Ultrastructure And Gene Expression Profile During Follicular Development And Application Of Human Oocyte In Vitro Maturation

Background:Knowledge of the mechanisms involved in the oocytes and follicles development and maturation is essential to fully understand the human reproductive processes. Since 1930s, with the development of electron microscopy and molecular biological techniques, researches on germinal cells have advanced to the levels of ultra-structure and biological molecules such as DNA, mRNA and protein. These improvements have provided powerful technical platform to research the origin and differentiation of germinal cells. Furthermore, it has also give us opportunities to identify the mechanisms underlying the stimulation the primordial follicles from the quiescent to the growth phase, morphologically changes from primary follicle to secondary follicle then to mature follicle, and the oocytes and follicular developmental environment associated with the endocrinal, autocrinal and paracrinal actions. Most important, the birth of the first in vitro fertilization and embryo transfer (IVF-ET) baby in 1978 enhanced profound researches on human reproduction; furthermore, the development of genetic molecular techniques combined with the emergence of DNA array techniques, provided more powerful technical supports to research the mechanisms involved in follicular development and oocytes maturation.To date, the implantation rate per cycle has still been close to 20%. In addition, only approximately 30%- 40% of the clinical pregnancy rate per IVF cycle and approximately 25% of living birth rate per IVF cycle have been observed in women who received assisted reproductive techniques. One of the important reasons is known to be the abnormal oocytes collected in the superovulatary environment during the controlled ovarian hyperstimulation procedure, resulting in low fertilization rate and poor quality embryo. On the other hand,since the availability of in vitro maturation (IVM) of oocytes in 1983, IVM has been becoming interesting alternatives to classical assisted reproductive technology approaches, especially in those at high risk for ovarian hyperstimulation syndrome (OHSS). More than for their clinical and biological indications, IVM of oocytes can also be considered as good social and economic alternatives to the classical IVF treatment, based on their financial cost-effectiveness with exclusion of expensive medications. However, due to the asynchronous development of the nucleus and cytoplasm of immature oocytes found in the IVM program, the clinical application of IVM in the recent 20 years has still been limited. Until 2005, there have been only 300 IVM babies. Thus, deeper knowledge of the biological and pathological mechanisms should improve poor developmental potential of in vitro maturation oocytes and the quality of derived embryos, their successful implantation, maintenance of pregnancy, and offspring health.Previous studies have shown that some ovarian factors are involved in the process of oocyte maturation. Researches had tried to add these factors to the medium to improve the outcome of oocyte matured in vitro; however, such IVM recipes are not comparable to physiological environment. Up to thousands of molecules are the process of follicle growth and oocyte maturation. Up to now, the interplay of growth factors and cytokines secreted by oocytes, cumulus cells and granulosa cells are unclear. Moreover, few studies work on the effects of hyperstimulation on ultrastructure of oocyte-cumulus complex (OCCs). Thus, we investigated the gene expression difference of granulosa cells collected from mature follicles and immature follicles and the ultrastructural changes of OCCs. These studies help to reveal the mechanisms of follicle growth and oocyte maturation, and are important for improvement of IVM culture system.Part I Changes in the ultrastructure of human oocyte cumulus complexes at different follicular developmental environment and stagesObjectivesTo examine the ultrastructural changes of of oocyte cumulus complexes (OCCs) when they matured in in vivo or in vitro environment, compare the ultrastructural differences of OCCs in natural status and conventional hyperstimulation treatment cycle, and investigate the effects of different cultural environment on the quality of oocytes. Methods The oocytes at different stages (GV, MI, and MII stages) were distinguished under lightmicroscopy. Samples (donated OCCs) were collected in natural cycles during laparoscopy