Zona pellucida

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Oocyte and developing zona pellucida in the ovary
Oocyte and developing zona pellucida in the ovary
Human blastocyst "hatching" from the zona pellucida
Human blastocyst "hatching" from the zona pellucida (week 1 Carnegie stage 3)

(Latin, zona pellucida = transparent zone) The zona pellucida (ZP) is a specialized extracellular matrix surrounding the developing oocyte (egg, ovum) within each follicle within the ovary. This thick matrix is thought to be formed by secretions from the oocyte and the follicle granulosa cells and in human oocytes consists of four types of zona pellucida glycoproteins ZP1, ZP2, ZP3 and ZP4 which have different roles in fertilization. Note that mice also have 4 ZP genes, but multiple stop and missense codons in ZP4 means that only three are produced. Polymers of ZP2 and ZP3 organized into extended filaments that are cross-linked by ZP1 homodimers.

Note that depending upon species and type of study, the zona pellucida can also be called the: oolemma, egg coat or vitelline membrane.

The zona pellucid has many different roles including in oocyte development, protection during growth and transport, fertilization, spermatozoa binding, preventing polyspermy, blastocyst development, and preventing premature implantation (ectopic pregnancy).

Human oocyte 11.jpg Early zygote.jpg
Human oocyte contained inside zona pellucida.[1] Early zygote inside zona pellucida

In human development, during the first week of development following fertilization the zona pellucida remains surrounding the blastocyst from which it "hatches" to commence implantation.

Fertilization Links: Fertilization | Oocyte | Spermatozoa | Cell Division - Meiosis | Menstrual Cycle | Zona pellucida | Zygote | Lecture - Fertilization | 2016 Lecture | Cell Division - Mitosis | Lecture - Week 1 and 2 | Hydatidiform Mole | Assisted Reproductive Technology | Lecture - Genital Development

Some Recent Findings

Human zygote inside the zone pellucida
Mouse germinal vesicle
Mouse germinal vesicle[2]
  • Perivitelline threads in cleavage-stage human embryos: observations using time-lapse imaging[3] "Time-lapse imaging of the human preimplantation embryo in vitro has revealed a transient phenomenon involving the appearance of perivitelline threads, commonly observed at the two-cell stage. These threads span the perivitelline space, arising at the specific area where the cytoplasmic membrane contacts the zona pellucida, before any perivitelline space is formed. The threads persist as the cytoplasmic membrane retracts from the zona pellucida to form the first cleavage furrow. In this observational report, these structures and their incidence are described. ...Presence or absence of threads did not affect embryo development. This descriptive study is limited; further characterization of these structures is needed to elucidate their potential role in early human embryo development."
  • A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans[4] "The extracellular zona pellucida surrounds ovulated eggs and mediates gamete recognition that is essential for mammalian fertilization. Zonae matrices contain three (mouse) or four (human) glycoproteins (ZP1-4), but which protein binds sperm remains controversial. ...These observations in transgenic mice document that the ZP2(51-149) sperm-binding domain is necessary for human and mouse gamete recognition and penetration through the zona pellucida."
  • Biosynthesis of hamster zona pellucida is restricted to the oocyte[5] "In the present work, the expression of ZP1, ZP2, ZP3 and ZP4 is carefully analyzed by in situ hybridization (ISH) in hamster ovaries. Our data suggest that the four hamster ZP genes are expressed in a coordinate and oocyte-specific manner during folliculogenesis. Furthermore, this expression is maximal during the first stages of the oocyte development and declines in oocytes from later development stages, particularly within large antral follicles."
  • Zona pellucida birefringence in in vivo and in vitro matured oocytes[6]
  • Zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis[7] "These studies revealed for the first time that in humans ZP1, in addition to ZP3 and ZP4, binds to capacitated spermatozoa and induces acrosomal exocytosis."
More recent papers  
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Search term: Zona pellucida Development

Ying Wang, Beibei Zhang, Jinyao Li, Adila Aipire, Yijie Li, Fuchun Zhang Enhanced contraception of canine zona pellucida 3 DNA vaccine via targeting DEC-205 in mice. Theriogenology: 2018, 113;56-62 PubMed 29455062

