Praca oryginalna Original paper
Expression of integrin beta 2 (ITGB2) and zona pellucida
glycoproteins (ZP3, ZP3α) in developmentally competent
and incompetent porcine oocytes*
)
Paweł antosik, Bartosz kemPisty*,**, Hanna Piotrowska***, Dorota Bukowska,
sylwia Ciesiolka*, miCHal Jeseta****, izaBela PiesCikowska*, kinga lange*****, Hieronim maryniak******, JęDrzeJ m. Jaśkowski, klaus P. Brüssow*******,
miCHał nowiCki*, maCieJ zaBel********
institute of Veterinary sciences, Poznan university of life science, 52 wojska Polskiego st., 60-628 Poznan, Poland *Department of Histology and embryology, Poznan university of medical science, 6 swiecickiego st.,
60-781 Poznan, Poland
**Department of anatomy, Poznan university of medical science, 6 swiecickiego st., 60-781 Poznan, Poland ***Department of toxicology, Poznan university of medical sciences, 30 Dojazd st., 60-631 Poznan, Poland ****Department of genetic and reproduction, reproductive Biotechnology unit, Veterinary research institute,
Brno, Czech republic
*****Department of inorganic and analytical Chemistry, karol marcinkowski university of medical sciences, 6 grunwaldzka str., 60-780 Poznan, Poland
******institute of zoology, Department of animal anatomy, Poznan university of life science, 71C wojska Polskiego st., 60-625 Poznan, Poland
*******institute of reproductive Biology, leibniz institute for Farm animal Biology, wilhelm-stahl-allee 2, 18196 Dummerstorf, germany
********Department of Histology and embryology, wroclaw medical university, 6a Chalubinskiego st., 50-368, wroclaw, Poland
Received 09.01.2014 Accepted 18.04.2014
*) This study was made possible due to grant number 2011/03/B/NZ4/02411“OPUS”from Polish Ministry of Scientific Research and Higher Education and
project no. 7AMB13PL052.
antosik P., kempisty B., Piotrowska H., Bukowska D., Ciesiolka s., Jeseta m., Piescikowska i., lange k., maryniak H., Jaśkowski J. m., Brüssow k. P., nowicki m., zabel m.
Expression of integrin beta 2 (ITGB2) and zona pellucida glycoproteins (ZP3, ZP3α) in developmentally competent and incompetent porcine oocytes
Summary
The fertilization potential of mammalian oocytes may be regulated at the molecular level by the expression of species-specific sperm-egg interaction molecules, whose activities and/or cellular distribution determine the recognition and fusion of gemetes. Although there exist studies on the expression of integrins (ITGs) and zona pellucida glycoproteins (ZPs) in developmentally fully competent oocytes, the mRNA levels encoding these proteins in immature and developmentally incompetent porcine oocytes have to be elucidated. Therefore, our aim was to determine the expression of ITGB2, ZP3, and ZP3α mRNAs in porcine oocytes before in vitro maturation (IVM), in oocytes stained with BCB test but colorless, and in BCB positive oocytes after IVM.
Porcine cumulus-oocyte complexes (COCs) were collected from 32 crossbred Landrace gilts, and then separated into three groups: (i) oocytes analyzed immediately after collection (n = 50), (ii) oocytes stained with brilliant cresyl blue (BCB+) and remained colorless (BCB–) (n = 50). After collection and/or staining and cultivation, all oocytes were denuded and analyzed regarding ITGB2, ZP3, and ZP3α by QT-PCR.
We found a higher expression of ITGB2, ZP3, and ZP3α in oocytes immediately after collection and in BCB+ oocytes compared to BCB– oocytes (P < 0.001, respectively). No differences in the ITGB2 and ZP3 mRNA levels were observed between oocytes after collection and BCB+ oocytes. In addition, BCB– oocytes revealed lower transcript expression of all the genes under study.
It is presumed that the similar mRNA levels of ITGB2 and ZP3 in oocytes after collection and in BCB+/ IVM oocytes may be related to (i) a toxic effect of BCB staining and/or to (ii) the degradation of accumulated maternal templates in porcine oocytes. Additionally, lower ITGB2, ZP3, and ZP3α transcript levels point to the down-regulation of the mRNA synthesis of stored maternal transcripts in developmentally incompetent porcine oocytes.
