Influence of Long Non-Coding RNAs on Human Oocyte Development.

Non-coding RNAs (ncRNAs) have long been regarded as transcriptional noise due to their lack of biological function. Nevertheless, mounting evidence supports the notion that long non-coding RNAs (lncRNAs) exhibit a diverse array of biological functions and participate extensively in RNA epigenetic regulation. These molecules have been identified as significant players in both normal human development and disease processes. LncRNAs can regulate signaling pathways or interact with miRNAs and mRNAs to regulate oocyte development and maturation. By influencing ovarian granulosa cell (GC) function, certain lncRNAs actively participate in physiological conditions and pathological processes associated with ovum development such as human oocyte maturation, fertilization, embryonic development, tumorigenesis, 1 and premature ovarian failure. 2 , 3 In this paper, we review the research on the influence of lncRNAs on the development of human oocyte.

NcRNAs can be divided into two main categories based on their length: small ncRNAs (less than 200 nucleotides; such as microRNA, piRNA, and snoRNA) and lncRNAs (more than 200 nucleotides). Through polyadenylation and RNA polymerase II catalysis, lncRNAs carry out a variety of biological tasks in both the nucleus and cytoplasm. 12 , 13 LncRNAs are a category of non-protein-coding RNA molecules that modulate the expression of target genes at both transcriptional and post-transcriptional levels. Many lncRNAs modulate the chromatin state by recruiting chromatin-modifying enzyme proteins to specific locations in genes. 14 By employing cis-regulation mechanisms that target specific genes, lncRNAs also impact the transcription and expression of genes located in close proximity. They also function as co-regulators to modulate transcription factor activity. Numerous studies have demonstrated interactions between various types of RNA molecules, such as lncRNAs and miRNAs, miRNAs and mRNAs, and lncRNAs and mRNAs. 15 The interactions among these molecules collectively create a dynamic regulatory network. They bind to microRNAs (miRNAs) to modulate the expression of messenger RNAs (mRNAs), consequently diminishing the miRNAs’ capacity to bind to mRNA targets. 16 , 17 LncRNAs have been discovered in a stable form in many tissues and body fluids, 18–20 and are widely involved in the regulation of gene expression networks, such as chromosome imprinting, cell growth, and tumorigenesis. 21–24 Research has shown that lncRNAs are expressed in oocytes and early embryos. Moreover, as the embryonic genome becomes active during the initial stages of human embryonic development, the expression levels of these lncRNAs undergo dynamic alterations. 25 , 26 LncRNAs are involved in various biological and developmental processes during early embryonic development. They are involved in the induction and maintenance of cell pluripotency, X chromosome inactivation, and gene imprinting. 27 In the regulatory process, several lncRNAs, including Xist, Tsix, and Xite, ensure the dosage regulation of X-expressed genes. 28 , 29 The expression levels of some lncRNAs in GCs are linked to the quality of oocytes and early embryos, and may play a variety of roles in early embryonic development. Consequently, these lncRNAs have the potential to serve as biomarkers for non-invasive prediction of oocyte development.