Hongqin Zhang, Qianqian Luo, Xueyan Lu, Na Yin, Dongli Zhou, Lianshuang Zhang, Wei Zhao, Dong Wang, Pengchao Du, Yun Hou, Yan Zhang, Wendan Yuan Effects of hPMSCs on granulosa cell apoptosis and AMH expression and their role in the restoration of ovary function in premature ovarian failure mice. Stem Cell Res Ther: 2018, 9(1);20 PubMed 29386068

Michal Zigo, Karl Kerns, Miriam Sutovsky, Peter Sutovsky Modifications of the 26S proteasome during boar sperm capacitation. Cell Tissue Res.: 2018; PubMed 29376192

Jinghua Hu, D Jo Merriner, Anne E O'Connor, Brendan J Houston, Luc Furic, Mark P Hedger, Moira K O'Bryan Epididymal cysteine-rich secretory proteins are required for epididymal sperm maturation and optimal sperm function. Mol. Hum. Reprod.: 2018; PubMed 29361143

Kaori Nozawa, Yuhkoh Satouh, Takao Fujimoto, Asami Oji, Masahito Ikawa Sperm-borne phospholipase C zeta-1 ensures monospermic fertilization in mice. Sci Rep: 2018, 8(1);1315 PubMed 29358633

Three-dimensional Structure

Mouse zona pellucida[8]

Electron microscopic studies have shown a variety of appearances from a porous, net-like structure to a nearly smooth and compact structure.

An scanning electron microscopic review[8] of both human and mouse zona pellucida structures at ovulation show:

  • spongy ZP appearance well correlates with mature oocytes.
  • a delicate meshwork of thin interconnected filaments
  • a regular alternating pattern of wide and tight meshes.
  • wide meshes correspond to "pores" of the "spongy" ZP.
    • tight meshes correspond to the compact parts of the ZP surrounding the pores.

Human Zona Pellucida

An immunochemistry study[9] of human ZP proteins expression during folliculogenesis within the ovary primordial follicle identified the presence of ZP1 and ZP3 in most follicles (93% and 95%, respectively), with ZP2 only in 32% of these follicles. The ZP proteins were also detected in the cytoplasm of cuboidal granulosa cells. This localization suggested to the authors that these proteins had been present since oogenesis.

Mouse Zona Pellucida

Mouse zona pellucida

Mouse zona pellucida development.[8]

In the mouse ovarian oocytes, and not granulosa cells, are the only source for mouse ZP glycoproteins.[10]

Zona Pellucida Glycoprotein Features

Human oocyte contained inside zona pellucida
Human oocyte contained inside zona pellucida.[1]
  1. A "ZP domain", which is a signature domain comprised of approximately 260 amino acid (aa) residues.
  2. An N-terminal hydrophobic signal peptide sequence.
  3. A potential N- and O-linked glycosylation sites.
  4. A C-terminal hydrophobic transmembrane-like domain (TMD)
  5. A potential consensus proprotein convertase (furin) cleavage site (CFCS) upstream of transmembrane-like domain (TMD).

Zona Pellucida glycoprotein 1

(ZP1) A 638 amino acid glycoprotein located in the zona pellucida, synthesized by and surrounding the oocyte.

Links: OMIM - ZP1

Zona Pellucida glycoprotein 2

Model of gamete recognition zona pellucida
Model of gamete recognition zona pellucida[11]

(ZP2) A 745 amino acid glycoprotein located in the zona pellucida, synthesized by and surrounding the oocyte. The protein acts as a secondary sperm receptor that binds sperm only after the induction of the sperm acrosome reaction. Before fertilization ZP2 binds spermatozoa. After fertilization ZP2 is proteolytically cleaved as an initial block to polyspermy.

Links: OMIM - ZP2

Zona Pellucida glycoprotein 3

(ZP3) A 424 amino acid glycoprotein located in the zona pellucida, synthesized by and surrounding the oocyte required for initial zona matrix formation and during fertilization for species-specific sperm binding. Now thought to exist in 2 isoforms ZP3A and ZP3B (a second polymorphic allele).

