The Role of Brain-derived Neurotrophic Factor in Mouse Oocyte Maturation in vitro

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation (IVM),but the role of BDNF in oocyte maturation at cellular level is not still clear.In this study,mouse cumulus-enclosed oocytes subjected to IVM were fertilized and cultured to blastocyst stage.Meiotic spindle configuration and cortical granules distribution during oocyte maturation in vitro were assessed by using immunofluorescence and laser confocal microscopy.The results showed that BDNF contributed to the complete preimplantation development of mouse oocytes compared to the control oocytes (13.78% vs.5.92%;P【0.05).Further,BDNF did not accelerate nuclear maturation of IVM oocytes.For the BDNF-treated oocytes at meiosis Ⅰ,Meiotic spindle areas were significantly smaller and the number of cytoplasmic microtubule organizing centers was greater than that in the control,and the percentages of oocytes showed spindles positioned near the oolemma and a well-formed cortical granule-free domain were significantly higher than that of the control.These morphological characteristics of the BDNF-treated oocytes were much closer to the oocytes matured in vivo than those of the control oocytes.In conclusion,BDNF can promote the developmental competence of mouse IVM oocytes,by improving the meiotic spindle configuration and location and cortical granules distribution at meiosis Ⅰ.

Protective effect of resveratrol against cadmium-induced toxicity on ovine oocyte in vitro maturation and fertilization

Background:Heavy metal cadmium(Cd)is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility.It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization,through oxidative stress induction.Resveratrol(Res)is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline.Here,we explored whether the addition of Res to in vitro maturation(IVM)medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization.Firstly,we evaluated the effect of supplementing IVM medium with two different Res concentrations(1and 2μmol/L)on nuclear maturation and fertilization of oocytes matured under CdCl2(2μmol/L)exposure.Therefore,the concentration of 1μmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels,mitochondrial(mt)distribution and activity,chromatin configuration,cytoskeleton morphology,cortical granules(CGs)distribution and mRNA expression of genes associated with cellular response to oxidative stress(i.e.SIRT1,SOD 1,GPX1,GSR,CAT)in Cd-exposed in vitro matured oocytes.Results:We found that 1μmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as,Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations.Moreover,we demonstrated that 1μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species(ROS)accumulation,preventing mt dysfunction,maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1,SOD1 and GPX1 genes.Conclusions:Taken together,our findings highlighted the beneficial influence exerted by Res in preventing Cdinduced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes.Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

In vitro Oocyte Maturation in the Zebrafish Brachydanio rerio and Fertilization and Development of the Mature Egg

Maturation process of zebrafish oocyte was investigated using in vitro incubation.In medium EM-199 containing 0.5 μg/ml of 17α-hydroxyprogesterone incubated under 80％ O_2 and at 25°C,germinal vesicles(GV)of oocytes in stage Ⅳ migrated from midway between the center and theperiphery ofoocytes to the periphery in 40 minutes and the oocytes went into stage V.Half an hourlater,the oocytes underwent germinel vesicle breakdown(GVBD)with a breakdown rate of 59％.Two more hours were needed for such oocytes to complete their final maturation.The mature eggscould not come off from the follicle layer surrounding them by themselves(ovulation).By removingthe follicle and adding active sperms for insemination,we could make the mature eggs fertilized.Thechorion was elevated and blastoderm formed on the animal pole.The cleavage and development ofthese fertilized eggs followed the same course as the naturally matured and fertilized eggs.Usingblastula formation as a marker of successful fertilization of the in vitro matured egg,the fertilizationrate was 78％.This is the first report on the successful in vitro incubation of mature oocytes inzebrafish.The establishment of this in vitro oocyte maturation technology has laid the foundationfor further investigation of the transfer of foreign genes in the germinal vesicles of oocytes.

Improvement of in vitro Cytoplasmic Maturation of Denuded Porcine Oocytes by Coculture with Follicular Mural Granulosa Cells

[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.