In patients with PCOS, the transcription level of lncRNA PWRN2 was significantly lower in the GCs of mature oocytes in comparison with immature oocytes. 43 Knocking out PWRN2 in KGN cells resulted in up-regulation of 118 lncRNAs and down-regulation of 58 lncRNAs. The results of the gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that PWRN2-regulated genes are engaged in a variety of metabolic processes. Several studies have identified specific lncRNAs that are up-regulated in the GCs of patients with PCOS. These lncRNAs have been found to regulate miRNAs, thereby influencing cellular development processes ( Table 1 ). 43–52 In individuals with PCOS, whether presenting with or without hyperandrogenism, the expression of lncRNAs exhibited significant alterations when compared to the normal control group. Notably, among these changes, lncRNA CRHBP displayed the most pronounced alteration in expression. The GO and KEGG pathway analysis revealed that CRHBP plays a crucial role in mitochondrial function by interacting with transcription factors YY1 and SIX5. 53 Few individual lncRNAs have been identified in the GCs of patients with PCOS, most of which are involved in the proliferation and apoptosis of GCs ( Table 2 ). 54–60 It can be seen that the dysfunction of lncRNAs may be related to abnormal follicles and hyperandrogen. Table 1 Up-Regulated lncRNAs That Regulate miRNA in GCs of Patients with PCOS LncRNA Regulated miRNA Functions Reference PWRN2 miR-92b-3p Influence oocyte nuclear maturation in PCOS [ 43 ] PVT1 miR-17-5p Regulate the proliferation and the apoptosis of GCs [ 44 ] ZFAS1 miR-129 Prevent the degradation of HMGB1 and affect the endocrine disorder, proliferation and apoptosis of GCs [ 45 ] NEAT1 miR-16, miR-483 and miR-324-3p Promote cell proliferation and inhibit cell apoptosis [ 46 ] HOTAIRM1 miR-433-5p Promote PIK3CD expression, thereby regulating the growth and apoptosis of GCs [ 47 ] PLAC2 miR-19a Participate in the occurrence of PCOS [ 48 ] XIST miR-30c-5p Inhibit cell proliferation and inducing apoptosis, and is associated with adverse pregnancy outcomes. [ 49 ] H19 miR-19b Regulate the proliferation and apoptosis of GCs [ 50 , 51 ] CDKN2B-AS1 miR-181a Affect the proliferation of GCs [ 52 ] Table 2 LncRNAs in GCs of Patients with PCOS LncRNA Up/Down regulation Functions Reference HCG26 Up Affect cell proliferation and cell cycle progression along with increased aromatase gene (CYP19A1) expression and estradiol production [ 54 ] BANCR Up Promote the expression of pro-apoptotic markers Bax and P53 to inhibit cell proliferation and induce cell apoptosis [ 55 ] HUPCOS Up Induce the excess of androgens in follicular fluid in PCOS patients [ 56 ] TUG1 Up Inhibit apoptosis and autophagy of GCs by ERK/MAPK pathway, thus leading to antral follicle increase [ 57 ] HAS2-AS1 Up Induce the hypermethylation in the promoter region of TGFBR2 to inhibit TGF-β signaling [ 58 ] MAP3K13-7:1 Up Resulted in cell cycle arrest at G0 / G1 phase and inhibit GCs proliferation [ 59 ] OC1 Up Affect the cell viability, apoptosis and production of estradiol [ 60 ] Up-Regulated lncRNAs That Regulate miRNA in GCs of Patients with PCOS LncRNAs in GCs of Patients with PCOS In patients with PCOS, the expression of LncRNA MALAT1 is reduced. Studies have confirmed that MALAT1 inhibits TGFβ signaling and causes cell cycle disruption by acting as a competitive endogenous RNA (ceRNA) and interacting with miR-125b and miR-203a. 61 Li et al also discovered that MALAT1 was down-regulated in GCs of patients with PCOS. MALAT1 bound to MDM2 and inhibited ubiquitination and degradation of p53, thereby increasing p53 protein levels. Knockdown of MALAT1 could increase apoptosis and decrease proliferation in KGN and primary GCs. 62 Contrarily, Tu et al reached a distinct conclusion, observing the up-regulation of MALAT1 in GCs from patients with PCOS. Additionally, they noted the up-regulation of both MALAT1 and phosphorylated SMAD 1/5 (Ser463/465) proteins following anti-Müllerian hormone (AMH) stimulation of KGN cells. Inhibition of MALAT1 can promote the proliferation of KGN cells in vitro. 