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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 2  |  Page : 84-88

Expression of target genes in cumulus cells derived from human oocytes with and without blastocyst formation


1 State Key Laboratory of Reproductive Medicine, Clinical Center for Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing 211166, China
2 Center for Reproductive Medicine, Shanghai 10th People's Hospital of Tongji University, Shanghai 200072, China
3 State Key Laboratory of Reproductive Medicine, Clinical Center for Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing 211166; Center for Reproductive Medicine, Shanghai 10th People's Hospital of Tongji University, Shanghai 200072, China

Date of Submission02-Feb-2019
Date of Web Publication9-Jul-2019

Correspondence Address:
Prof. Ri-Cheng Chian
Center for Reproductive Medicine, Shanghai 10th People's Hospital of Tongji University, 301 Yanchang Road, Shanghai 200072
China
Yu-Gui Cui
Clinical Center for Reproductive Medicine, State Key Laboratory of Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 211166
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.262387

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  Abstract 


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.

Keywords: Cumulus Cells; Developmental Competence; In vitro Maturation; Oocyte Quality; Oocyte


How to cite this article:
Zhou TP, Zhang D, Cai LB, Xu YX, Shao L, Liu KL, Liu JY, Cui YG, Wang L, Chian RC. Expression of target genes in cumulus cells derived from human oocytes with and without blastocyst formation. Reprod Dev Med 2019;3:84-8

How to cite this URL:
Zhou TP, Zhang D, Cai LB, Xu YX, Shao L, Liu KL, Liu JY, Cui YG, Wang L, Chian RC. Expression of target genes in cumulus cells derived from human oocytes with and without blastocyst formation. Reprod Dev Med [serial online] 2019 [cited 2019 Jul 15];3:84-8. Available from: http://www.repdevmed.org/text.asp?2019/3/2/84/262387




  Introduction Top


Assisted reproductive technologies (ARTs), especially in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), have helped millions of infertile couples to have healthy babies. In conventional IVF or ICSI treatment, gonadotropins are administered to stimulate the ovaries, with the goal of generating many oocytes and therefore more embryos available for transfer. It is commonly thought that transferring more embryos will increase the chance of pregnancy. Although transferring several embryos in one treatment cycle does increase the rate of live births, it is also more likely to result in multiple births, which increases the risk of pregnancy loss and low-body-weight newborns. Therefore, elective single-embryo transfer is desirable to reduce the chance of multiple births. This approach requires the production of a high-quality embryo from developmentally competent oocytes.

Although ARTs have improved over the decades, treatment efficiency still needs to be optimized. Even with best-prognosis donor oocytes, the live birth rates by oocyte retrieval and embryo transfer are 7.3% and 24.6%, respectively.[1] The most important factor for the success of IVF and ICSI treatments is embryo quality, which primarily depends on oocyte quality. Currently, oocyte developmental competence is assessed using morphological characteristics.[2] However, these criteria may not accurately predict oocyte developmental competence after fertilization. If the developmental competence or quality of oocytes can be assessed before insemination, it could enhance IVF and ICSI treatment efficiency in terms of pregnancy and live birth rates.

Communication between oocytes and the surrounding cumulus cells (CCs) is critical for oocyte maturation and developmental competence.[3],[4] CCs are considered target cells for evaluating oocyte quality and developmental competence.[5],[6] Interestingly, a previous study reported that the difference in SPSB2 and TP53I3 expression in human CCs from normal and chromosomally abnormal oocytes was highly significant,[7] suggesting that both genes might potentially serve as noninvasive markers of oocyte aneuploidy. In addition, it has been suggested that the expression of some specific genes in human CCs may be used to select oocytes or predict oocyte embryonic developmental competence in patients undergoing ICSI treatment,[8],[9],[10] but the results were inconsistent and could not be verified due to the limited size of samples. Moreover, some reports have investigated the gene expression differences between oocyte-surrounding CCs with high or low developmental competence using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and microarray analysis, but these studies failed to exclude other confounding factors.[11],[12]

