Effects of Cumulus Cells on in vitro Fertilization of Bovine

[Objective] The aim was to explore the effect of cumulus cells on the in vitro fertilization of in vitro matured bovine oocytes. [Method] The in vitro matured oocytes were divided into three groups of cumulus cells removal, partial removal and no removal. [Result] In the co-culture with cumulus cells, the oocytes of the removal group had higher cleavage rate and blastocyst rate （74.4%±4.1, 53.7%±5.1） than those of the no removal group （72.7%±5.1, 52.4%±3.5）, but the difference was not significant （P〉0.05）, while both groups had better performances than the re- moval group （39.6%±4.5, 18.8%±4.6） with the difference reaching the significant level （P〈0.05）. All the three groups showed significant difference with the control. The combination of cumulus cells and melatonin achieved the best effects as the cleavage rate and blastocyst rate of the partial removal group （79.8%±3.7, 56.5%±5.1） were better than those of the no removal group （78.2%±2.6, 55.8%±4.6）, and the difference was not significant, while both group had better performances than the removal group （48.3%±5.5, 22.7%±4.3） and the control group with the differences reaching the significant level （P〈0.05）. [Conclusion] The study provided technical support for the production of dairy cows and beef cattle.

Heat exposure impairs porcine oocyte quality with suppressed actin expression in cumulus cells and disrupted F-actin formation in transzonal projections

Background:Transzonal projections(TZPs)constitute a structural basis for the communication between the oocyte and its surrounding cumulus cells(CCs),which play critical roles in promoting the oocyte maturation.Previously we found that heat stress(HS)causes loss of TZPs in porcine cumulus-oocyte complexes(COCs)with decreased density of filamentous actin(F-actin).However,the time-course responses of F-actin and its monomeric actins(β-actin andγ-actin)during the in vitro maturation of oocytes remain unclear.Results:In this study,excised porcine ovaries were exposed to HS at 41.5°C for 1 h before COCs were isolated and matured in vitro for 44 h.HS significantly reduced oocyte quality,characterized by impaired cumulus expansion,delayed meiotic resumption and lower survival rate and polar body extrusion rate,as well as decreased expression of mitochondrial DNA-encoded genes and elevated mitochondrial reactive oxygen species concentration.Expression ofβ-actin andγ-actin in CCs increased gradually with oocytes maturation,which was significantly reduced in HS group,especially at 24 h and/or 44 h of in vitro maturation.By contrast,the number of TZPs and the fluorescence intensity of F-actin in zona pellucida decreased gradually during oocytes maturation,which were significantly reduced by HS at 24 h of in vitro maturation.Moreover,colocalization analyses revealed bothβ-actin andγ-actin contribute to the F-actin formation in porcine TZPs,and the colocalization of F-actin with GJ protein connexin 45 was significantly reduced in heat-exposed COCs.Conclusions:The results indicate that the suppression of actin expressions in CCs,which may lead to the F-actin unstabilization in TZPs,will subsequently contribute to the compromised quality of oocytes under HS.

Expression of Target Genes in Cumulus Cells Derived from Human Oocytes with and without Blastocyst Formation

Objective:The transcriptional profile of cumulus cells(CCs)during oocyte maturation provides information for predicting oocyte developmental competence.Our previous study using a mouse model indicated that there were nine different genes related to oocyte development potential expressed in CCs during oocyte maturation.The purpose of this study was to elucidate whether gene expression levels of CCs during oocyte maturation are associated with oocyte developmental competence.Methods:The human CCs collected from each oocyte were divided into two groups after tracking depending on whether or not they developed to the blastocyst stage:(1)the oocytes were developed to blastocyst stage after fertilization(B+)and(2)the oocytes were not developed to blastocyst stage after fertilization(B−).The expression levels of the nine selected genes(ARRB1,ATP2C1,CDH5,CNTNAP1,LGR4,MKLN1,RHOBTB1,SIX2,and SMC2)were examined.CCs were obtained from 29 women who were undergoing intracytoplasmic sperm injection treatment cycles.Quantitative reverse transcriptase-polymerase chain reaction analysis was performed on cumulus masses collected before insemination.Each sample was run three times.Statistically significant differences in mRNA expression of the target genes in independent samples were evaluated by two-tailed Student’s t-test,and P<0.05 was considered significantly different.Results:There were significant differences in the mRNA expression levels of ARRB1(P=0.016),LGR4(P=0.025),and SMC2(P=0.013)between the groups B+and B−.Gene expression of ARRB1,LGR4,and SMC2 in CCs is related to blastocyst development.Conclusions:Analysis of expression of ARRB1,LGR4,and SMC2 genes in CCs as biomarkers may provide predictive information on oocyte developmental competence before insemination and fertilization.

The Cumulus Cells and Oocytes: A Systematic Review of Extended Culture for Intracytoplasmic Sperm Injection

Currently-placed protocols for extended culture for in vitro fertilization(IVF)and intracytoplasmic sperm injection(ICSI)are not uniformly standardized in determining the optimal stage of oocyte maturation for maximizing clinical outcomes.The objective of this systematic review is to elucidate the relationship between extended cumulus-oocyte culture and its effect on the clinical outcomes of IVF/ICSI.We included an electronic search on PubMed Central as well as the Journal of Fertility and Sterility to yield seven studies on extended oocyte culture for IVF/ICSI.Four of the seven investigations illustrate the promising beneficial relationship of extended culture with conditioned or supplemented media to mimic physiological uterine conditions.Three studies did not capture beneficial relationships between extended oocyte culture and clinical outcomes with unconditioned,unsupplemented maturation medium.Improvement in fertilization rates,oocyte development,and live birth rates may be achieved by extended culture with the addition of supplemental biochemicals.The usage of follicular fluid,cumulus cells,and meiotic inhibitors imitates the physiological in vivo conditions,whereas extended oocyte culture imitates in vivo temporal conditions.The conjunction of extended oocyte culture with supplemented metabolites,either added in maturation media manually or secreted by cumulus-oocyte complexes,mimics natural uterine physiological conditions to improve clinical outcomes for patients seeking IVF/ICSI.