63 In summary, based on these studies, it is evident that lncRNAs play significant roles in regulating cell proliferation and apoptosis in GCs of patients with PCOS. A notable down-regulation of lncRNA MALAT1 was observed in GCs of patients diagnosed with endometriosis. This down-regulation was found to be correlated with the number of antral follicles. Furthermore, the expression level of MALAT1 was significantly lower in cancer tissues from patients with endometriosis-associated infertility compared to the normal group. 64 In comparison to healthy controls, paraffin blocks of patients with endometriosis had up-regulated levels of lncRNA SNHG4 expression. 65 Most of the differentially expressed lncRNAs of patients were associated with the number of oocytes retrieved, the number of 2PN zygotes, and the number of embryos in the cleavage stage. These results suggest that the abnormal expression of lncRNAd is not only related to follicular development, but also to subsequent fertilization and embryonic development. A total of 37 mRNAs, 51 lncRNAs, and 101 circRNAs were detected to be differentially expressed in GCs of patients with ovarian endometriosis. A network comprising lncRNA-miRNA-mRNA interactions, primarily governing cell differentiation and the cell cycle, was constructed through the analysis of 10 differentially expressed lncRNAs and 22 mRNAs. 66 MUC20-OT1 was also discovered to be highly correlated with the expression of PRKAR2A, GPC6, SMC5, EIF3A, ITGAV, and PGRMC2. PGRMC2 was closely linked to the expressions of AC123912SMC5, EIF3A, PRKAR2A, ITGAV, and PGRMC2. LERFS was co-expressed with SMC5, PRKAR2A, ITGAV, and PGRMC2. 64 LncRNAs interact with miRNAs and mRNAs to regulate the expression of target genes to regulate oocyte development. In POI, the cortical tissue of ovaries contained 20 lncRNAs with variable expression, several of which were co-regulated. 67 Patients with limited ovarian reserve (DOR) had 244 up-regulated and 222 down-regulated lncRNAs in their GCs. According to bioinformatics analysis, “cell adhesion and apoptosis”, “steroid biosynthesis”, “immune system”, and other biological processes were considerably enriched in the differentially expressed lncRNAs genes. 68 Patients with DOR had lower expression of lnc-GULP1-2:1, but patients with PCOS had significantly higher reserve. 69 Duan et al discovered 78 lncRNAs differentially expressed in GCs of patients with premature ovarian failure (POF), among these, the significantly down-regulated lncRNA ZNF674-AS1 was positively correlated with serum basal estradiol and AMH levels, and negatively correlated with basal FSH levels. 70 H19 was moderately positively correlated with AMH in serum. H19 expression was 3.7-fold higher in GCs of patients with high ovarian response than those with low ovarian response. 71 It can be seen that the dysfunction of lncRNAs in GC may be related to abnormal follicular development. Some lncRNAs found in patients’ GCs have been found to regulate or interact with miRNAs, and they mainly regulate cell proliferation, apoptosis, cell cycle, and ovarian function ( Table 3 ). 69 , 70 , 72–77 Table 3 LncRNAs in GCs of Patients with Ovarian Insufficiency LncRNA Up/Down Regulation Regulate or Interact with miRNA or protein Functions Reference GULP1-2:1 Down COL3A1 Affect the proliferation of GCs [ 69 ] ZNF674-AS1 Down PCNA and ALDOA Inhibit glycolysis and proliferation of GCs. [ 70 ] DLEU1 Up miR-146b-5p Promote cell proliferation [ 72 ] BBOX1-AS1 Up miR-146b Affect the cell apoptosis [ 73 ] HCP5 Down YB1 Regulate the transcription of MSH5 and DNA damage repair [ 74 ] PVT1 Down Foxo3a Promote the apoptosis of GCs [ 75 ] DANCR Down hNRNPC-p53 interaction Inhibit cell growth and cause a G1 arrest in the cell cycle [ 76 ] H19 Down Let-7 Regulate AMH expression [ 77 ] In POI, the cortical tissue of ovaries contained 20 lncRNAs with variable expression, several of which were co-regulated. 67 Patients with limited ovarian reserve (DOR) had 244 up-regulated and 222 down-regulated lncRNAs in their GCs. According to bioinformatics analysis, “cell adhesion and apoptosis”, “steroid biosynthesis”, “immune system”, and other biological processes were considerably enriched in the differentially expressed lncRNAs genes. 68 Patients with DOR had lower expression of lnc-GULP1-2:1, but patients with PCOS had significantly higher reserve. 