An increasing number of data proves that the presence of the zona pellucida is not essential to mammalian embryo production, including maturation, fertilization, and embryo culture. In fact, the structure of the zona pellucida of in vitro-produced embryos differs significantly from its in vivo counterpart, influencing metabolism and requiring disproportionate efforts to crack open at the time of hatching. This review aims to focus attention on this field and stimulate research in zona-free embryo culture. In domestic animals, extensive application of purpose-designed culture systems for zona-free embryos proved the feasibility of this approach. It may open new possibilities and increase efficiency in both transgenic research and human-assisted reproduction.
Get full access to this article
View all access options for this article.
References
1.
AlteriA., ViganòP., Abu MaizarA., JovineL., GiacominiE., and RubinoP. (2018). Revisiting embryo assisted hatching approaches: A systematic review of the current protocols. J. Assist. Reprod. Genet. 35, 367–391.
2.
AmstrongS., AtkinsonM., MacKenzieJ., PaceyA., and RarquharC. (2019). Add-ons in the laboratory: Hopeful, but not always helpful. Fertil. Steril. 112, 994–999.
3.
AssidiM., MontagM., and SirardM.-A. (2014). Analysis of correlations between zona pellucida birefringence and molecular markers of oocyte developmental competence. BMC Genom. 15, P48,1471-2164-15-S2-P48.
4.
AzambujaR.M., KraemerD.C., and WesthusinM.E. (1998). Effect of low temperatures on in-vitro matured bovine oocytes. Theriogenology, 49, 1155–1164.
5.
BehrB., IvakhnenkoV., TranC., and TanT. (2003). Empty zona pellucida as a container for preservation of blastomeres in preimplantation genetic diagnosis. Fertil. Steril 79,11.
6.
BhaktaH.H., RefaiF.H., and AvellaM.A. (2019). The molecular mechanisms mediating mammalian fertilization. Development 146,dev176966.
7.
BleilJ.D., and WassarmanP.M. (1980). Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell, 20, 873–882.
8.
BragaD.P., deA.F., FigueiraR. deC.S., QueirozP., MadaschiC., IaconelliJr., A., and Borges JrE. (2010). Zona pellucida birefringence in in vivo and in vitro matured oocytes. Fertil. Steril. 94, 2050–2053.
9.
BrownC.R., ClarkeN., AikenM., and BavisterB.D. (1990). Changes in the composition of the hamster zona pellucida after fertilization in vivo but not in vitro. Reproduction, 90, 447–454.
10.
BuemoC.P., GambiniA., MoroL.N., HiriartM.I., Fernández-MartínR., CollasP., and SalamoneD.F. (2016). Embryo aggregation in pig improves cloning efficiency and embryo quality. PLoS One 11,e0146390.
11.
CallesenH., LiuY., PedersenH.S., LiR., and SchmidtM. (2014). Increasing efficiency in production of cloned piglets. Cell. Reprogram. 16, 407–410.
12.
CarrollJ., DepypereH., and MatthewsC. D. (1990). Freeze-thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen- thawed mouse oocytes. J. Reprod. Fert. 90, 547–553.
13.
CerveraR.P., and Garcia-XiménezF. (2003). Vitrification of zona-free rabbit expanded or hatching blastocysts: A possible model for human blastocysts. Hum. Reprod. 18, 2151–2156.
14.
CohenJ. (1991). Assisted hatching of human embryos. J. In Vitro Fertil. Embryo. Transfer. 8, 179–190.
15.
CohenJ., AlikaniM., TrowbridgeJ., and RosenwaksZ. (1992). Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum. Reprod. 7, 685–691.
16.
DattaA.K., CampbellS., DevalB., and NargundG. (2015). Add-ons in IVF programme—Hype or hope? Facts Views Vis. Obgyn. 28, 241–250.
17.
DeanJ. (1992). Biology of mammalian fertilization: Role of the zona pellucida. J. Clin. Invest. 89, 1055–1059.
18.
DingJ., RanaN., and DmowskiW.P. (1999). Case Report: Intracytoplasmic sperm injection into zona-free human oocytes results in normal fertilization and blastocyst development. Hum. Reprod. 14, 476–478.
19.
DuY., LiJ., KraghP.M., ZhangY., SchmidtM., BøghI.B., ZhangX., PurupS., KuwayamaM., JørgensenA.L., PedersenA. M., VillemoesK., BolundL., and VajtaG. (2007). Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer. Cloning Stem Cells, 9, 469–476.