operation, IVF/ICSI cycles, and IVM cycles. The ultrastructures of immatured oocytes in natural cycles and IVF/ICSI cycles, matured oocytes in IVF/ICSI cycles and IVM cycles were studied by transmission-electron microscopy. ResultsThe difference in the structure and distribution of cumulus cells, zona pellucida, microvilli, cortical granule, mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, lipoid drop vesicles and centrioles was observed in OCCs that were developed in different developmental environment and were at different developmental stages.1. Difference of the ultrastructures was observed in the GV stage OCCs which were collected in natural cycle and conventional ovarian hyperstimulation cycle. In OCCs of natural cycle, there were abundant lipoid drop vesicles and Golgi apparatus, no perivitelline space, dense and thin zona pellucida, and clear nucleoli. Each oocyte and its surrounding cumulus cells share a communication system, the gap junction, which embraced these cells as an intact functional system. The gap junction turned to be loose between the oocyte and its surrounding cumulus cells with no structure space at GV stage in IVF/ICSI treatment cycle. The number of cortical granule in oocyte increased; microvilli were long and thin and protruded into zona pellucida; the number of lipoid drops decreased, the membrane of nucleoli was less arranged. These changes suggested that the follicles were luteinized earlier.2. Abundant organelles such as mitochondria, lysosomes, rough endoplasmic reticulum, Golgi apparatus, large lipoid drops were found in the cumulus cells of OCCs at MI stage in IVF/ICSI treatment cycle. Occasionally medulla-like body was detected. Cellular organelles and cortical granules were increased in oocytes; organelles such as mitochondria and lysosomes migrated toward cortical region gradually; a perivitelline space between cytoplasm membrane and zona pellucida emerged with vesicular structures; the nucleus disappeared; the thickness of zona pellucida increased to 11～12 um; and the microvilli became longer and protruded into zona pellucida.3. A large mount of vesicular structures were distributed in the cytoplasm of matured oocytes at MII stage in IVF/ICSI cycle; the zona pellucida became wider and looser; the perivitelline space appeared and turned to spread, especially at the site of first pole body expelling; the microvilli retracted from the zona pellucida; the number of cortical granules increased; cortical granules migrated toward the margin of cells and arranged along oolemma linearly; the number of rough endoplasmic reticulum and Golgi apparatusdecreased; a "transparent" area with no organelle appeared in the centre of the oocyte; the number and structure of mitochondria were not changed. On the other hand. the number of mitochondria in the IVM oocytes decreased and the structure became less arranged; smooth endoplasmic reticulum with vesicular structures could be observed: the number of cortical granule decreased; mitochondria in the cumulus cells became swollen; some nuclei in granulosa cells disappeared; apoptosis happened in some granulosa cells. Conclusions1. The GV stage OCCs collected from IVF/ICSI cycles, which mainly generated mature oocytes, had loose junction between oocytes and cumulus cells due to the overdose of FSH and hCG. Thus, nutrition transported through the gap junction to the oocytes was depleted. The impairment of communication between oocytes and cumulus cells and earlier distribution of cortical granules probably lead to decrease of fertilization rate and developmental potentials. The mature capacity of these oocytes was impaired by hyperstimulation hormone environment. Thus, the GV oocytes from IVF ICSI cycles were not suitable for further culture and embryo transplantation.2. The cumulus cells surrounding oocytes at MI stage in hyperstimulation cycle showed active proliferation and function; the changes of zona pellucida and cortical granule of oocytes were fully ready to undergo further development.3. The smooth endoplasmic reticulum was swollen in IVM oocytes, while the number of mitochondria decreased and their structure became less arranged; lipoid drops and cortical granules decreased. The cumulus cells showed obvious signs of apoptosis. It indicated that these changes were responsible for the poor quality of oocytes and impairment of developmental potential.Part II The different expressions of gene profile of granulose cell from mature ovarian follicles and immature ovarian folliclesObjectivesTo examine