In our previous microarray analysis, we found significant changes in gene expression in mouse CCs from the germinal vesicle and metaphase-II stages and some potential target genes were selected.[13] Although real-time PCR confirmed the validity and reproducibility of the differential expression of these selected genes, it was unclear whether these genes could be used as biomarkers of human oocyte developmental competence following fertilization. Recently, we used next-generation RNA sequencing (RNA-seq) to profile each in vitro-matured (IVM) mouse oocyte following IVF to identify which oocytes developed to the blastocyst stage and then grouped the CCs based on the developmental competence of their corresponding oocyte.[14] RNA-seq yielded approximately 60,000 detected transcripts, but only 103 and 97 were significantly upregulated and downregulated, respectively, in the CCs associated with the oocytes that developed to the blastocyst stage. After comparison with the positive reference group, nine genes with the most significantly changed transcript profiles were selected: Arrb1, Atp2c1, Cdh5, Cntnap1, Mkln1, Lgr4, Rhobtb1, Smc2, and Six2. qRT-PCR confirmed upregulation of all nine genes. Furthermore, we confirmed that the expression of Arrb1, Atp2c1, Cdh5, Mkln1, Lgr4, Rhobtb1, and Smc2 increased significantly in the CCs associated with the mouse oocytes that developed to the blastocyst stage in the study.

In the present study, we compared the expression profiles of the nine selected genes in human CCs following ICSI to identify which oocytes developed to the blastocyst stage, in order to confirm that these differentially expressed genes were related to human oocyte quality and developmental competence.


  Methods Top


Patients

Twenty-nine women undergoing ICSI due to male factor infertility signed informed consent forms and were enrolled in this study at the Clinical Center for Reproductive Medicine (CCRM) of the First Affiliated Hospital of Nanjing Medical University, China. The patient management and research procedures were approved by the Ethics Committees of the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital), with the file code of 2016-SR-028. The patients were between 24 to 38 years of age with an average of 31 years, and their body mass index ranged from 17.9 to 24.9. Patients were excluded if they had a history of endometriosis, polycystic ovary syndrome, uterine fibroids, or ovarian cysts.

Culture media and reagents

The embryo culture media were from Cook Company (Australia). Oligo-dT and real-time PCR kits were from Takara (Japan), the RNeasy Mini Kit and Reverse Transcription Kit were from Qiagen (USA), and RNAlater was from Ambion (USA).

Collection of cumulus cells with embryo development tracking

Conventional ovarian stimulation was performed in each patient, and oocyte retrieval was performed 36 h after HCG injection. The collected cumulus–oocyte complexes (COCs) were cultured in an incubator for at least 1 h before the oocyte was denuded from the CCs. Each COC was placed into a 35-μL droplet of hyaluronidase solution (80 U/mL), which was prewarmed at 37°C for 30 min. The COC was pipetted gently several times using a fine pipette until the oocyte was denuded completely from the CCs. CCs from each mature oocyte were collected under a microscope, transferred into a labeled PCR tube separately, and then centrifuged at 6,000 ×g for 10 min. The supernatant was then discarded, 15 μL RNAlater was added to the tube, and the samples were stored at −80°C after adequate mixing. The denuded mature oocytes were rinsed and placed into labeled droplets of fertilization medium prior to ICSI. After ICSI, each oocyte was cultured individually in a 10-μL droplet of fertilization medium covered with mineral oil at 37°C in a trigas incubator.

Sixteen to eighteen hours after ICSI, each fertilized oocyte (with 2 pronuclei, 2PN) was transferred to 10 μL cleavage medium for culture. On day 3, according to the patient's demands, the cleaved embryos that needed to be cultivated to blastocyst stage were transferred to blastocyst media and cultured until day 5 or day 6.

Although a total of 340 COCs were collected from 29 patients, only 131 individual oocytes were available to be tracked for blastocyst development. The collected CCs derived from oocytes that developed to the blastocyst stage (n = 70) were defined as group B+, and those derived from oocytes that did not develop to the blastocyst stage (n = 61) were defined as group B−.