The fertilization potential of mammalian oocytes is one of the factors that determine the further develop-mental competence of female gametes. After success-ful maturation in vivo or in vitro and reaching the MII stage, oocytes can be successfully fertilized by a single spermatozoon (3, 32). However, in many mammalian species, including pigs, polyspermic fertilization be-comes the main problem of in vitro fertilization (IVF) and in vitro embryo production (IVP) (30). The activ-ity of glucose-6-phosphate dehydrogenase (G6PDH) is evaluated by the Brilliant Cresyl Blue (BCB) test. In growing oocytes, the level of synthesized enzyme (G6PDH) is higher, but in oocytes that have completed their growth phase the enzyme is inactivated (25, 27). Fertilization is a complex process that includes the species-specific recognition, interaction, and fusion of male and female gametes. It is accepted that all of these steps are regulated at the molecular level by a proper or abnormal expression of gamete interaction-dependent molecules, which include proteins of both oocytes and the surrounding somatic cumulus cells (4, 29).
It has been shown that both sperm- and oocyte-specific proteins are involved in gamete interaction (5, 18, 28). The most common proteins involved in fertilization include zona pellucida glycoproteins, which form the zonal structure. In several mammalian species, the most important role during fertilization is played by the zona pellucida glycoproteins 1 (ZP1), 2 (ZP2), and 3 (ZP3). However, in other species, in-cluding pigs, there is also an additional zona pellucida glycoprotein 3α, called ZP4 (35). It has been shown that ZP3 plays the role of a primary receptor, whereas ZP2 functions as a secondary sperm receptor (2, 9, 11, 16). ZP1 forms the main structure of the zona matrix and, therefore, presumably plays a structural/integral role in the zona pellucida rather than functions as an important factor regulating the interaction of gametes during fertilization (10). It has been shown that the expression of ZPs may be regulated by several intrinsic and extrinsic factors, including the follicular size and morphology of female gametes (13).
The role of zona pellucida proteins in the interaction of mammalian gametes was intensively investigated in several studies (23, 24). However, the role of ZP3 and ZP4 in sperm-egg binding still needs further investiga-tion. In a similar study, Yonezawa et al. (35) used the baculovirus-Sf9 cell expression system for porcine ZP glycoproteins to investigate the interaction of porcine ZP3 and ZP4 and their ability to form a heterocomplex consisting of ZP3 and ZP4. Previously, these authors found that a mixture of recombinant ZP3 (rZP3) and ZP4 (rZP4) displayed sperm-binding activity towards bovine spermatozoa but not towards porcine sperma-tozoa, probably because of differences in the carbo-hydrate structure between native and recombinant ZP glycoproteins. Moreover, they observed that a mixture of porcine rZP3 and native ZP4 (nZP4) down-regulates the binding of porcine spermatozoa to the zona
pel-lucida. A mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine spermatozoa, although the mixture inhibited the binding of bovine spermato-zoa. They conclude that nZP4, but not rZP4, is neces-sary for the binding activity of the porcine ZP3/ZP4 complex towards porcine spermatozoa. Furthermore, they suggest that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-ZP binding activity.
It has been found that proteins responsible for cell recognition and/or adhesion may also regulate im-portant stages of fertilization (14). Integrins, which belong to the large family of adhesion molecules, are found to a large extent in several types of cancer cells (6). However, the expression of integrins, especially of integrin beta 2 (ITGB2), and the distribution of pro-teins have also been observed within porcine and bitch oocytes. ITGB2 belongs to the family of leucocyte adhesion glycoproteins, which includes beta subunit (90 to 110 kD). Hynes (12) showed that the diversity of formation of alpha-beta heterodimers is specifically restricted to the type of cells in which integrins are overexpressed.
Although some data have been published regarding the role of ZP glycoproteins and integrins during fer-tilization in many mammalian species, there are still few studies indicating a differential expression of these genes in relation to the developmental competence of female gametes. Therefore, the aim of this study was to compare the expression patterns of ZP3, ZP3α, and ITGB2 mRNAs in BCB+ and BCB– porcine oocytes.
Material and methods
Animals and collection of porcine ovaries and cumulus-oocyte complexes (COCs). A total of 32 crossbred Landrace
gilts with a median age of 160 days (range 140-180 days) and a median weight of 100 kg (95-120 kg) were used in this study. The experiments were approved by the local ethical committee.