Effects of Ages of Donors and Conditions of Preserving Ovaries on Porcine Oocytes Maturation in vitro and Efficiency of Parthenogenetic Activation

Effects of different ages of donors and different conditions of preserving ovaries onporcine oocytes maturation in vitro and efficiency of parthenogenetic activation werestudied. The experiments included: 1) effects of different temperatures (22, 30, 37, 38.5and 40℃) of preserving ovaries on porcine oocytes maturation in vitro and developmentalpotential; 2) effects of periods of preserving ovaries on porcine oocytes maturation invitro and development in vitro; 3) effects of different ages of donors on porcine oocytesmaturation in vitro and developmental potential. The results of the experiment showed:1) There were no statistical differences (p>0.05) of the parthenogenetic cleavage rate(79.64% vs 76.18%) and blastocyst rate (18.11% vs 33.82%) between oocytes from ovariespreserved at 38.5℃ and those preserved at 37℃. When the preserving temperature wasincreased to 40℃, the cleavage rate (21.68%) and the blastocyst rate (0) were greatsignificantly lower than those at 37℃(p<0.01). The cleavage rate (80.79% vs 76.18%) andblastocyst rate (29.61% vs 33.82%) were not different between 30 and 37℃(p>0.05). Whenthe preserving temperature was decreased to 22℃, the rate of cleavage was not different,but the rate of blastocyst was significantly lower, compared with that at 37℃; 2) Thecleavage and blastocyst rates of the porcine oocytes collected after slaughter 2 or 6hwere not different (p>0.05); 3) The cleavage rate of oocytes from gilts and sows aftermaturation was not different, but the blastocyst rate of the sow group was significantlyhigher than that of gilt group (p<0.05). The blastocyst cell number of sows and giltshowed no difference (p>0.05).

Effect of Dynein Inhibitor on Mouse Oocyte in vitro Maturation and Its Cyclin B1 mRNA Level

Objective To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level. Methods Immature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level. Results In dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 mmol/L SOV and control groups (P<0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 mmol/L. In incon- tinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 mmol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P<0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed. Conclusion Dynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or MⅠto anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.

Evaluation of pig oocyte in vitro maturation and fertilization using three gonadotropin-based hormonal compounds

Objective:To evaluate the effect of human chorionic gonadotropins(hCG)and equine chorionic gonadotropins(eCG)on in vitro gilt oocyte maturation and embryonic development,using frozen semen for fertilization.Methods:Two independent experiments(6 replicates each)were carried out to evaluate gilt oocyte maturation,and fertilization and embryonic development by using ovaries from a local abattoir.Totally,712 oocytes were randomly distributed in four-well dishes to receive Novormon(eCG 5.0 IU),PG600(eCG 5.0 IU and hCG 2.5 IU),Chorulon(hCG 5.0 IU),or no hormones.Oocytes were incubated with 5%CO2,95%air and saturation humidity at 39℃for 44 h.Maturation of the oocytes to metaphaseⅡwas assessed by using the aceto-orcein technique.In addition,741 oocytes were used and randomly distributed in four-well dishes,and then oocyte maturation was carried out as mentioned,but matured oocytes were washed and placed in fertilization medium with frozen-thawed sperm.Gametes were co-incubated for 7 h,and then washed and placed in development medium,and incubated for further 7 days,at which time embryonic development was evaluated.Fertilization and embryo development media were not supplemented with the studied hormones.Results:Novormon(eCG)and PG600(eCG+hCG)treatments significantly improved the percentages of metaphaseⅡoocytes compared to the control group(P<0.05).Furthermore,a significant increase was also observed in the young blastocyst stage between the control group and the PG600 treatment group(P<0.05).Conclusions:Hormonal products Novormon(eCG)and PG600(eCG+hCG)can obtain the highest percentages of in vitro maturation in gilt oocytes;however,this effect is not transferred to fertilization rates.

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.