20.
EbnerT., BalabanB., MoserM., SheblO., UrmanB., AtaB., and TewsG. (2010). Automatic user-independent zona pellucida imaging at the oocyte stage allows for the prediction of preimplantation development. Fertil. Steril. 94, 913–920.
21.
ElsheikhA.S.I. (2007). Handmade cloning of mammals. Afr. J. Biotechnol. 6, 1862–1868.
22.
EvansJ.P. (2020). Preventing polyspermy in mammalian eggs-contributions of the membrane block and other mechanisms. Mol. Reprod. Dev. 87, 341–349.
23.
FongC.Y., BongsoA., NgS.C., AnandakumarC., TrounsonA., RatnamS. (1997). Ongoing normal pregnancy after transfer of zona-free blastocysts: Implications for embryo transfer. Hum. Reprod. 12, 557–560.
24.
FongC.Y., BongsoA., NgS.C., KumarJ., TrounsonA., RatnamS. (1998). Blastocyst transfer after enzymatic treatment of the zona pellucida: Improving in-vitro fertilisation and understanding implantation. Hum. Reprod. 13, 2926–2932.
25.
GambiniA., JarazoJ., OliveraR., and SalamoneD.F. (2012). Equine cloning: In vitro and in vivo development of aggregated embryos. Biol. Reprod. 87, 1–9.
26.
GonzalesD.S., and BavisterB.D. (1995). Zona pellucida escape by hamster blastocysts in vitro is delayed and morphologically different compared with zona escape in vivo. Biol. Reprod. 52, 470–480.
27.
GookD.A., EdgarD.H., BorgJ., and MarticM. (2007). Detection of zona pellucida proteins during human folliculogenesis. Hum. Reprod. 23, 394–402.
28.
GreveJ.M., and WassarmanP.M. (1985). Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat. J. Mol. Biol. 181, 253–264.
29.
HaddadM., StewartJ., XieP., CheungS., TroutA., KeatingD., ParrellaA., LawrenceS., RosenwaksZ., and PalermoG.D. (2021). Thoughts on the popularity of ICSI. J. Assist. Reprod. Genet. 38, 101–123.
30.
HerrlerA., and BeierH.M. (2000). Early embryonic coats: Morphology, function, practical applications. Cells Tissues Organs, 166, 233–246.
31.
HiraokaK. (2004). Case report: Successful pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida on day6. Hum. Reprod. 19, 988–990.
32.
HiraokaK., and KinutaniK. (2007). Vitrification of human hatched blastocysts. J. Reprod. Med. 52, 413–415.
33.
HsiehY.Y., ChangC.C., and TsaiH.D. (2001). Zona-free oocyte fertilized with intracytoplasmic sperm injection and underwent further division: Case report and literature review. Chang Gung Med. J. 24, 599–602.
34.
HuY., and TroliceM.P. (2016). Live birth of twins derived from zona-free oocytes. Fertil. Steril. 105, 1232–1235.
35.
JinM., LarssonA., and Ove NilssonB. (1996). Monoclonal antibodies against unfertilized zona-free mouse oocytes: Characterization and effects on fertilization. Mol. Reprod. Dev. 43, 47–54.
36.
KapurR.P., and JohnsonL.V. (1986). Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos. J. Exp. Zool. 238, 249–260.
37.
KirienkoK.V., ApryshkoV.P., NaumovaA. A., KharitonovaM. A., KlepukovA. A., BoltA. I., ErmilovaI. Y., MironovaA. G., BozinaY. V., LebedevaE. B., SimonenkoE. Y., VajtaG., and YakovenkoS. (2019). Mechanical zona pellucida removal of vitrified/warmed human blastocysts does not affect the clinical outcome. Reprod. Biomed. Online 39,749.
38.
KorkmazC., SakinciM., Bayoglu TekinY., and ErcanC.M. (2014). Do quantitative birefringence characteristics of meiotic spindle and zona pellucida have an impact on implantation in single embryo transfer cycles? Arch. Gynecol. Obstet. 289, 433–438.
39.
LagutinaI., LazzariG., DuchiR., TuriniP., TessaroI., BrunettiD., ColleoniS., CrottiG., and GalliC. (2007). Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep. Theriogenology, 67, 90–98.
