Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

Reprofit International Clinic of Reproductive Medicine Brno Czech Republic

Zobrazit více v PubMed

Stitzel ML, Seydoux G. Regulation of the oocyte-to-zygote transition. Science  2007; 316:407–408. PubMed

Mtango NR, Potireddy S, Latham KE. Oocyte quality and maternal control of development. Int Rev Cell Mol Biol  2008; 268:223–290. PubMed

Lu X, Gao Z, Qin D, Li L. A maternal functional module in the mammalian oocyte-to-embryo transition. Trends Mol Med  2017; 23:1014–1023. PubMed

Coticchio G, Dal Canto M, Mignini Renzini M, Guglielmo MC, Brambillasca F, Turchi D, Novara PV, Fadini R. Oocyte maturation: Gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization. Hum Reprod Update  2015; 21:427–454. PubMed

Rienzi L, Balaban B, Ebner T, Mandelbaum J. The oocyte. Hum Reprod  2012; 27:i2–i21. PubMed

Watson AJ. Oocyte cytoplasmic maturation: A key mediator of oocyte and embryo developmental competence. J Anim Sci  2007; 85:E1–E3. PubMed

Kline D. Attributes and dynamics of the endoplasmic reticulum in mammalian eggs. Curr Top Dev Biol  2000; 50:125–154. PubMed

Brevini TA, Cillo F, Antonini S, Gandolfi F. Cytoplasmic remodelling and the acquisition of developmental competence in pig oocytes. Anim Reprod Sci  2007; 98:23–38. PubMed

Ferreira EM, Vireque AA, Adona PR, Meirelles FV, Ferriani RA, Navarro PA. Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence. Theriogenology  2009; 71:836–848. PubMed

Mao L, Lou H, Lou Y, Wang N, Jin F. Behaviour of cytoplasmic organelles and cytoskeleton during oocyte maturation. Reprod Biomed Online  2014; 28:284–299. PubMed

Holubcová Z, Blayney M, Elder K, Schuh M. Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes. Science  2015; 348:1143–1147. PubMed PMC

Sakakibara Y, Hashimoto S, Nakaoka Y, Kouznetsova A, Höög C, Kitajima TS. Bivalent separation into univalents precedes age-related meiosis I errors in oocytes. Nat Commun  2015; 6:7550. PubMed PMC

Zielinska AP, Holubcova Z, Blayney M, Elder K, Schuh M. Sister kinetochore splitting and precocious disintegration of bivalents could explain the maternal age effect. Elife  2015; 4:e11389. PubMed PMC

Gruhn JR, Zielinska AP, Shukla V, Blanshard R, Capalbo A, Cimadomo D, Nikiforov D, Chi-Ho Chan A, Newnham LJ, Vogel I, Scarica C, Krapchev M  et al.  Chromosome errors in human eggs shape natural fertility over reproductive life span. Science  2019; 365:1466–1469. PubMed PMC

Roeles J, Tsiavaliaris G. Actin-microtubule interplay coordinates spindle assembly in human oocytes. Nat Commun  2019; 10:4651. PubMed PMC

Zamboni L, Thompson RS, Smith DM. Fine morphology of human oocyte maturation in vitro. Biol Reprod  1972; 7:425–457. PubMed

Sathananthan HA. Maturation of the human oocyte in vitro: Nuclear events during meiosis (an ultrastructural study). Gamete Res  1985; 12:237–254.

Sundström P, Nilsson BO, Liedholm P, Larsson E. Ultrastructure of maturing human oocytes. Ann N Y Acad Sci  1985; 442:324–331. PubMed

Szöllösi D, Mandelbaum J, Plachot M, Salat-Baroux J, Cohen J. Ultrastructure of the human preovulatory oocyte. J In Vitro Fert Embryo Transf  1986; 3:232–242. PubMed

Suzuki S, Kitai H, Tojo R, Seki K, Oba M, Fujiwara T, Iizuka R. Ultrastructure and some biologic properties of human oocytes and granulosa cells cultured in vitro. Fertil Steril  1981; 35:142–148. PubMed

Motta PM, Nottola SA, Micara G, Familiari G. Ultrastructure of human unfertilized oocytes and polyspermic embryos in an IVF-ET program. Ann N Y Acad Sci  1988; 541:367–383. PubMed

Yang YJ, Zhang YJ, Li Y. Ultrastructure of human oocytes of different maturity stages and the alteration during in vitro maturation. Fertil Steril  2009; 92:396.e1–396.e6. PubMed

Nottola SA, Coticchio G, Sciajno R, Gambardella A, Maione M, Scaravelli G, Bianchi S, Macchiarelli G, Borini A. Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop. Reprod Biomed Online  2009; 19:17–27. PubMed

