2- Mercaptoethanol Reduces the Damaging Effect of Hydrogen Peroxide During in Vitro Maturation of Nili Ravi Buffalo Denuded Oocytes

Bovine herpesvirus 1 can impact the bovine oocyte development during in vitro maturation

Bovine herpesvirus 1 (BoHV-1) disseminates easily, is difficult to control, and is widely spread in cattle herds worldwide. BoHV-1 causes a broad range of losses to the cattle industry, mainly concerning reproduction. Previous studies involving experimental infection of BoHV-1 in an in vitro embryo production system have reported impairment of embryonic development by BoHV-1. In this study, we evaluated the interference of BoHV-1 in the in vitro maturation system of cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) cultured with a cumulus cell suspension. Blood samples and ovaries were collected from slaughterhouse cows unvaccinated against BoHV-1. Using virus neutralization assays, the seropositive animals were classified according to their antibody titers. The oocytes were recovered by follicular aspiration and divided into two groups, COCs and DOs, which were evaluated for their nuclear maturation capacity using immunofluorescence assays by laser scanning confocal microscopy. Two experiments were carried out: (I) in vitro maturation of COCs and DOs after artificial infection of seronegative animals and (II) in vitro maturation of COCs and DOs of seropositive animals. In experiment I, a difference (P < 0.01) was observed between the maturation rates of the control group COCs (78.2%) and the infected COCs (43.6%). In experiment II, there was a difference (P < 0.01) in the maturation rate between animals with antibody titers ≥16 (56.9%) and the control group (79.4%). Immunofluorescence assays identified BoHV-1 in the COCs and DOs. Therefore, it was concluded that BoHV-1 affects the in vitro maturation process in both in vitro and natural infections.

32 Vitrification of in vitro-matured bovine oocytes in triacetate cellulose hollow fibres

A prospective method of vitrification in triacetate cellulose hollow fibres (HF) introduced by Matsunari et al. (2012 J. Reprod. Dev. 58, 599-608) allowed significant simplification and standardization of vitrification/warming procedures and was successfully used for group cryopreservation of various pre-implantation mammalian embryos. The goal of the current work was to evaluate the effectiveness of the HF vitrification method for cryopreservation of in vitro-matured bovine oocytes. The base medium for all the vitrification and rewarming solutions was calcium-free TBP-like protein-HEPES supplied with 20% of fetal bovine serum. Groups of 15 morphologically normal in vitro-matured bovine oocytes were equilibrated with 3% (vol/vol) ethylene glycol for 15min, loaded into HF, and transferred into vitrification solution containing 30% ethylene glycol and either 0.5M (Group 1) or 1.0M (Group 2) sucrose. Hollow fibres were incubated for either 60s (Group 1) or 30s (Group 2) and immediately plunged into LN. Rewarming was conducted at 39°C. Oocytes within HF were placed in decreasing concentrations of sucrose solutions to remove cryoprotectants. Then, oocytes were subjected to IVF. Non-vitrified denuded oocytes were used as a control. Survival rates were evaluated at 21h post-rewarming. Part of the presumptive zygotes were fixed and stained with acetolacmoid for fertilization rate. The remaining zygotes were cultured for 10 days. Developmental rates were evaluated at 44h and 7 and 10 days post-IVF. All results are presented as mean percentage±standard deviation. Data were analysed using Mann-Whitney U test. Significance was set at P&amp;lt;0.05. Survival rate was significantly lower in Group 1 (79.0±8.0%) and Group 2 (75.0±5.0%) compared with the control group (97.0±4.0%). Fertilization rate in Group 1 differed significantly from the control (80.5±18.3% v. 95.5±9.1%). Cleavage rates in Groups 1 and 2 did not differ significantly from the control (42.5±15.7% v. 60.7±11.1% v. 63.0±15.8%, respectively). Blastocyst yields at 7 days post-IVF were 0.9±2.3 (1/116) and 9.6±5.4% (6/65) in Groups 1 and 2, respectively. The former was significantly lower than in the control group (17.0±10.3%, 23/154). It should be noted that hatching in the control group started at 8 days post-IVF and was delayed in Groups 1 and 2 for at least 24h. Day 10 blastocyst yields were 3.0±3.3 (P&amp;lt;0.05), 20.9±13.8, and 30.4±9.6% in Groups 1 and 2 and the control group, respectively. All obtained Day 10 blastocysts (3/116) in Group 1 hatched. Hatching rate in Group 2 was significantly lower than in the control group. Both Groups 1 and 2 showed relatively high survival and fertilization rates, but embryo development rates in both groups had a tendency to be lower than in the control. However, the obtained results indicate that the modifications of the protocol may increase the effectiveness of HF vitrification. The HF vitrification method remains a prospective option for simultaneous cryopreservation of a group of bovine oocytes.