LH Plays a Role to Enhance the Nuclear and Cytoplasm Synchronization During In vitro Maturation of Ovine Oocytes

This study is aimed to investigate the effect of luteinizing hormone （LH） on maturation and fertilization in vitro of ovine oocytes. The ovine oocytes were cultured in vitro in medium with or without LH, and then checked by confocal laser scanning microscope to observe the distribution of cortical granules stained with FITC-LCA during different maturation periods. Similarly, some in vitro matured oocytes were also fertilized in vitro for analysis of their developmental potentiality further. After being cultured in vitro for 4 h, there were significant differences about the rate of germinal vesicle break down （GVBD） between the treatment （with LH） and the control groups （without any hormones） （36.76% vs 18%, P〈0.05）. Further, there were also significant differences of the cleavage and blastocyst rates between these two groups （67.15% vs 42.37%, 21.9% vs 12.71%, P〈0.05, respectively）. The distribution of cortical granules appeared to spread from the edges to the central site of sheep oocytes following their delaying durations of maturation in vitro. It can be concluded that LH may play a role to delay the occurence of GVBD, prolong the maturation duration of cytoplasm, and enhance the nuclear and cytoplasm synchronization of ovine oocytes matured in vitro and finally improve their in vitro developmental potentiality.

Effects of Chemical Activation on the Parthenogenetic Development of Porcine in vitro Maturation Oocytes

The objective of this study was to determine the effects of ionomycin combined with cytochalasin B （CB）, cycloheximide （CHX）, or 6-dimethylaminopurine （6-DMAP） on the activation of porcine oocytes. In Experiment 1, in vitro matured oocytes were activated with 15, 20, 25 or 30 mmol L -1 ionomycin separately. Activation rates of 20, 25 mmol L-1 and 30 mmol L treatments were higher （P〈0.05） than that of 15 mmol L-1 treatment. In Experiment 2, in vitro matured oocytes were activated with 20 mmol L-1 ionomycin for 10, 20, 30, 40 or 50 rain and then incubated with 2 mmol L-1 6-DMAP for 6 h. Cleavage and blastocyst rates [（72.40 ± 13.02）%, （25.37 ± 11.43）%] after treatments for 40 min were higher （P〉0.05） than those of the other treatments. In Experiment 3, matured oocytes were activated with ionomycin and then incubated with 7.5 mg mL-1 CB, 10 mg mL-1 CHX, 2 mmol L-1 6-DMAP, 7.5 mg mL-1 CB ＋ 10 mg mL-1 CHX or 7.5 mg mL-1 CB ＋ 2 mmol L-1 6-DMAP for 6 h. The rates of activation, cleavage and blastocyst of 2 mmol L -1 6-DMAP treatment [（86.05 ± 4.29）%, （61.77 ±8.10）% and （21.62± 3.31）%] were higher （P〈0.05） than those of 7.5 mg mL-1 CB treatment. In Experiment 4, matured oocytes were activated with ionomycin and then incubated with 2 mmol L-1 6-DMAP for 3.5, 5.5 or 7.5 h. Cleavage rates and blastocyst rates of 5.5 h treatment [（66.59 ± 14.36）% and （25.40 ± 10.16）%] were higher （P〉0.05） than those of other treatments. In conclusion, activation of porcine oocytes appears to be most successful using the combination of ionomycin （20 mmol L-1, 40 min） followed by 6-DMAP （2 mmol L-1, 5.5 h）.

Expression of Interleukin-6 and Interleukin-6 Receptor in Ovine Oocytes During In vitro Maturation