RNA extraction and quantitative polymerase chain reaction for nine target genes

Total RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. Three extractions were performed for each group, and the CCs from 10 COCs were pooled in one extraction. RNA quality was assessed using a NanoDrop ND-1000 spectrophotometer, and then RNA samples were reverse transcribed using the Sensiscript Reverse Transcription Kit (#205211) according to the manufacturer's instructions. Reverse transcription was performed at 37°C for 1 h. The generated cDNAs were used as templates in q-PCR reactions to quantify the selected candidate genes.

Real-time PCR was performed using SYBR Premix ExTaq (2X), 0.8 μL each of forward and reverse primer (5 pmol/μL), 0.4 μL ROX, and 1 μL DNA template in a total volume of 20 μL; the following two-step PCR amplification protocol was used: 95°C for 10 s, followed by 60°C for 31 s, for a total of 40 cycles. The specific primer sequences are listed in [Table 1], and GAPDH was used as an internal reference. The amplification curves and melting curves were analyzed, and the expression of the selected genes was normalized to GAPDH expression and calculated using the 2−ΔΔCt method.
Table 1: Specific primer sequences used in this study

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Statistical analyses

Data were analyzed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA), and mRNA expression levels of the target genes are presented as the mean ± standard deviation. Statistical significance was evaluated using an independent-samples t- test, and P < 0.05 was considered statistically significant.


  Results Top


The expression levels of the nine target genes in human CCs derived from the oocytes that developed to the blastocyst stage (B+) and the oocytes that did not develop to the blastocyst stage (B−) are shown in [Figure 1]. The mRNA levels of ARRB1 (P = 0.016), LGR4 (P = 0.025), and SMC2 (P = 0.013) were significantly lower in the B+ group than that in the B− group. However, there were no significant differences in the mRNA levels of ATP2C1, CNTNAP1, CDH5, MKLN1, RHOBTB1, and SIX2 between the two groups.
Figure 1: Expression of nine target genes in cumulus cells derived from oocytes that developed to the blastocyst stage and oocytes that did not develop to the blastocyst stage. *The mRNA levels of ARRB1 (P = 0.016), LGR4 (P = 0.025), and SMC2(P = 0.013) were significantly lower in the B+ group than that in the B− group. However, there were no significant differences in the mRNA levels of ATP2C1, CNTNAP1, CDH5, MKLN1, RHOBTB1, and SIX2 in the blastocyst and nonblastocyst groups.

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  Discussion Top


Each oocyte is surrounded by a group of CCs, and these cells provide energy substrates for oocyte metabolism during the maturation process and support oocyte developmental competence following fertilization. Bidirectional communication between the oocyte and the surrounding CCs is essential for oocyte maturation and development. Therefore, many genes expressed in CCs have been considered as potential biomarkers for predicting oocyte developmental competence.[15],[16],[17] Such genes are involved in many important cellular events, including the cell cycle, growth factor signaling, extracellular matrix production, metabolism, apoptosis, and signal transition.[18],[19],[20] A previous analysis compared the genomic differences in human CCs according to the ability of oocytes to reach the blastocyst stage.[21] Three genes were selected as potential biomarkers, and only RGS2 (regulator of G-protein signaling 2) was associated with oocyte developmental competence in a generalized linear mixed model. However, the results are inconsistent, and these studies failed to exclude other confounding factors.[22],[23],[24] A study by Yerushalmi et al.[25] investigated the transcriptome of human CCs during IVM via RNA-seq, but they focused on the changes between immature and mature CCs in a limited sample size. A recent RNA-seq study on the links between corona cell gene expression and oocyte competence revealed enriched transcript levels of genes involved in Wnt signaling, MAPK signaling, ATP generation, and focal adhesion,[26] which is consistent with the results of our previous study.[14] Although some RNA-seq or miRNA sequencing studies have investigated intrafollicular somatic cells,[27],[28],[29] to the best of our knowledge, our previous study is the only one that used next-generation RNA-seq to study the entire transcriptome of mouse CCs in the context of blastocyst-forming ability.[14] Based on that study, nine genes with the most significantly changed transcript profiles (ARRB1, ATP2C1, CDH5, CNTNAP1, MKLN1, LGR4, RHOBTB1, SMC2, and SIX2) were selected for the present study as potential markers for predicting oocyte developmental competence. Our results in this study support that the expression levels of ARRB1, LGR4, and SMC2 mRNAs in human CCs could be potential markers of oocyte quality and developmental competence.