INFLUENCE OF GAMETE CO-INCUBATION TIME, SIRE AND SPECIAL ADDITIVES ON THE IN VITRO FERTILIZATION OF CUMULUS-ENCLOSED OR DENUDED BUFFALO OOCYTES

The present study was designed to investigate the influence of cumulus removal before the in vitro fertilization step and the impact of co-incubation time, sire, and additives to the fertilization medium on the efficiency of in vitro fertilization of buffalo oocytes. In vitro matured oocytes were fertilized either as cumulus-oocyte complex (COCs) or after removal of cumulus cells (denuded). Cumulus-enclosed or denuded oocytes were co-incubated with sperm cells for 6, 12 or 18 h (experiment 1), fertilized with sperm cells from one of three sires (experiment 2) or fertilized in medium supplemented with 20μg∕ml heparin, 5 mM theophylline or a mixture 20μM penicillamine, 10μM hypotaurine and 1 μM epinephrine (PHE) (experiment 3). In all experiments, a group of oocytes was fixed and stained to evaluate the fertilization pattern (penetration, normal and abnormal fertilization rates) and the rest were cultured up to 8 days to assess the developmental competence (cleavage and blastocyst yield). In all experiments, removal of cumulus cells before fertilization step significantly retarded the fertilization pattern and the developmental competence. Various co-incubation times did not significantly influence the fertilization pattern or the developmental competence of denuded or COCs. However, 6 h tended to decrease the abnormal fertilization (15.74±1.70 vs 28.46±6.06, P=0.069) and to improve the blastocyst/oocyte (11.70±3.41 vs 5.53±1.75) and the blastocyst/cleavage (27.14±6.19 vs 11.98±3.81, P=0.082) when compared with 18 h. Sperm cells from the three sires resulted in similar fertilization pattern and developmental competence in COCs and denuded oocytes. In COCs and denuded oocytes, PHE tended to improve the blastocyst/oocyte (7.44±2.58 vs 14.67±4.29) and blastocyst/cleavage (18.43±6.08 vs 33.13±9.27) in comparison to heparin (P˃0.05). Thus, none of the investigated factors could counteract the adverse effect of cumulus removal otherwise, the addition of PHE showed promising results but it need further investigations.

Buffalo oocyte-secreted factors promote cumulus cells apoptosis and the rate of cGMP production but not steroidogenesis.