Shahedi A, Hosseini A, Khalili MA, Norouzian M, Salehi M, Piriaei A, Nottola SA. The effect of vitrification on ultrastructure of human in vitro matured germinal vesicle oocytes. Eur J Obstet Gynecol Reprod Biol  2013; 167:69–75. PubMed

Coticchio G, Dal Canto M, Fadini R, Mignini Renzini M, Guglielmo MC, Miglietta S, Palmerini MG, Macchiarelli G, Nottola SA. Ultrastructure of human oocytes after in vitro maturation. Mol Hum Reprod  2016; 22:110–118. PubMed

Segovia Y, Victory N, Peinado I, García-Valverde LM, García M, Aizpurua J, Monzó A, Gómez-Torres MJ. Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation. J Reprod Dev  2017; 63:377–382. PubMed PMC

Holubcová Z, Kyjovská D, Martonová M, Páralová D, Klenková T, Kloudová S. Human egg maturity assessment and its clinical application. J Vis Exp  2019; 150:e60058. doi: 10.3791/60058. PubMed DOI

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ  et al.  Fiji: An open-source platform for biological-image analysis. Nat Methods  2012; 9:676–682. PubMed PMC

Otsuki J, Nagai Y. A phase of chromosome aggregation during meiosis in human oocytes. Reprod Biomed Online  2007; 15:191–197. PubMed

Mehlmann LM, Terasaki M, Jaffe LA, Kline D. Reorganization of the endoplasmic reticulum during meiotic maturation of the mouse oocyte. Dev Biol  1995; 170:607–615. PubMed

FitzHarris G, Marangos P, Carroll J. Changes in endoplasmic reticulum structure during mouse oocyte maturation are controlled by the cytoskeleton and cytoplasmic dynein. Dev Biol  2007; 305:133–144. PubMed

Yu Y, Dumollard R, Rossbach A, Lai FA, Swann K. Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation. J Cell Physiol  2010; 224:672–680. PubMed PMC

Sathananthan AH. Ultrastructural changes during meiotic maturation in mammalian oocytes: Unique aspects of the human oocyte. Microsc Res Tech  1994; 27:145–164. PubMed

Longo FJ, Chen DY. Development of cortical polarity in mouse eggs: Involvement of the meiotic apparatus. Dev Biol  1985; 107:382–394. PubMed

Gulyas BJ. Cortical granules of mammalian eggs. Int Rev Cytol  1980; 63:357–392. PubMed

Sathananthan AH, Ng SC, Chia CM, Law HY, Edirisinghe WR, Ratnam SS. The origin and distribution of cortical granules in human oocytes with reference to Golgi, nucleolar, and microfilament activity. Ann N Y Acad Sci  1985; 442:251–264. PubMed

Ducibella T, Kurasawa S, Rangarajan S, Kopf GS, Schultz RM. Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida. Dev Biol  1990; 137:46–55. PubMed

Otsuki J, Nagai Y, Chiba K. Lipofuscin bodies in human oocytes as an indicator of oocyte quality. J Assist Reprod Genet  2007; 24:263–270. PubMed PMC

Liu J, Lu G, Qian Y, Mao Y, Ding W. Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles. Fertil Steril  2003; 80:447–449. PubMed

Shu Y, Gebhardt J, Watt J, Lyon J, Dasig D, Behr B. Fertilization, embryo development, and clinical outcome of immature oocytes from stimulated intracytoplasmic sperm injection cycles. Fertil Steril  2007; 87:1022–1027. PubMed

Piqueras P, Gallardo M, Hebles M, Jiménez JM, Migueles B, Montero L, Sánchez-Martín F, Sánchez-Martín P. Live birth after replacement of an embryo obtained from a spontaneously in vitro matured metaphase-I oocyte. Syst Biol Reprod Med  2017; 63:209–211. PubMed

Holubcová Z, Kyjovská D, Martonová M, Páralová D, Klenková T, Otevřel P, Štěpánová R, Kloudová S, Hampl A. Egg maturity assessment prior to ICSI prevents premature fertilization of late-maturing oocytes. J Assist Reprod Genet  2019; 36:445–452. PubMed PMC

Gu NH, Zhao WL, Wang GS, Sun F. Comparative analysis of mammalian sperm ultrastructure reveals relationships between sperm morphology, mitochondrial functions and motility. Reprod Biol Endocrinol  2019; 17:66. PubMed PMC

Beuchat A, Thévenaz P, Unser M, Ebner T, Senn A, Urner F, Germond M, Sorzano COS. Quantitative morphometrical characterization of human pronuclear zygotes. Hum Reprod  2008; 23:1983–1992. PubMed