The ovaries and reproductive tracts were recovered at slaughter and transported to the laboratory within 30 min at 38.5°C in 0.9% NaCl.
There, the ovaries were placed in a 5% fetal bovine se-rum solution (FBS) (Sigma-Aldrich Co. St. Louis, MO) in phosphate buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA). The follicles with a size of 3-5 mm were opened by puncturing individually in a sterile petri dish, and COCs were recovered. These were washed three times in modi-fied PBS supplemented with 36 µg/ml pyruvate, 50 µg/ml gentamycin, and 0.5 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA). The COCs were then visualized under a stereoscopic microscope, counted, and carefully evaluated morphologically with the use of a four-grade scale, as described previously (13). Only the four-grade I COCs, with a homogeneous cytoplasm and a complete and compact cumulus oophorus, were used in further steps of the experiment. COCs were denuded, and oocytes were subjected to different treatment: group I (n = 50) were analyzed imme-diately after collection without BCB staining, group II (n = 50) were stained by BCB but remained colorless (BCB–),
and group III (n = 50) were stained blue by BCB (BCB+) and underwent in vitro maturation (IVM).
Assessment of oocyte developmental competence by the brilliant cresyl blue (BCB) test. Oocytes from groups
II and III were washed two times in modified Dulbecco phos-phate buffered saline (PBS-DPBS) (Sigma-Aldrich Co. St. Louis, MO) supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin (Sigma-Aldrich), 0.4% [w/v] BSA, 0.34 mM pyruvate, and 5.5 mM glucose (DPBSm). The oocytes were treated with 26 µM BCB (Sigma-Aldrich) diluted in DPBSm at 38.5°C in 5% CO2 in air for 90 min. After treatment, the oocytes were transferred to DPBSm and washed two times. During the washing procedure, they were examined under an inverted microscope (Zeiss, Axiovert 35, Lübeck, Germany) and classified as either stained blue (BCB+) or colorless (BCB–). Only BCB+ oocytes, which had completed their growth phase and potentially had a higher developmental competence, were used for IVM.
In vitro maturation of porcine oocytes. BCB+ oocytes
were cultured in Nunclon™Δ 4-well dishes in 500 µl stan-dard porcine IVM culture medium; TCM 199 (tissue culture medium) with Earle’s salts and L-glutamine (Gibco BRL Life Technologies, Grand Island, NY, USA), supplemented with 2.2 mg/ml sodium bicarbonate (Nacalai Tesque, Inc., Kyoto, Japan), 0.1 mg/ml sodium pyruvate (Sigma-Aldrich), 10 mg/ml BSA, (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mg/ml cysteine (Sigma-Aldrich), 10% (v/v) filtered por-cine follicular fluid and gonadotropin supplements at a final concentration of 2.5 IU/ml hCG (Ayerst Laboratories, Inc. Philadelphia, PA, USA) and 2.5 IU/ml eCG (Intervet, Whitby, ON, Canada). Wells were covered with a mineral oil overlay, and the cells were cultured for 44 h at 38°C in 5% CO2 in air.
Real-time quantitative PCR (QT-PCR) analysis of
ITGB2, ZP3, and ZP3α mRNA expression. Total RNA was
isolated from the oocytes of all experimental groups (13). The RNA samples were resuspended in 20 µl of RNase-free water and stored in liquid nitrogen. RNA samples were treated with DNase I and reverse-transcribed into cDNA (RT). QT-PCR was conducted in a LightCycler real-time PCR detection system (Roche Diagnostics GmbH, Mannheim, Germany) using SYBR® Green I as the detection dye, and target cDNA
was quantified by the relative quantification method. For amplification, 2 µl of the total (20 µl) cDNA solution was added to 18 µl of QuantiTect® SYBR® Green PCR (Master
Mix Qiagen GmbH, Hilden, Germany) and primers. One RNA sample of each preparation was processed without the RT (reverse transcription) reaction to provide a negative control in the subsequent PCR. To ensure that granulosa cells did not contaminate the oocytes, we demonstrated the absence of cytochrome P450 aromatase transcript by RT and QT-PCR.
The housekeeping genes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ACTB were amplified as ref-erencesfor mRNA quantification.