Links: OMIM - ZP3

Zona Pellucida glycoprotein 4

(ZP4) A 540 amino acid glycoprotein located in the zona pellucida, synthesized by and surrounding the oocyte required for initial zona matrix formation and along with ZP3 during fertilization for inducing the acrosome reaction and inhibited the binding of spermatozoa to zona pellucida in a time- and dose-dependent reaction. Associated with several mammalian species (human, rat, hamster and rabbit) but not all mammalian species.

Links: OMIM - ZP4

Zona Pellucida Binding Protein

(ZPBP) Not a zona pellucida protein, but a spermatozoa protein found located on the acrosome surface and one of several proteins that participate in secondary binding between acrosome-reacted sperm and the zona pellucida.

Links: OMIM - ZPBP

Zona Pellucida Birefringence

(ZPB) Optical property of the zona pellucida using polarization imaging when viewed microscopically. This property has been used to qualitatively predict the developmental potential of a in vitro matured metaphase-II (MII) oocytes.

High birefringence, compared to low, has been associated with oocytes contributing to conception cycles when compared with those of nonconception cycles and higher implantation, pregnancy, and live birth rates from transferred oocytes.[12][6] This work follows on from the earlier discovery of an increased birefringence in the meiotic spindle an early indicator of oocyte activation[13]

Oocyte zona birefringence intensity is associated with embryonic implantation potential in ICSI cycles[12]

"Using zona birefringence as the only selection criterion, two fertilized oocytes, preferably derived from high birefringence (HZB) oocytes, were selected for further culture and transfer. The required criteria were met by 135 ICSI cycles (124 patients; 34.9 ± 4.1 years of age). Embryos for transfer were used in 20 cycles derived from HZB/HZB oocytes, in 50 cycles from HZB/LZB oocytes and in 65 from LZB/LZB oocytes. The corresponding implantation (P < 0.025), pregnancy (P < 0.005) and live birth (P < 0.025) rates were significantly different between HZB/HZB and HZB/LZB versus LZB/LZB group. Embryo development was superior in embryos derived from HZB oocytes."

Zona pellucida birefringence in in vivo and in vitro matured oocytes[6]

The percentage of high birefringence oocytes was higher in immature than in mature oocytes (40.1 vs. 23.6%). Among immature oocytes, an increased percentage of HB in prophase-I stage oocytes compared to metaphase I stage oocytes was also observed (50.7 vs. 25.0%). However, the percentage of HB oocytes did not change when comparing oocytes before and after in vitro maturation for both prophase I and metaphase I oocytes. No influence of ZPB was observed on the spontaneous in vitro maturation potential. Exclusively for metaphase II retrieved oocytes, a positive influence of ZPB on fertilization (odds ratio [OR], 1.78; 95% confidence interval [CI], 1.27-2.49) and embryo quality (OR, 2.28; 95% CI, 1.04-4.99) was noted.

Birefringence - Defined as the double refraction of light in a transparent, molecularly ordered material, which is manifested by the existence of orientation-dependent differences in refractive index.

Links: Olympus - Optical Birefringence


  • acrosome reaction - The chemical change within the spermatozoa following binding to the zona pellucida, that leads to the release of acrosomal enzymatic contents. These enzymes degrade the zona pellucida and allow a spermatozoa to penetrate an oocyte.
  • granulosa cell - A specific cell type that proliferates in association with the oocyte within the developing follicles of the ovary. These cells form the follicle stratum granulosa and are also given specific names based upon their position within the follicle. In the antral follicle, membrana granulosa sits on the follicular basal lamina and lines the antrum as a stratified epithelium. The cumulus oophorus is a column of granulosa cells that attaches the oocyte to the follicle wall. The corona radiata are the granulosa cells that directly surround the oocyte, and are released along with it at ovulation. Following ovulation the corona radiata provide physical protection to the oocyte and granulosa cells within the ovulating follicle contribute to corpus luteum.