The objectives of this study were to investigate the effect of buffalo oocyte-secreted factors (OSFs) on cumulus cells (CCs) functions, apoptosis and cGMP generation, and whether the direct contact between oocyte and CCs is essential for oocyte-mediated regulation of CCs functions. Buffalo CCs were cultured during IVM within three groups: (a) intact cumulus-oocyte complexes (COCs), (b) CCs cocultured with denuded oocytes (DOs) (CCs+DOs) and (c) CCs monolayer cultured alone (CCsM). After 24hr of IVM, CCs were harvested for evaluation of the relative mRNA abundance of the genes encoding gap junction (GJA1), glycolysis (PFKP and LDHA), apoptosis (CASPASE-3 and BCL-2) and steroidogenesis (ER-β and PGR) by QRT-PCR, and CASPASE-3 proteins, using western blot. Intracellular cGMP content was also assessed by ELISA. Results showed that the relative abundance of LDHA, PFKP and BCL-2 significantly increased (p<0.05) in COCs, whereas GJA1 and CASPASE-3 exhibited lower expression (p<0.05) compared to CCs+DOs and CCsM groups. However, the expression levels of CASPASE-3, both mRNA and protein, were significantly (p<0.05) downregulated in CCs+DOs compared to CCsM. There was no significant difference in the expression level of PGR and ER-β between the groups. The intracellular content of cGMP was notably (p<0.05) higher in COCs compared to CCs+DOs and CCsM groups. In conclusion, this study demonstrated, for the first time, that buffalo OSFs protect CCs against apoptosis and stimulate their cGMP production; however, the regulation of cumulus glycolysis and gap junction is confined to those in close contact with the oocyte. Neither OSFs from COCs nor those from DOs have any effect on CCs steroidogenesis.

Follicular fluid and supernatant from cultured cumulus-granulosa cells improve in vitro maturation in patients with polycystic ovarian syndrome

To study the effectiveness of a new invitro maturation (IVM) approach based on heterologous follicular fluid (HFF) and supernatant of cumulus-granulosa cells (CGCs) mimicking the intact follicular microenvironment to rescue immature denuded oocytes (IDOs) of patients with polycystic ovary syndrome (PCOS) whose IVM or IVF outcomes remain poor. Randomized controlled trial. University-affiliated private center. One hundred fifty-nine IDOs were obtained from 47 patients with PCOS. First, a simple IVM system (S-IVM; 40 IDOs; control group) was compared with different protocols based on the addition of autologous follicular fluid (AFF-IVM; 44 IDOs), HFF (HFF-IVM; 42 IDO), or HFF with CGC isolated from seven women without PCOS and presenting 100% invivo oocyte maturation (HFF/CGC-IVM; 33 IDOs). None. IVM outcomes were compared among the four groups (S-IVM, AFF-IVM, HFF-IVM, HFF/CGC-IVM); then the vitro and invivo maturation results (from controlled ovarian stimulation of PCOS patients) were compared for each group. The HFF/CGC-IVM method gave the best yield of developed blastocysts per IDO compared with S-IVM, AFF-IVM, and HFF-IVM (27% vs. 2%, 2%, and 12%, respectively). The IVM rate with the HFF/CGC-IVM method was even higher than that compared with the invivo maturation rate (79% vs. 42%), with significant improvement in the cleavage rate (71% vs. 61%). This adapted IVM system could be used to reach an acceptable result in meiotic competence and competent metaphase II oocytes capable of developing into intact embryos after fertilization and before transfer.

Urea influences amino acid turnover in bovine cumulus-oocyte complexes, cumulus cells and denuded oocytes, and affects in vitro fertilization outcome

High-protein diets often lead to an increase in urea concentration in follicular fluid of dairy cows, which may reduce oocyte competence. In the present study, maturation media were supplemented with urea (0, 20, 40 mg/dl), and amino acids (AAs) turnover was evaluated in the 24-h spent media of specimens (cell types), bovine cumulus-oocyte complexes (COCs), cumulus cells (CCs), or denuded oocytes (DOs). The main effects of urea and cell type, and their interaction were significant on the individual turnover (expect threonine, glycine, and tyrosine) and total turnover, depletion, and appearance of AAs. The results showed a high level of urea and DOs increased the depletion of all AAs and that of essential and non-AAs, respectively. Sensitivity analysis revealed the highest sensitivity of isoleucine, lysine, and tryptophan to urea, especially in DOs. Principal component analysis (PCA) evaluated the strong correlations between the turnover of: (1) glutamine, aspartic acid or glycine, and developmental competence and fertilization of COCs; (2) serine, isoleucine, valine or glutamic acid, and cleavage rate of DOs; and (3) serine, glutamine, aspartic acid or alanine, and CCs viability. In conclusion, urea significantly changed the turnover of AAs by COCs, CCs and DOs, and reduced the subsequent developmental competence of bovine oocytes.