40.
LambertH., CitinoS., CollazoI., and JeyendranR.S. (1991). Penetration of zona-free hamster oocytes by ejaculated cryopreserved gorilla spermatozoa. Fertil. Steril. 56, 1201–1203.
41.
LarmanM.G., SheehanC.B., and GardnerD.K. (2006). Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes. Reproduction, 131, 53–61.
42.
LauferN. (1991). Assisted hatching of human embryos. J. In Vitro Fertilization Embryo Transfer 8,12.
43.
LiJ., VajtaG., and CallesenH. (2009). Comparison of cell numbers of zona-intact and zona-free parthenogenetically activated porcine embryos cultured in vitro. Reprod. Fertil. Dev. 22,234.
44.
LiR., LiuY., PedersenH.S., KraghP.M., and CallesenH. (2013). Development and quality of porcine parthenogenetically activated embryos after removal of zona pellucida. Theriogenology, 80, 58–64.
45.
LitscherE.S., and WassarmanP.M. (2020). Zona pellucida proteins, fibrils, and matrix. Ann. Rev. Biochem. 89, 695–715.
46.
LiuT., DouH., XiangX., LiL., LiY., LinL., PangX., ZhangY., ChenY., LuanJ., XuY., YangZ., YangW., LiuH., LiF., WangH., YangY., BoludL., VajtaG., and DuY. (2015). Factors Determining the Efficiency of porcine somatic cell nuclear transfer: Data analysis with over 200,000 reconstructed embryos. Cell. Reprogram. 17, 463–471.
47.
LiuY., Lucas-HahnA., PetersenB., LiR., HermannD., HasselP., ZieglerM., LarsenK., NiemannH., and CallesenH. (2017). Developmental competence and epigenetic profile of porcine embryos produced by two different cloning methods. Cell. Reprogr. 19, 171–179.
48.
MannaC., RienziL., GrecoE., SbraciaM., RahmanA., PoveriniR., SiracusaG., and De FeliciM. (2001). Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques. Zygote, 9, 201–210.
49.
MansourR.T., RhodesC.A., AboulgharM.A., SerourG.I., and KamalA. (2000). Transfer of zona-free embryos improves outcome in poor prognosis patients: A prospective randomized controlled study. Hum. Reprod. 15, 1061–1064.
50.
MassipA., and MulnardJ. (1980). Time-lapse cinematographic analysis of hatching of normal and frozen-thawed cow blastocysts. J. Reprod. Fert. 58, 475–478.
51.
MatsuuraK. (2014). Numerical calculations for diffusion effects in the well-of-the-well culture system for mammalian embryos. Reprod. Fertil. Dev. 26,742.
52.
McRaeA.C., and ChurchR.B. (1990). Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocysts. Reproduction, 88, 31–40.
53.
MetwalleyA., BrashaN., EstevesS.C., FawzyM., BrashaH., HellaniA., El HamsharyM., and KhamissO. (2020). Role of diagnostic intracytoplasmic sperm injection (ICSI) in the management of genetically determined zona pellucida-free oocytes during in vitro fertilization: A case report. Zygote, 28, 519–523.
54.
MichelmannH., RathD., Töpfer-PetersenE., and SchwartzP. (2007). Structural and functional events on the porcine zona pellucida during maturation, fertilization and embryonic development: A scanning electron microscopy analysis. Reprod. Domest. Anim. 42, 594–602.
55.
MintzB. (1962). Experimental study of the developing mammalian egg: Removal of the zona pellucida. Science, 138, 594–595.
56.
MontagM., and van der VenH. (2008). Oocyte assessment and embryo viability prediction: Birefringence imaging. Reprod. BioMed. Online, 17, 454–460.
57.
MurayamaY., MizunoJ., KamakuraH., FuetaY., NakamuraH., AkaishiK., AnzaiK., WatanabeA., InuiH., and OmataS. (2006). Mouse zona pellucida dynamically changes its elasticity during oocyte maturation, fertilization and early embryo development. Human Cell, 19, 119–125.
58.
NaitoK., ToyodaY., and YanagimachiR. (1992). Production of normal mice from oocytes fertilized and developed without zonae pellucidae. Hum. Reprod. 7, 281–285.
59.
O'NeillC. (2008). The potential roles for embryotrophic ligands in preimplantation embryo development. Hum. Reprod. Update, 14, 275–288.