the gene expression profiles of granulose cells isolated from mature and immature ovarian follicles, and to attempt an understanding of the underlying molecular mechanism of granulosa cells involved in the maturation of ovarian follicle/oocyte.MethodsThe granulosa cells were isolated from the immature follicles in IVM cycle and mature follicles in conventional ovarian stimulation cycle. Total RNA was isolate and purify from granulosa cells. The granulosa cell from immature follicles were regarded as study group, while the granulosa cells from matured ones were regarded as control group. The expressive difference of study group and control group was investigated with the oligonucleotide microarray containing 8300 human genes. Ten of the genes were further confirmed by real-time RT PCR for their expression characteristics in granulosa cells. Results1. Changes in patterns of 462 genes expression in granulose cells were observed between mature and immature follicles. Among them. the expressions of 190 genes were up-regulated and 270 genes were down-regulated in granulose cells of immature follicles compared with mature ones. These genes included oncogene (15), ion channel and transport proteins (13), cellular cyclic protein (15). stress reaction protein (1) , proteins responsible for cellular structure and movement (18), cellular apoptosis related protein (4), DNA synthesis and repair, reconstruction protein (8), DNA binding proteins and transcription factors (15), cellular receptors (11), immunological related protein (20), cellular signal and transduction protein (54), proteins involved in metabolism (66), factors responsible for protein translation and synthesis (35), development related proteins (9), and others with unknown function (92). Some of the genes have 2 or more than 2 types of functions. 166 genes were remained to be unclassified.2. According to the rate of up-regulated genes and down-regulated genes, the expression ration of 6 down-regulated genes (STAR, MPHOSPH6, CALM1, MAML1, CD53, RRAGA) and 4 up-regulated genes (ICAM1, CYR61, ZNF264, STMN1) were selected and further confirmed by real-time RT-PCR. The ratio of difference gene expression examined by microarray of the 6 down-regulated genes were 0.16, 0.219, 0.214, 0.319, 0.183 and 0.411, respectively; while the ratio by real-time RT-PCR were 0.166, 0.086, 0.213, 0.186, 0.177 and 0.345, respectively. Furthermore, the ratio of difference gene expression examined by microarray of the 4 up-regulated genes were 2.385, 3.619, 3.992 and 10.245, respectively; while the ratio by real-time RT-PCR were2.28, 2.023, 2.30 and 5.07, respectively. The trend of the differences of gene expression between microarray and real-time RT-PCR was similar. The difference of gene expression between the two groups of these ten genes was statistically analyzed, resulting in only gene with no significant change, while the other genes being significantly changed between the two groups(P<0.001).3. The microarray results showed that genes related to proliferation, mitochondria duplication, gap junction, estrogen secretion, energy synthesis were up-regulated in the granulosa cells from immature follicles. These up-regulated genes included: growth factors: CTGF, TIEG, PDGFC, FGFR-1, CGR19, et al; cell cycle modulation protein and apoptosis related factors: SMC1L1, HMG2, PTP4A1, cyclin B, CDK9, STMN1, melanoma antigen, et al; genes related to signaling transduction and gap junction: PRSS11, PTP4A1, CYR61, GAB1, ICAM1 (CD54), Integrins -β8, connexin 43, et al. genes related to cystoskeleton, such as microtubule protein were also dominant in expression: XPO1, GPAT, SPP1, LOC51174, MIP-T3, et al. Another group of up-regulated genes were modulation factors stimulating estrogen secretion: CYP19, IRS1 , ADRBK2, CRHBP, et al.4. The microarray results showed that genes related to steroid synthesis, mitochondria synthesis, energy transportation, cellular signaling transduction, extracellular matrix formation, mucus secretion were up-regulated in the granulosa cells from mature follicles. These genes included: genes related to signaling transduction: PTPRG, PRKCD, KIAA010, FDPS, IMPA1, TPI1, PSPHL, PIP5K2B, et al; genes related to extracellular matrix formation and mucus secretion: PTX3, CALM1, MAML1, FN1, Lumican, COL5A2, FCN1, TIMP1, et al; genes related to mitochondria and energy supply: COX6C, COX8, MPP1, MRPL9, MRPL15, MRPL20, PMPCB, TIMM8B, ATP6J, ATP6M, ATP5H, ATP6H, et al; genes related to steroid synthesis: StAR, LOC51144, DHCR24, HSD11B1, HSD17B4, 