Developmental competence of buffalo (Bubalus bubalis) denuded oocytes cocultured with cumulus cells: Protective role of cumulus cells

The objectives of the present study were to evaluate the developmental competence of buffalo denuded oocytes (DOs) cocultured with cumulus cells (CCs) during in vitro maturation, and to investigate the mechanisms by which CCs promote oocyte maturation and development. Buffalo oocytes were matured in vitro for 24 h in three groups: (1) intact cumulus–oocyte complexes (COCs) (2) DOs cocultured with CCs (DOsCC), and (3) DOs cultured alone (DOs). Matured oocytes were used to determine the relative mRNA abundance of Gdf-9, Bmp15, Zar1, Caspase-3, Bcl-2, Zp2, Zp3, Cd9 and Pde3a by Rt-qPCR and CASPASE-3 protein expression by immunofluorescence. The intracellular content of cGMP, cAMP and MPF activity and the rate of embryonic development were also assessed. Results of the present study showed that in DOs, the relative mRNA abundance of Gdf-9, Bmp15, and Cd9 significantly (P < 0.05) decreased, whereas Caspase-3 (mRNA and protein levels), Bcl-2, and Pde3a exhibited higher expression than DOsCC and COCs. However, there was no significant difference among the groups in the expression level of Zar-1, Zp2, and Zp3. The intracellular content of cAMP and MPF activity was notably higher (P < 0.05) in DOs compared to COCs and DOsCC. There was no significant difference between COCs and DOsCC in cGMP content, which was significantly lower (P < 0.05) in DOs. Moreover, the cleavage and blastocyst rates were 58.4 ± 1.8%, 43.7 ± 1.1%, 18.4 ± 0.9% and 18.0 ± 1.3%, 11.0 ± 0.9% and 4.5 ± 0.6% in COCs, DOsCC and DOs groups, respectively. In conclusion, the presence of CCs protects buffalo DOs from apoptosis and promotes maturation through regulation of the intracellular content of cAMP and MPF activity and improves the fertilizing capacity of oocytes through modulation of the gamete fusion gene, Cd9.

Inhibitory Role of the Mud Crab Short Neuropeptide F in Vitellogenesis and Oocyte Maturation via Autocrine/Paracrine Signaling.

Neuropeptides, in addition to their classical role in the nervous system, act on intraovarian factors to regulate reproductive functions in vertebrates. However, this function of neuropeptides has not been characterized in crustaceans. Short neuropeptide F (sNPF), a highly conserved invertebrate neuropeptide, has been reported to be involved in feeding, metabolism, and in differentiation processes including reproduction. Although sNPF and its receptor (sNPFR) have been detected in the ovary in different species, ovarian colocalization of sNPF/sNPFR has not been investigated. In this study, we identified Scylla paramamosain (mud crab) sNPF (Sp-sNPF) as an endogenous ligand for the S. paramamosain orphan G protein-coupled receptor NPY2R in mammalian cell line HEK293T. We designated this receptor as Sp-sNPFR. RNA in situ hybridization in pre-vitellogenic ovary and reverse transcription-PCR on isolated denuded oocytes and follicle layers showed that Sp-sNPF was exclusively localized to the follicle cells, whereas Sp-sNPFR was detected in both follicle cells and oocytes. We also found that Sp-sNPF partly suppressed spontaneous maturation of denuded oocytes and caused intracellular cAMP accumulation and Ca2+ mobilization. Moreover, injection of synthetic Sp-sNPF peptides inhibited the expression of vitellogenin and vitellogenin receptor genes in vivo. These combined results suggest for the first time that Sp-sNPF may have inhibitory functions in vitellogenesis and oocyte maturation possibly via the autocrine/paracrine pathway.