60.
OrsiniM.W. (1963). Morphological evidence on the intrauterine career of the ovum. In Delayed Implantation. A.C. Enders, ed. (University of Chicago Press, Chicago, IL) pp. 155–166.
61.
ParkC.-H., JeongY.-H., LeeD.-K., HwangJ.Y., UhK.-J., YeomS.-C., AhnC., and LeeC.-K. (2015). Availability of empty zona pellucida for generating embryonic chimeras. PLoS One 10,e0123178.
62.
PazG. (2004). Case Report: Pregnancy outcome following ICSI of oocytes with abnormal cytoplasm and zona pellucida. Hum. Reprod. 19, 586–589.
63.
PeuraT.T., LewisI.M., and TrounsonA.O. (1998). The effect of recipient oocyte volume on nuclear transfer in cattle. Mol. Reprod. Dev. 50, 185–191.
64.
PeuraT.T., and VajtaG. (2003). A comparison of established and new approaches in ovine and bovine nuclear transfer. Cloning Stem Cells, 5, 257–277.
65.
QuintansC., DonaldsonM., BertolinoM., and PasqualiniR. (2003). Birth resulting from transfer of blastocysts cryopreserved with propanediol after spontaneous hatching. Reprod. Biomed. Online, 6, 66–68.
66.
RajuG.R., PrakashG., KrishnaK., and MadanK. (2007). Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: A preliminary study using PolScope imaging. Reprod. Biomed Online, 14, 166–174.
67.
RienziL., VajtaG., and UbaldiF. (2011). Predictive value of oocyte morphology in human IVE: A systematic review of the literature. Reprod. Update, 17, 34–45.
68.
RitchieW.A., TaylorJ.E., GardnerJ.O., WilmutI., CarlisleA., NeilC., KingT., and WhitelawC.B.A. (2005). Live lambs born from zona pellucida-denuded embryos. Cloning Stem Cells, 7, 178–182.
69.
RothT.L., SwansonW.F., and WildtD. E. (1994). Developmental competence of domestic cat embryos fertilized in vivo versus in vitro. Biol. Reprod. 51, 441–451.
70.
SavyV., AlberioV., Vans LandschootG., MoroL.N, OleaF.D, Rodrıguez-AlvarezL., and SalamoneD.F. (2021). Effect of embryo aggregation on in vitro development of adipose-derived mesenchymal stem cell-derived bovine clones. Cell. Reprogramming 23,5.
71.
SchieveL.A., MeikleS.F., PetersonH.B., JengG., BurnettN.M., and WilcoxL.S. (2000). Does assisted hatching pose a risk for monozygotic twinning in pregnancies conceived through in vitro fertilization? Fertil. Steril. 74, 288–294.
72.
SchieweM.C., AraujoE., AschR.H., and BalmacedaJ.P. (1995). Enzymatic characterization of zona pellucida hardening in human eggs and embryos. J. Assist. Reprod. Genet. 12, 2–7.
73.
SeshagiriP.B., Sen RoyS., SireeshaG., and RaoR.P. (2009). Cellular and molecular regulation of mammalian blastocyst hatching. J. Reprod. Immunol. 83, 79–84.
74.
ShuY., PengW., and ZhangJ. (2010). Pregnancy and live birth following the transfer of vitrified–warmed blastocysts derived from zona- and corona-cell-free oocytes. Reprod. BioMed. Online. 21, 527–532.
75.
SinghE.L. (1987). The disease control potential of embryos. Theriogenology, 27, 9–20.
76.
SinowatzF., Topfer-PetersenE., KolleS., and PalmaG. (2001). Functional morphology of the zona pellucida. Anat. Histol. Embryol. 30, 257–263.
77.
SoupartP., and Ann StrongP. (1975). Ultrastructural observations on polyspermic penetration of zona pellucida-free human oocytes inseminated in vitro. Fertil. Steril. 26, 523–537.
78.
StangerJ.D., StevensonK., LakmakerA., and WoolcottR. (2001). Pregnancy following fertilization of zona-free, coronal cell intact human ova. Hum. Reprod. 16, 164–167.
79.
SturmeyR.G., and LeeseH.J. (2003). Energy metabolism in pig oocytes and early embryos. Reproduction, 126, 197–204.
80.