Tesarik J, Martinez F, Rienzi L, Ubaldi F, Iacobelli M, Mendoza C, Greco E. Microfilament disruption is required for enucleation and nuclear transfer in germinal vesicle but not metaphase II human oocytes. Fertil Steril  2003; 79:677–681. PubMed

Zhang J. Revisiting germinal vesicle transfer as a treatment for aneuploidy in infertile women with diminished ovarian reserve. J Assist Reprod Genet  2015; 32:313–317. PubMed PMC

Reznichenko AS, Huyser C, Pepper MS. Mitochondrial transfer: Implications for assisted reproductive technologies. Appl Transl Genom  2016; 11:40–47. PubMed PMC

Koch GL. The endoplasmic reticulum and calcium storage. Bioessays  1990; 12:527–531. PubMed

Sá R, Cunha M, Silva J, Luís A, Oliveira C, Teixeira da Silva J, Barros A, Sousa M. Ultrastructure of tubular smooth endoplasmic reticulum aggregates in human metaphase II oocytes and clinical implications. Fertil Steril  2011; 96:143–9.e7. PubMed

Motta PM, Nottola SA, Makabe S, Heyn R. Mitochondrial morphology in human fetal and adult female germ cells. Hum Reprod  2000; 15:129–147. PubMed

Youle RJ, Bliek AM. Mitochondrial fission, fusion, and stress. Science  2012; 337:1062–1065. PubMed PMC

Paula WB, Agip AN, Missirlis F, Ashworth R, Vizcay-Barrena G, Lucas CH, Allen JF. Female and male gamete mitochondria are distinct and complementary in transcription, structure, and genome function. Genome Biol Evol  2013; 5:1969–1977. PubMed PMC

Dvoŕak M, Tesarík J, Pilka L, Trávník P. Fine structure of human two-cell ova fertilized and cleaved in vitro. Fertil Steril  1982; 37:661–667. PubMed

Pereda J, Croxatto HB. Ultrastructure of a seven-cell human embryo. Biol Reprod  1978; 18:481–489. PubMed

Mohr LR, Trounson AO. Comparative ultrastructure of hatched human, mouse and bovine blastocysts. J Reprod Fertil  1982; 66:499–504. PubMed

Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril  2001; 76:1175–1180. PubMed

Cho YM, Kwon S, Pak YK, Seol HW, Choi YM, Park DJ, Park KS, Lee HK. Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells. Biochem Biophys Res Commun  2006; 348:1472–1478. PubMed

Tobias IC, Khazaee R, Betts DH. Analysis of mitochondrial dimensions and cristae structure in pluripotent stem cells using transmission electron microscopy. Curr Protoc Stem Cell Biol  2018; 47:e67. PubMed

Uraji J, Scheffler K, Schuh M. Functions of actin in mouse oocytes at a glance. J Cell Sci  2018; 131:jcs218099. PubMed

Duan X, Sun SC. Actin cytoskeleton dynamics in mammalian oocyte meiosis. Biol Reprod  2019; 100:15–24. PubMed

Holubcová Z, Howard G, Schuh M. Vesicles modulate an actin network for asymmetric spindle positioning. Nat Cell Biol  2013; 15:937–947. PubMed PMC

Aida T, Oda S, Awaji T, Yoshida K, Miyazaki S. Expression of a green fluorescent protein variant in mouse oocytes by injection of RNA with an added long poly(A) tail. Mol Hum Reprod  2001; 7:1039–1046. PubMed

Sousa Martins JP, Liu X, Oke A, Arora R, Franciosi F, Viville S, Laird DJ, Fung JC, Conti M. DAZL and CPEB1 regulate mRNA translation synergistically during oocyte maturation. J Cell Sci  2016; 129:1271–1282. PubMed PMC

Tetkova A, Susor A, Kubelka M, Nemcova L, Jansova D, Dvoran M, Del Llano E, Holubcova Z, Kalous J. Follicle-stimulating hormone administration affects amino acid metabolism in mammalian oocytes. Biol Reprod  2019; 101:719–732. PubMed

Danev R, Yanagisawa H, Kikkawa M. Cryo-electron microscopy methodology: Current aspects and future directions. Trends Biochem Sci  2019; 44:837–848. PubMed

Briggman KL, Bock DD. Volume electron microscopy for neuronal circuit reconstruction. Curr Opin Neurobiol  2012; 22:154–161. PubMed

Tachibana M, Kuno T, Yaegashi N. Mitochondrial replacement therapy and assisted reproductive technology: A paradigm shift toward treatment of genetic diseases in gametes or in early embryos. Reprod Med Biol  2018; 17:421–433. PubMed PMC

High-Resolution 3D Reconstruction of Human Oocytes Using Focused Ion Beam Scanning Electron Microscopy