To study the effects of interleukin-6 （IL-6） and its receptor （IL-6R） during in vitro maturation of ovine oocytes, the mRNA and protein expression levels of IL-6 and IL-6R, along with their localization, were examined during ovine oocytes maturation in vitro through real-time PCR, Western blotting, and immunohistochemistry. Specific patterns of expression of IL-6 and IL-6R were observed at both mRNA and protein levels at each stage of ovine oocytes maturation. IL-6 and IL-6R were distributed primarily on the surface of the cell membrane, with little expression in the cytoplasm or nucleus. IL-6 and IL-6R were expressed significantly at higher levels in the maturation around 4 h, and then decreased dramatically. However the level slightly elevated at 20-24 h. The role of IL-6 and IL-6R on oocytes maturation was studied through in vitro addition of recombinant human IL-6 in different concentrations. The addition of 10 ng mL-1 IL-6 significantly increased the rates of oocytes maturation （P〈0.05）, but did not affect the rates of development of the subsequence IVF ovine embryos. In summary, IL-6 is likely to play an important role in the early ovine oocytes maturation. The expression patterns of the IL-6 and IL-6R on the ovine oocytes maturation open up the possibility of regulatory role of the cytokine in ovine oocytes maturation.

Effects of Medium on in Vitro Maturation of Pig Oocytes and in Vitro Early Development of Parthenogenetic Embryos

[ Objective] To improve quality of oocytes maturing in vitro and to optimize in vitro culture system of porcine early embryos. [ Method ] Oocytes were cultured in the improved TCM199 and NCSU-23 basic media, which were added pig follicular fluid （PFF） or high-quality fetal bovine serum （FBS） both at a proportion of 10% （V/V）. After in vitro maturation and development, effects of medium on maturation of pig oocytes and development of eady parthenogenetic embryos were investigated using maturing rate of pig oocytes and development rate of parthenogenetic embryos as indicators. [ Result] After 42 h culture, the maturing rates of pig oocytes respectively cultured in the TCM199, TCM199 added FBS, TCM199 added PFF, NCSU-23, NCSU-23 added FBS and NCSU-23 added PFF were （54.2 ±3.5）%, （68.5 ±3.2）%, （69.3 ±3.7）%, （51.6 ±3.3）%, （63.2 ±3.1 ）% and （65.5 ±3.5）%, respectively. The pig oocytes cultured in the TCM199 or NCSU-23 that was added FBS or PFF had significantly higher maturing rate （P 〈 0.05）. The development rates of parthenogenetic embryos were not significantly different between these six experimental media. However, the parthenogenetic embryos which developed in the TCM199 added PFF （36.5 ±4.8） had significantly more blastomeres than those developed in the TCM199 or NCSU-23 （ 18.7 ± 3.2 and 15.5 ± 2.4, respectively） （ P 〈 0.05）. [ Conclusion ] The improved TCM199 and NCSU-23 added PFF or FBS can largely promote in vitro maturation of pig oocytes and in vitro development of early parthenogenetic embryos.

Effect of Different Reproductive Stages and Culture Times on Domestic Cat <i>in Vitro </i>Oocyte Maturation

The domestic cat has been used as a model to carry out comparative research in assisted reproduction, to be applied in wild cats. The efficiency in domestic cat IVM concerning the reproductive status and/or cultivation times has previously been investigated;however, the studies were carried out separately. The objective of this research was to evaluate the maturation of oocytes of domestic cats of different reproductive stages using two different in vitro culture times. The ovaries were obtained by Ooforo-Salpingo-Hysterectomy of cats that were of the following groups: 1) prepubertal, 2) follicular, 3) pregnant or 4) in anestrus. Maturation was carried out with TCM199 medium supplemented with BSA for 24 h and 48 h. On average, 29 ± 25, 20 ± 15, 17 ± 9 and 17 ± 13 oocytes/cat were recovered from the prepubertal follicular, pregnant, and anestrus stages, respectively, but did not show a significant difference (P > 0.05). Also, meiotic maturation did not show a significant difference between the different reproductive stages at 24 h and 48 h, respectively (P > 0.05). However, in the prepubertal and follicular stages, greater oocyte maturation numbers were observed at 48 h compared to 24 h (P < 0.05). In contrast, the aforementioned result was not observed in the pregnant and anestrus stages (P > 0.05), indicating that the in vitro culture duration is an important factor during in vitro maturation of domestic cat oocytes.