TakahashiK., ArakiY., and MotoyamaM. (1999). Normal development of a zona-free oocyte to the blastocyst stage following ICSI. Hum. Reprod. 14,2677.
81.
ThouasG.A., JonesG.M., and TrounsonA.O. (2003) The ‘GO’ system—A novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage. Reproduction, 126, 161–169.
82.
TrounsonA.O., and MooreN.W. (1974). The survival and development of sheep eggs following complete or partial removal of the zona pellucida. Reproduction, 41, 97–105.
83.
TruelsonS.L., GrahamJ.K., MortimerR.G., and FieldT.G. (1996). In vitro penetration into bovine oocytes and zona-free hamster oocytes by bull spermatozoa treated with liposomes. J. Dairy Sci. 79, 991–999.
84.
UenoS., BodriD., UchiyamaK., OkimuraT., OkunoT., KobayashiT., and KatoK. (2014). Developmental potential of zona pellucida-free oocytes obtained following mild in vitro fertilization. Fertil. Steril. 102, 1602–1607.
85.
VajtaG., PeuraT.T., HolmP., PaldiA., GreveT., TrounsonA.O., and CallesenH. (2000). New method for culture of zona-included or zona-free embryos: The Well of the Well (WOW) system. Mol. Reprod. Dev. 55, 256–264.
86.
VajtaG., RienziL., and BavisterB.D. (2010). Zona-free embryo culture: Is it a viable option to improve pregnancy rates? Reprod. BioMed. Online. 21, 17–25.
87.
Van SoomA., WrathallA.E., HerrlerA., and NauwynckH.J. (2010). Is the zona pellucida an efficient barrier to viral infection? Reprod. Fertil. Dev. 22,21.
88.
VitthalaS., GelbayaT.A., BrisonD.R., FitzgeraldC.T., and NardoL.G. (2009). The risk of monozygotic twins after assisted reproductive technology: A systematic review and meta-analysis. Hum. Reprod. Update, 15, 45–55.
89.
WangW.-H., MachatyZ., AbeydeeraL.R., PratherR.S., and DayB.N. (1998). Parthenogenetic activation of pig oocytes with calcium ionophore and the block to sperm penetration after activation. Biol. Reprod. 58, 1357–1366.
90.
WassarmanP.M. (2008). Zona pellucida glycoproteins. J. Biol. Chem. 283, 24285–24289.
91.
WassarmanP.M., and LitscherE.S. (2008). Mammalian fertilization is dependent on multiple membrane fusion events. Methods Mol. Biol. 475, 99–113.
92.
WatanabeM., HoshiK., YazawaH., YanagidaK., and SatoA. (1995). Use of the artificial zona pellucida made of calcium alginate in the development of preimplantation mouse embryo. J. Mamm. Ova Res. 12, 95–100.
93.
WolfE., ArnoldG.J., BauersachsS., BeierH.M., BlumH., EinspanierR., FröhlichT., HerrlerA., HiendlederS., KölleS., PrelleK., ReichenbachH-D., StojkovicM., WenigerkindH., and SinowatzF. (2003). Embryo-maternal communication in bovine—Strategies for deciphering a complex cross talk. Reprod. Domest. Anim, 38, 276–289.
94.
WoodS.A., AllanN.D., RossantJ., AuerbachA., and NagyA. (1993). Non-injection methods for the production of embryonic stem cell-embryo chimeras. Nature 365(6441),87–89.
95.
WuG.-M., LaiL., MaoJ., McCauleyT.C., CaamañoJ.N., CantleyT., RiekeA., MurphyC.N., PratherR.S., DidionB.A., and DayB. N. (2004). Birth of piglets by in vitro fertilization of zona-free porcine oocytes. Theriogenology, 62, 1544–1556.
96.
YamatoyaK., ItoC., ArakiM., FuruseR., and ToshimoriK. (2011). One-step collagenase method for zona pellucida removal in unfertilized eggs: Easy and gentle method for large-scale preparation. Reprod. Med. Biol. 10, 97–103.
97.
ZhaoM., and DeanJ. (2002). The zona pellucida in folliculogenesis, fertilization and early development. Rev. Endocr. Metab. Disord. 3, 19–26.
98.
ZuccottiM., UrchU.A., and YanagimachiR. (1991). Collagenase as an agent for dissolving the zona pellucida of hamster and mouse oocytes. Reproduction, 93, 515–520.