Cumulus Cells and their Associations with Immune Functions

The recent progress in the association of cumulus cells with immune functions is a largely ignored area. With over 350 million new sexually transmitted infections occurring annually in adults of reproductive age, we feel the need to explore more about how the cumulus cells defend themselves and protect the oocytes during the development through the ovulation period. Application of assisted reproductive technologies allows scientists to study and better understand cumulus cells. There are still many immune factors to be taken into consideration to optimize the oocyte quality besides ovarian stimulations. The objective of this review is to summarize the key elements of cumulus cells and their association with the immune function.

Effect of Mitochondrial Transplantation from Cumulus Granular Cells to the Early Embryos of Aged Mice

Objective To assess the role of mitochondria in the early embryonic development of ageing mice.Methods Mitochondria isolated from cumulus granular cells of aged mice were microinjected into oocytes or zygotes of aged mice. In the setting of oocyte injection, mitochondria were transferred via intracytoplasmic sperm injection （ICSI＋MIT）, and ICSI without mitochondrial transfer. In the setting of zygote injection, mitochondria were directly microinjected into fertilized oocytes （MIT）, and those injected with buffer alone （mock injection） or not injected （uninjected） served as controls.Results Although the rates of oocyte cleavage between ICSI and ICSI＋MIT groups were not statistically different （P〉0.05）, the rate of blastocyst in the ICSI＋MIT group was significantly higher than that in ICSI group （P〈0.05）. Although both the cleavage and blastocyst rates of mock injection group were significantly lower than those of uninjected group （P〈0.05）, likely due to mechanical damages of the cells by microinjection, the decrease of these rates was prevented by mitochondrial transfer. After mitochondrial transfer, the rates of both cleavage and blastocyst were significantly improved over the mock-iniection group （P〈0.05）.Conclusion Mitochondrial transplantation can improve the developmental potential of early embryos of aged mice.

Oocyte Transfer as an Analytical Tool:Vintage Experiments in Domestic Farm Animals

Four transplant studies are described that focus on fertilisation and early development or the progression of unfertilised oocytes （eggs） in the oviduct. （1） Pig eggs transplanted from ovulations induced during the luteal phase of the estrous cycle were fertilised in the oviducts of inseminated recipient animals in estrus. By contrast, pig eggs from donors in estrus became highly polyspermic when transplanted to the oviducts of animals force-mated during the luteal phase. （2） Pig embryos at the stage of hatched blastocysts （ days 7 and 8） could be transplanted successfuUy to synchronous recipients and full embryonic development demonstrated to between days 19 and 23 of pregnancy. Thus, the exposed trophectoderm of developing embryos could withstand the physical ma- nipulation of recovery and transplantation, and the li-fespan of corpora lutea in the unmated recipients could be prolonged by transfer of day 7 and 8 blastocysts. （3） Bovine oocytes aspirated from 2 to 6 mm diameter Graafian follicles and matured in vitro were fertilized normally in the oviducts of inseminated recipient heifers, demonstrating the potential of slaughterhouse ovaries for the generation of embryos. （4） Transplanting equine eggs to a pig oviduct, in which egg descent to the uterus requires only 46 to 48 h, did not reveal a retarded progress of degenerating unferfil- ised horse eggs, suggesting the involvement of nonphysical factors in equine embryo progression to the uterus. Prostaglandins of embryonic origin are now known to be a key. A final section examines the postovulatory role of ovarian follicular cells on the secretory activity of the oviductal epithelium.

Advances of Long Noncoding RNAs-mediated Regulation in Reproduction

Objective： Advances in genomics and molecular biology have led to the discovery of a large group of uncharacterized long noncoding RNAs （lncRNAs）. Emerging evidence indicated that many lncRNAs function in multiple biological processes and its dysregulation otten causes diseases. Recent studies suggested that almost all regulatory lncRNAs interact with biological macromolecules such as DNA, RNA, and protein. LncRNAs regulate gene expression mainly on three levels, including epigenetic modification, transcription, and posttranscription, through DNA methylation, histone modification, and chromatin remodeling. LncRNAs can also affect the development of diseases and tllerefore be used to diagnose and treat diseases. With new sequencing and microarray techniques, hundreds oflncRNAs involved in reproductive disorders have been identified, but their functions in these disorders are undefined. Data Sources： This review was based on articles published in PubMed databases up to July 10, 2017, with the tbllowing keywords： ＂long noncoding RNAs＂, ＂＇LncRNA＂, ＂placentation＂, and ＂＇reproductive diseases＂. Study Selection： Original articles and reviews on the topics were selected. Results： LncRNAs widely participate in various physiological and pathological processes as a new class of important regulatory/＇,actors. In spermatogenesis, spermatocytes divide and differentiate into mature spermatozoa. The whole process is elaborately regulated by the expression of phase-specific genes that involve many strains of lncRNAs. Literature showed that IncRNA in reproductive cumulus cells may contribute to the regulation of oocyte maturation, fertilization, and embryo development. Conclusions： LncRNA has been found to play a role in the development of reproduction. Meanwhile, we reviewed the studies on how lncRNAs participate in reproductive disorders, which provides a basis fbr the study of [ncRNA in reproduction regulation.