3β-HSD, enolase 1, SECRET, et al.Conclusions1. The expression difference of 462 genes between the granulosa cells from immature and mature follicles was detected by microarray. It suggests that the growth and mature of human oocytes are modulated by factors secreted by granulosa cells of follicles at different developmental stages.2. Ten of the genes showing expression difference were confirmed by real-time RT-PCR. Except one gene showed no difference by real-time RT-PCR, the real-time RT-PCR results of other gene were consistent with results of microarray. It suggested that the results of microarray are reliable. We can further investigate the gene expression pattern and do some functional assay according to the results of microarray.3. The expressions of 190 up-regulated genes and 270 down-regulated genes were observed in granulose cells from immatured follicles compared with matured follicles, suggesting that more regulatory factors are involved in the developmental process as the follicles grewand matured, and the function of granulose cells of these follicles turn to be more complex.4. We hypothesize that the roles of granulose cells at different growth stages were different. During early stage of follicular development, the granulose cells are mainly responsible for cellular proliferation, estrogen secretion and gap junction; while follicles became more mature, the granulose cells transfer to mediate progestogen secretion, signal transduction, substance transportation, extracellular matrix formation and mucus secretion.5. The results of microarray indicated that the signaling transduction factors, growth factors, cell cycle modulation factors and paracrine factors secreted by granulosa cells are somewhat different for follicles at different stages. These factors modulate oocyte maturation, or in turn, the functions of granulosa cells are modified by the secretion from oocytes.6. Although the results of microarray suggested us dominant gene expression during the process of follicle development, the detailed function of the genes are remained to be investigated. Further study of these genes will lead to better understanding of the mechanisms of oocyte/follicle growth and maturation.Part III In vitro maturation—in vitro fertilization—embryo transfer—frozen-thawed of human oocytesObjectivesTo investigate the influence of ovarian follicle diameter, IVM culture media and culture duration on the number of retrieved immature oocytes, maturation rate, fertilization rate, cleavage rate, number of high-quality embryo, implantation rate, pregnancy rate, delivery rate, survival and development of frozen-thawed germinal vesicle stage and in vitro matured human oocytes. MethodsThe oocytes were obtained by follicular aspiration from follicle-stimulating hormone-treated women undergoing oocyte retrieval for in vitro maturation between July 2002 and July 2004. IVM program was performed due to the possible risk of ovarian hyperstimulation in IVF-ET program. All the oocytes retrieved from follicles with 10-13.5 mm in diameter were allowed to remain in medium M-199 (TCM 199) or HTF supplemented with 10% humanplasma protein or 10% synthesized serum substance, and 0.075 IU/ml r-FSH +0.51U/mL hCG+1μg/mlβE2. Then the effects of follicle diameter, IVM culture medium and culture duration on the retrieval rate, maturation rate, fertilization rate, cleavage rate, number of high-quality embryo, implantation rate, clinical pregnancy rate, delivery rate and outcome of derived embryos transfer of in vitro matured human oocytes was investigated. Results1. When the diameters of follicles varied and the diameter of largest oocyte exceeded 12 mm, the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased;2. Using different culture medium (TCM 199 and HTF) had no effects on the maturation rate, fertilization rate, and cleavage rate of oocytes, however, the high-quality embryos formation rate was higher for the oocytes cultured in HTF medium (P <0.01) .3. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy.Conclusions1. When the diameters of follicles varied and the diameter of largest oocyte exceeded 12 mm. the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased;2. Although TCM199-based medium could improve the developmental potential and implantation rate of embryos derived from in vitro matured oocytes, the implantation rate of these embryos is still lower than the implantation rate of embryos generated in conventional IVF or ICSI cycles.3. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy.