| Objectives: Assisted reproductive technology has become an important treatment to address fertility requirements for patients with infertility.Conventional in vitro fertilization(IVF)is a technique in which the oocytes and sperms of a couple with infertility are removed from the body,fertilized,and developed into embryos in the culture system.In vitro maturation(IVM)is an assisted reproductive technology based on traditional IVF,where immature oocytes are removed from sinus follicles,matured in vitro with little or no exogenous gonadotropin stimulation,fertilized,and the embryos are transferred to mother.IVM is primarily indicated for infertile patients with impaired oocyte maturation,particularly those at risk of polycystic ovary syndrome,follicular hypoplasia,and ovarian hyperstimulation syndrome(OHSS).In addition,it is recommended for patients with a contraindication to a persistent high estrogen status and for cancer patients with a desire of fertility preservation.The technique is considered to be a safer,cheaper and,simpler method of treatment.However,the mechanisms of oocyte maturation are not fully understood and IVM technology is not widely accepted.Compared to oocytes matured in vivo,oocytes matured in vitro have lower maturity,developmental capacity,blastocyst formation,implantation,pregnancy,and live birth rates,as well as a higher incidence of abnormal embryo development and embryo arrest.The oocyte microenvironment is critical for oocytes to acquire developmental competence,and inadequate culture conditions are the major reason for reduced oocyte developmental competence.Therefore,the construction of a three dimensional(3D)culture system that mimics the in vivo oocyte development environment to improve oocyte development potential and pregnancy outcomes in vitro will hopefully bring benefits to patients seeking assistance with assisted reproductive technologies.During the development of follicles,oocytes communicate with the surrounding granulosa cells(GCs)through gap junctions and paracrine signals,which play an important role in oocyte metabolism,resumption of meiosis,and fertilization.The connection between oocytes and the surrounding GCs is disrupted during in vitro culture of oocytes,which results in oocytes being deficient in some unknown nutrients and functional signals.One study found that co-culture of GCs with immature oocytes increased oocyte maturation rates,improved the quality of embryos,and altered their gene expression to resemble the gene expression profile of oocytes matured in vivo.Extracellular matrix(ECM)provides structural support for follicles and initiates a variety of intracellular signaling cascades through binding to cell surface integrin receptors,modulating cell proliferation,survival,differentiation,steroid formation,and promoting follicular development and maturation.One study revealed that the major components of ECM,fibronectin and laminin,are capable of facilitating oocyte meiosis.Follicle development is related to the physical pressure and tissue rigidity.Several studies have used materials such as alginate,gelatin,hyaluronic acid,and matrigel to create 3D models of follicles in vitro culture,but these materials are complicated to use,require changes in temperature and p H to create a 3D model,use enzymes to obtain mature follicles,which cannot provide a stable environment for follicle culture thereby affecting follicle development.Hydrogel,due to the balance of biochemical and physical properties,are able to simulate the structure of ECM in vivo,while modifying functional molecules,facilitating the establishment of 3D culture systems for oocytes,which have promising applications in the field of tissue engineering.Therefore,in this study,we established an in vitro follicle culture system based on laminin peptide-modified hydrogel and an IVM 3D culture system using laminin peptide-modified hydrogel and human granulosa cells(h GCs)to determine the effects of laminin peptide-modified hydrogel on the biological functions of h GCs and oocytes,and to initially investigate the molecular mechanisms.Methods:(1)The physico-chemical properties of laminin peptide-modified hydrogel were examined,and the optimal dilution ratio of laminin peptide-modified hydrogel was determined by culturing follicles in vitro to observe their growth and development,detecting the live cell rate and oocyte meiosis,compared with two-dimensional(2D)culture.(2)The in vitro 3D culture system of h GCs was established based on laminin peptide-modified hydrogel.The experimental techniques of flow cytometry,immunofluorescence,PCR,Western Blot,and ELISA were applied to verify the effects of laminin peptide-modified hydrogel on the cell cycle,apoptosis,hormone secretion,cell senescence,oxidative stress,and mitochondrial membrane potential of h GCs in 3D culture,and to preliminarily elucidate the molecular mechanisms.(3)2D culture,Laminin peptide-modified hydrogel 3D culture without h GCs,and Laminin peptide-modified hydrogel-h GCs 3D culture system were constructed to verify the effects of the culture system on the biological functions of oocytes by immunofluorescence staining with fluorescence microscopy and confocal microscopy to observe changes in oocyte morphology,spindle,chromosome,cortical granules,endoplasmic reticulum,oxidative stress levels,and mitochondrial membrane potential.Results: The data of the first part showed that the rate of gelation of the Laminin peptide-modified hydrogel varied according to the different hydrogel dilution ratios(1:3 > 1:5 > 1:7),with a storage modulus of 144 Pa(1:3),63.4 Pa(1:5),and 20.8 Pa(1:7),respectively.The data showed that the highest follicle survival rate,follicle viability,pseudo-antrum follicle formation rate,oocyte maturation rate,and largest diameter were achieved at a dilution ratio of 1:5 for the Laminin peptide-modified hydrogel.By measuring the follicle diameters of each group,the follicle diameters of the 2D group(191 ± 11 μm),the Laminin peptide-modified hydrogel dilution ratio 1:5 group(214 ± 22μm)and the 1:7 group(207 ± 22 μm)were significantly higher than that of the 1:3 group(158 ± 10 μm,p< 0.05)up to day 6,and the follicle diameters of the hydrogel dilution ratio 1:5 group(338 ± 15 μm)were the largest and the smallest in the 1:3 group(180 ±25 μm,p< 0.05)up to day 10.Comparing the survival rates of the four groups,we found that the survival rates of follicles in the Laminin peptide-modified hydrogel dilution ratio1:3 group were significantly lower than those in the other groups(p< 0.05);the survival rates of follicles in the Laminin peptide-modified hydrogel dilution ratio 1:5 and 1:7groups were higher than those in the 2D group by day 6 and 10(p< 0.001).In addition,the rates of live cells and pseudo-antrum follicle formation were higher in the group with1:5 hydrogel dilution ratio up to day 10 than in the remaining groups.Furthermore,the oocyte maturation rates of follicles in the 2D,Laminin peptide-modified hydrogel dilution ratio 1:5 and 1:7 groups after IVM treatment were 14.3%,34.7%,and 18.5%,respectively,with the highest oocyte formation rate in the Laminin peptide-modified hydrogel dilution ratio 1:5 group(p< 0.05).Based on the above results,the Laminin peptide-modified hydrogel dilution ratio of 1:5 provided the most suitable physical environment for follicle growth and development,so all subsequent experiments were conducted using the 1:5 dilution ratio.The data from the second part of the study showed that Laminin peptide-modified hydrogel 3D culture improved the biological function of h GCs.The cell viability assay revealed that the proportion of live cells in 2D cultured h GCs was significantly lower than that in 3D cultured h GCs on days 4 and 6 in vitro(p< 0.01).The cell cycle assay showed that the proportion of S-phase(p< 0.001)and G2/M-phase(p< 0.05)cells was significantly higher in 3D cultured h GCs than in the 2D group.The results of the apoptosis assay showed that the percentage of early apoptotic cells and the expression of apoptosis-related genes caspase3,caspase8,and BAX were significantly lower in 3D cultured h GCs than in 2D cultured h GCs on days 2 and 4(p< 0.05).The hormone secretion was significantly higher in 3D cultured h GCs than in 2D cultured h GCs by day4(p< 0.05).In addition,the rate of senescent cells and the expression of senescence-related proteins P16 and P21 were significantly lower in 3D cultured h GCs than in 2D cultured h GCs(p< 0.05).HGCs in 3D culture had significantly lower levels of oxidative stress and expression of antioxidant-related proteins and genes SOD1 and GPX4(p< 0.01).JC-1 staining and flow cytometry showed that 3D cultured h GCs had weaker JC-1 monomer signals and stronger JC-1 multimer signals,and significantly higher relative mitochondrial membrane potential ratios in 3D cultured h GCs(p< 0.05).Transmission electron microscopy demonstrated that the percentage of abnormal mitochondria in 2D cultured h GCs reached 74.5 ± 21.3% after 48 h of in vitro culture.3D culture significantly reduced the percentage of abnormal mitochondria to 28.54 ±11.4%.Further,3D cultured h GCs showed significantly higher expression of cumulus expansion-related genes and proteins COX2,HAS2,and PTX3 than h GCs cultured in 2D,increased expression levels of TGFβ1,phospho-SMAD3,and phospho-ERK1(p< 0.05),and activation of TGFβ-SMAD3-ERK pathway.The results of the third part of the study indicated that Laminin peptide-modified hydrogel 3D culture increased oocyte nuclear and cytoplasmic maturation rates,did not affect spindle and chromosome arrangement and distribution,reduced oocyte oxidative stress,increased oocyte mitochondrial membrane potential,and increased the normal distribution of cortical granules and endoplasmic reticulum.Compared to the 2D group,oocyte maturation rates were significantly higher in the Laminin peptide-modified hydrogel and Laminin peptide-modified hydrogel-h GCs culture groups(61.1 ± 7.4% vs.72.1 ± 11.0% vs.73.4 ± 10.2%;p< 0.05).In addition,DCFH-DA fluorescent probe staining showed that oocytes in the Laminin peptide-modified hydrogel and Laminin peptide-modified hydrogel-h GCs culture groups had significantly lower levels of oxidative stress than the 2D group(p< 0.05).JC-1 fluorescent probe staining showed that oocytes in the Laminin peptide-modified hydrogel and Laminin peptide-modified hydrogel-h GCs culture groups had weaker JC-1 monomer signal intensity and stronger JC-1 multimer signal intensity,and mitochondrial fluorescence in the Laminin peptide-modified hydrogel 3D and Laminin peptide-modified hydrogel-h GCs culture groups intensity ratio(red/green)was significantly higher(p< 0.001).Furthermore,the distribution of cortical granules and endoplasmic reticulum reflected the cytoplasmic maturation of oocytes.87.0% of the oocytes in the Laminin peptide-modified hydrogel-h GCs culture group had class III cortical granules,which was significantly higher than that of oocytes in the 2D and Laminin peptide-modified hydrogel 3D culture groups(p< 0.05).The proportion of mature endoplasmic reticulum in the oocytes of the Laminin peptide-modified hydrogel-h GCs culture group was 84.6%,which was significantly higher than that of the other groups(p< 0.05),and the spindle and chromosome arrangement and distribution were normal in all three groups.Conclusions: 1.The Laminin peptide-modified hydrogels were stable and provided a durable physical environment suitable for follicle growth and development.2.The Laminin peptide-modified hydrogel 3D culture contributed to the biological function of h GCs and activated the TGFβ-SMAD3-ERK signaling pathway.3.The Laminin peptide-modified hydrogel-h GCs IVM system contributed to the biological function of oocytes and improved the maturation efficiency of oocytes.In this study,we established a 3D culture system for IVM using Laminin peptide-modified hydrogels and h GCs by simulating the environment of oocyte development in vivo,providing a new approach to improve the utilization of immature oocytes in clinical treatment. |
PDF Full Text Request