Study of Estrus Cow Serum （ECS） in Maturation Media on in vitro Maturation Rate of Bovine Oocytes

The objective of this research was to study the effect of ECS on cumulus expansion and rate of nucleus maturation of bovine oocytes. Media maturation were used： （1） TCM 199 ＋ FCS 10%; （2） TCM 199 ＋ FCS 10% ＋ ECS 5%; （3） TCM 199 ＋ FCS 10% ＋ ECS 7, 5%; （4） TCM 199 ＋ FCS 10% ＋ ECS 10%; （5） TCM 199 ＋ ECS 10%. Supplementation of ECS had significantly difference （P 〈 0.05） on expansion of cumulus cells and rate of nucleus maturation. Supplementation of ECS 5% was the best result in expanded cumulus cells and metaphase II rate： 82% and 72% respectively. It was concluded that medium of TCM 199 ＋ FCS 10%o ＋ ECS 5% was the best maturation medium

Effect of Lamb Age and in vitro Culture System of Oocytes on JIVET Technology

[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed Han lambs aged 5 to 10 weeks were induced to superovulate via i. p. injection of pregnant mare's serum gonadotropin( PMSG). The oocytes were matured in basal maturation solution or modified maturation solution,which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then,the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum( ESS) or fertilization medium II containing 3 mg/ml bull serum albumin( BSA). Finally,the number of oocytes,oocyte maturation rate and cleavage rate of the lambs of different ages were determined. [Results]The average number of oocytes recovered per lamb was( 111. 00 ± 16. 97),( 139. 50 ± 28. 99),( 108. 50 ± 17. 68) and( 42. 00 ± 11. 31) for5-,7-,8-and 10-week-old Dorper × small-tailed Han lambs,respectively. The number of oocytes obtained from 5-,7-and 8-week-old lambs was significantly higher than that from 10-week-old lambs( P < 0. 05),but there was no significant difference among 5-,7-and 8-week-old lambs( P > 0. 05). The maturation rate of oocytes cultured in modified maturation solution was 3. 64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II( P < 0. 01). [Conclusions] The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks,adding a certain amount of cysteine into oocyte maturation solution,and a certain amount of ESS into fertilization medium.

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa

Aim: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). Methods: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. Results: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P < 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. Conclusion: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

L-carnitine improves developmental competence of buffalo oocytes in vitro

Objective:To investigate the effect of L-carnitine on in vitro maturation and subsequent in vitro embryo production of buffalo oocytes.Methods:Cumulus oocyte complexes(COCs)were aspirated from ovaries of slaughtered buffaloes.COCs were classified into good and fair qualities based on morphological observation of numbers and integrity of cumulus cells surrounding the oocyte.Both categories of COCs were placed in in vitro maturation medium with supplementation of different concentrations(0,0.250,0.375 or 0.500 mg/mL)of L-carnitine.Oocytes from both qualities were in vitro fertilized and in vitro cultured for 7 days,to examine the developmental competence.Results:Supplementation of L-carnitine to in vitro maturation medium increased the cumulus cell expansion rate of COCs to grade A,and reduced the cumulus cell expansion of COCs to grade B and grade C in both good and fair quality oocytes.Similarly,L-carnitine induced the in vitro meiotic progression of buffalo oocytes to metaphaseⅡin both good and fair quality oocytes.Additionally,L-carnitine reduced the rate of oocyte degeneration in both good and fair quality oocytes.L-carnitine increased the rate of cleaved formation at day 2 and blastocyst formation at day 7 during in vitro culture in both qualities of oocytes.Moreover,a higher rate of blastocyst production was observed in L-carnitine-treated fair quality oocytes,which was higher than the results in the untreated good quality oocytes.Conclusions:L-carnitine enhances meiotic maturation and subsequent embryo development from both good and fair quality buffalo oocytes.