69 Duan et al discovered 78 lncRNAs differentially expressed in GCs of patients with premature ovarian failure (POF), among these, the significantly down-regulated lncRNA ZNF674-AS1 was positively correlated with serum basal estradiol and AMH levels, and negatively correlated with basal FSH levels. 70 H19 was moderately positively correlated with AMH in serum. H19 expression was 3.7-fold higher in GCs of patients with high ovarian response than those with low ovarian response. 71 It can be seen that the dysfunction of lncRNAs in GC may be related to abnormal follicular development. Some lncRNAs found in patients’ GCs have been found to regulate or interact with miRNAs, and they mainly regulate cell proliferation, apoptosis, cell cycle, and ovarian function ( Table 3 ). 69 , 70 , 72–77 LncRNAs in GCs of Patients with Ovarian Insufficiency High-throughput sequencing was used to analyze the differences in lncRNAs in GCs from high- and low-risk women with OHSS. A total of 23,815 lncRNAs were discovered, of which 482 were differentially expressed, 205 were up-regulated and 277 were down-regulated. Among the 7 lncRNAs with the most obvious differential expression, lnc-SEC16B.1–6, lnc-SNURF-13, lnc-LGR6-6, lncH2AFY2-2 were up-regulated, while lnc-BRD2-2, lnc-HSPA6-2, lnc-CLIC6-5 were down-regulated. 78 So, lncRNA may be involved in the occurrence of OHSS.

Several lncRNAs participate in multiple biological processes and their dysregulation frequently causes disease. According to recent studies, most regulatory lncRNAs interact with biological macromolecules such as DNA, RNA, and proteins. LncRNAs primarily regulate gene expression through DNA methylation, histone modification, chromatin remodeling, including epigenetic modification, transcription, and post-transcription. Studies on lncRNAs and oocyte development have resulted in positive findings. LncRNAs have the potential to influence GCs, ovaries, and disease development through diverse pathways. Consequently, they may find utility in the diagnosis and treatment of various diseases.

Oocytes are the largest cells in the body, and the complex process of oocyte development is called oogenesis. This complex process involves numerous interactions between the oocyte and surrounding granulosa and cumulus cells. The four stages of follicle development are: original follicle, primary follicle, secondary follicle, and mature follicle, which span over a period of several months. During the early phases of follicular development, the oocyte undergoes enlargement, and granulosa and cumulus cells (GCs) begin to surround the oocyte. As cytoplasmic content increases within oocytes during this phase, proliferation and differentiation of GCs drive formation of sinus cavities within initial follicles. Follicle-stimulating hormone (FSH) stimulates the development of these structures from early-stage sinus to pre-ovulation stage. With the formation of the follicular antrum, the GCs gradually differentiate into two different types: mural GCs (MGCs), which are distributed around the follicular antrum, and cumulus cells (CCs), which directly envelop the oocyte. MGCs predominantly perform ovarian secretory hormone/cytokine functions while regulating oocyte development through autocrine/paracrine mechanisms. In addition to oocytes, granulosa cells are the most important type of cells in the follicle. There is extensive communication between GCs and oocytes, and they work together as a single functional unit. The secretory function of GCs can be influenced by apoptosis or a decline in quality, subsequently affecting oocyte development and the quality of subsequent embryos. 4 In addition, maturation signaling for oocyte meiosis is accomplished through oocyte-granule intercellular junctions. 5 , 6 The expression of GC genes has also been linked in numerous studies to different therapeutic outcomes such as oocyte maturation, fertilization, embryo development, implantation, and pregnancy. 7–9 The transcriptomes of GCs have also been investigated to determine gamete quality 10 and to explore the possibility of clinical application for infertility treatment. 11 LncRNAs in GCs play a significant role in normal folliculogenesis, but they also alter in function and expression in response to pathological situations. Therefore, GCs can be selected as a noninvasive indicator of oocyte competence and ovarian function.