The special blastocyst formation tracking system with human CCs was used in this study, and the corresponding oocytes were used to demonstrate significant differences in the mRNA levels of ARRB1 (P = 0.016), LGR4 (P = 0.025), and SMC2 (P = 0.013) in CCs from the B+ and B− groups [Figure 1]. Due to understandable limitations in protein sample volume, it is very difficult to directly measure the protein levels of these target genes in human CCs. ARRB1, LGR4, and SMC2 are relevant to the G protein-coupled receptor (GPCR) or Wnt signaling pathway. ARRB1 is a member of the arrestin protein family, which is thought to regulate the agonist-mediated desensitization and internalization of GPCRs. This multivalent adaptor protein displays broad specificity for GPCRs. Thus, ARRB1 is involved in the cellular responses to certain hormones and growth factors such as insulin.[30] Furthermore, ARRB1 can decrease cAMP levels, indicating that it may play a critical role in oocyte development.[23],[30] ARRB1 was reported to be required for MAPK activation as a signaling scaffold,[31] regulate gene transcription via its nuclear function,[32] and participate in metabolic reprogramming.[33],[34]

LGR4 encodes a GPCR in the secreted R-spondin (RSPO1-4) family. LGR4 is a functional activator of the canonical Wnt/β-catenin signaling pathway and is required for the development of various organs.[35],[36] Within the leucine-rich repeat-containing GPCR family, LGR4 and LGR5 are global markers of many stem cells and progenitor cells.[37] LGR4 also has high homology with the follicle-stimulating hormone and luteinizing hormone receptors, and it is abundantly expressed in both male and female reproductive organs.[38],[39] LGR4 conditional knockout mice are subfertile, and LGR4 regulates the expression of steroidogenic receptors in female rats.[40],[41] In addition, LGR4 was recently shown to have a surprising role in triggering the formation of long actin-rich, cytoneme-like membrane protrusions.[42] Cytonemes are a type of filopodium that provide a platform for the exchange of signaling proteins between cells. The functions of cytonemes were elucidated in sea urchin embryos and the Drosophila germarium, and a recent study demonstrated actin-based filopodial transport of Wnt8a during vertebrate tissue patterning.[43]

The SMC2 protein is a subunit of chromosome condensation complexes. Condensin is not only essential for cell division but also critical for DNA repair in mammals.[44],[45] The accumulation of DNA damage is widely thought to be associated with poor oocyte quality.[46] A study revealed a novel transcriptional regulation of SMC2 by Wnt signaling, in which β-catenin is coupled on the SMC2 promoter.[47]

In conclusion, we found significantly lower mRNA levels of the ARRB1, LGR4, and SMC2 target genes in human CCs corresponding to oocytes that developed to the blastocyst stage compared to their levels in CCs from oocytes that did not develop to blastocyst after fertilization via ICSI. Therefore, the expression of these three target genes (ARRB1, LGR4, and SMC2) in human CCs could be related to blastocyst formation. Determining the expression of these target genes in human CCs may also provide information for predicting oocyte developmental competence prior to insemination, leading to enhanced implantation rate during IVF infertility treatment.

Acknowledgments

A special thanks to the embryology laboratory members and Mr. Chao Gao and Ms. Li Gao for their assistance during the sample collection and laboratory work at CCRM, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.

Financial support and sponsorship

Supported by The Key Project from Chinese Technology Department (Project No. 2017YFC1001601).

Conflicts of interest

There are no conflicts of interest.



 
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