Impact of Luveris and Gonal-F on bovine immature oocytes developmental competence in vitro

Objective: To investigate the gonadotrophin drug Luveris? (L) and Gonal-F? (G) effects on bovine immature oocyte in vitro maturation culture. Methods: In total, 2000 bovine cumulus-oocyte complexes (COCs) were purchased commercially and cultured in media with ovulation stimulation drugs Gonal-F and Luveris (0, 5, 10, 20, 40 IU/mL), individually, for maturation in vitro. 2000 Oocytes were divided evenly into two groups, Luveris and Gonal-F groups, individually, 1000 Oocytes. After 24 and 48h culture, cumulus expansion was assessed under dissecting microscopes. Oocyte maturation (GV, MI, MII) was determined by examining nuclear maturation and polar body formation under inverted microscopes. Results: Luveris and Gonal-F enhanced oocyte maturation in vitro in a dose-dependently manner. Culture media supplemented with L or G significantly improved MⅡ Oocyte maturation rates after culture for 24 h and 48 h in comparison with the control group. Oocyte maturation rates were 33.3% and 37.5%(20 IU/mL), individually, 33.3% and 53.6% (40 IU/mL);20 IU/mL G was 15.1% and 16.2%;40 IU/mL G was 38.9% and 39.5%, respectively. Conclusion: LH (Luveris) can promote prophase bovine immature oocytes developmental competence in vitro, which is mostly likely stimulated by FSH(Gonal-F ). Furthermore, LH can delay oocytes GVBD that is very useful for IVF clinical medication guidance.

Research Progress of In vitro Oocyte Maturation

In vitro maturation(IVM)has been used in clinical settings for 30 years.The merits of IVM include that it needs a relatively small amount of hormones and short treatment period.However,because the effectiveness of IVM is lower than that of controlled ovarian hyperstimulation,there are few centers routinely use IVM,and it is only applicable to a few special populations.In this article,several oocyte sources related to IVM have been discussed and the effects of gonadotropin priming and triggering on IVM are described.Furthermore,we have reviewed the optimization of IVM culture conditions in recent years along with the effects of IVM on genes of oocytes and cumulus cells and the obstetric and neonatal outcomes.We aim to provide indications for future improvement of IVM technology so that the success rates of IVM technology in special populations can be improved.We hope that this mild and natural protocol can be applied to more populations,including individuals with normal ovulation.

Kisspeptin stimulates oocyte maturation,and food deprivation modulates the abundance of kisspeptin system in zebrafish gonads

Kisspeptin is a hormone involved in the neuroendocrine control of reproduction in fish.We hypothesized that kisspeptin stimulates oocyte maturation and modulates other reproductive hormones in zebrafish;and the gonadal kisspeptin system in zebrafish is affected during energy unavailability.The main goals of this research were to test in vitro effects of kisspeptin on oocyte maturation and mRNA abundance in zebrafish ovarian follicles and determine how short-term feed restriction affects kisspeptin and its receptors in zebrafish testis and ovary.This study demonstrates the presence of kisspeptin and its receptors in zebrafish(Danio rerio)gonads and its direct action on ovarian follicles in vitro.Kisspeptin(10 ng/mL)induced oocyte maturation,as indicated by germinal vesicle break down at 18-and 24-hours post-treatment.Kisspeptin significantly increased the abundance of mRNAs encoding reproductive hormones and its receptors in zebrafish oocytes.This suggests that kisspeptin-10 affects ovarian functions by modulating other hormones.Reproduction is a process that requires energy.Therefore,whether energy availability affects the kisspeptin system in zebrafish gonads was determined.Food deprivation modulated kisspeptin expression differently in zebrafish testis and ovary.Kiss2 and kiss1ra were upregulated while kiss1rb was downregulated in the testis post-food deprivation.Meanwhile,no changes in kiss in the ovary were found after food deprivation.However,kiss1rb was downregulated in unfed fish at 3-and 7-days post-food deprivation.Overall,our results suggest sex-and tissue-specific changes in the gonadal abundance of the kisspeptin system in zebrafish.The fine tuning of reproduction during energy fluctuations in fish is likely mediated via changes in hormones,including kisspeptin as shown in this research.