  1. 1.0 1.1 Wei Shi, Bo Xu, Li-Min Wu, Ren-Tao Jin, Hong-Bing Luan, Li-Hua Luo, Qing Zhu, Lars Johansson, Yu-Sheng Liu, Xian-Hong Tong Oocytes with a dark zona pellucida demonstrate lower fertilization, implantation and clinical pregnancy rates in IVF/ICSI cycles. PLoS ONE: 2014, 9(2);e89409 PubMed 24586757 | PMC3933533 | PLoS One
  2. Hong-Xia Zhou, Yu-Zhen Ma, Ying-Lei Liu, Ying Chen, Cheng-Jie Zhou, Sha-Na Wu, Jiang-Peng Shen, Cheng-Guang Liang Assessment of mouse germinal vesicle stage oocyte quality by evaluating the cumulus layer, zona pellucida, and perivitelline space. PLoS ONE: 2014, 9(8);e105812 PubMed 25144310 | PLoS One.
  3. Louise Kellam, Laura M Pastorelli, Angel M Bastida, Amy Senkbeil, Sue Montgomery, Simon Fishel, Alison Campbell Perivitelline threads in cleavage-stage human embryos: observations using time-lapse imaging. Reprod. Biomed. Online: 2017; PubMed 29074360
  4. M J Izquierdo-Rico, L Gimeno, C Jiménez-Cervantes, J Ballesta, M Avilés Biosynthesis of hamster zona pellucida is restricted to the oocyte. Theriogenology: 2011, 75(3);463-72 PubMed 21074836
  5. M J Izquierdo-Rico, L Gimeno, C Jiménez-Cervantes, J Ballesta, M Avilés Biosynthesis of hamster zona pellucida is restricted to the oocyte. Theriogenology: 2011, 75(3);463-72 PubMed 21074836
  6. 6.0 6.1 6.2 Daniela Paes de Almeida Ferreira Braga, Rita de Cássia Savio Figueira, Priscila Queiroz, Camila Madaschi, Assumpto Iaconelli, Edson Borges Zona pellucida birefringence in in vivo and in vitro matured oocytes. Fertil. Steril.: 2010, 94(6);2050-3 PubMed 20079896
  7. Anasua Ganguly, Antonin Bukovsky, Raj K Sharma, Pankaj Bansal, Beena Bhandari, Satish K Gupta In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis. Hum. Reprod.: 2010, 25(7);1643-56 PubMed 20504872
  8. 8.0 8.1 8.2 Giuseppe Familiari, Michela Relucenti, Rosemarie Heyn, Giulietta Micara, Silvia Correr Three-dimensional structure of the zona pellucida at ovulation. Microsc. Res. Tech.: 2006, 69(6);415-26 PubMed 16703610
  9. Debra A Gook, D H Edgar, J Borg, M Martic Detection of zona pellucida proteins during human folliculogenesis. Hum. Reprod.: 2008, 23(2);394-402 PubMed 18033806
  10. Majid El-Mestrah, Philip E Castle, Girum Borossa, Frederick W K Kan Subcellular distribution of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes. Biol. Reprod.: 2002, 66(4);866-76 PubMed 11906903
  11. Matteo A Avella, Boris Baibakov, Jurrien Dean A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans. J. Cell Biol.: 2014, 205(6);801-9 PubMed 24934154
  12. 12.0 12.1 M Montag, T Schimming, M Köster, C Zhou, C Dorn, B Rösing, H van der Ven, K Ven der Ven Oocyte zona birefringence intensity is associated with embryonic implantation potential in ICSI cycles. Reprod. Biomed. Online: 2008, 16(2);239-44 PubMed 18284880
  13. L Liu, J R Trimarchi, R Oldenbourg, D L Keefe Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes. Biol. Reprod.: 2000, 63(1);251-8 PubMed 10859266


Paul M Wassarman Zona pellucida glycoproteins. J. Biol. Chem.: 2008, 283(36);24285-9 PubMed 18539589



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Cite this page: Hill, M.A. (2018, February 20) Embryology Zona pellucida. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Zona_pellucida

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