In order to quantify specific gene expression in oocytes, the levels ofthe expression of specific mRNAs in each sample were calculated relative to those of GAPDH and ACTB. To ensure theintegrity of these results, an additional housekeeping gene of18S rRNA was used as an internal standard to ensure that GAPDH and ACTB mRNAs were not regulated in the three groups of oocytes. These genes have been identified as appropriate housekeeping genes for use
in quantitative PCR studies (31). The expression of GAPDH and ACTB did not vary when normalized against 18S rRNA (results not shown).
Statistical analysis. ANOVA followed by the Tukey
post-test was used to compare the results of real-time QT-PCR quantification. The experiments were carried out in at least three replicates. The results quantifying the relative abun-dance (RA) of investigated mRNAs were expressed as the mean of the transcripts GAPDH/ACTB/18S rRNA ratio. The differences were considered to be significant at *P < 0.05, **P < 0.01, and ***P < 0.001. The software program Graph-Pad Prism version 4.0 (GraphGraph-Pad Software, San Diego, CA) was used for statistical calculations.
Results and discussion
The QT-PCR analysis revealed a 4-fold and 20-fold higher expression pattern of ZP3 mRNA compared to ITGB2 and ZP3α, respectively. Regarding the ITGB2 mRNA level, we found similar expression profiles in oocytes immediately after collection and in BCB+ oocytes, but their expression in BCB– oocytes was different (P < 0.001). No differences in ZP3 mRNA expression profiles were observed in oocytes of groups I and III, but the mRNA level in BCB– oocytes was lower (P < 0.001). Regarding ZP3α, an augmented mRNA expression was observed in oocytes of group I compared to both BCB– and BCB+ oocytes (P < 0.01, P < 0.001, respectively). The ZP3α mRNA level, as well, was much lower in BCB– oocytes compared to oocytes of the other groups (P < 0.001).
The fertilization potential of mammalian oocytes is determined by extrinsic and intrinsic factors, including follicular environment, oviductal fluid composition, conformation and number of oocyte surrounding so-matic cell layers, cell-to-cell interaction, and gamete interaction. The interaction between female and male gametes is regulated on several levels, which includes the specific recognition of gametes, spermatozoon-cumulus cell interaction, and gamete fusion (8). It has been shown that these processes are regulated on a molecular level by the expression of species-specific “fertilization potential” genes and proteins, which mainly include zona pellucida proteins (ZPs), integrins (ITGs), and adhesion molecules, such as CD9 (15). The expression of mRNAs and the distribution of oocyte-specific proteins within the cytoplasm of the gamete, zona pellucida and/or oocyte membrane are thought to be determined by extrinsic and intrinsic factors, such as follicular size, COC morphology, and (steroid) hormone milieu (1, 20-22, 31).
Since most of the studies used fully developmen-tally competent oocytes (BCB+) before and/or after IVM, our study was aimed at examining the influence of the IVM procedure on ITGB2, ZP3, and ZP3α mRNA expressions by comparing BCB+ oocytes after maturation (developmentally competent) and oocytes before BCB staining or those that remained colorless (BCB–) (developmentally incompetent). Our study showed similar patterns of ITGB2, ZP3
and ZP3α mRNAs expression in all three groups of porcine oocytes. Regarding ITGB2 and ZP3, which are important regulators of molecular mechanisms of fertilization, we did not observe differences in the expression of these transcripts between oocytes before staining and BCB+/IVM oocytes. This indicates that porcine oocytes immediately after recovering from
the follicle and oocytes stained positive in the BCB test have a different potential for in vitro maturation and fertilization than BCB– oocytes or that the BCB staining has some effect on oocytes and, therefore, it leads to a degradation of mRNAs accumulated during folliculo- and oogenesis. Our hypothesis related to a “block” of maternal template synthesis following the attainment of the MII stage by growing oocytes, was confirmed by the observations of Yu et al. (36). They demonstrated that MSY2, described as a regulator of maternal translation and mRNA stability during oogen-esis in mice and used as the Msy2 hairpin dsRNA in growing oocytes in the oocyte-specific ZP3 promoter, leads to a significant reduction (by 60%) of the MSY2 protein product in fully grown oocytes. Furthermore, they observed that the fertility potential was signifi-cantly reduced, the oocytes did not manifest normal
Ca2+ oscillations, failed to resume meiosis properly
or to undergo normal cortical granule exocytosis. They also did not manifest ZP2 cleavage to ZP2f. In transgenic mice, an abnormal chromatin configuration and spindle structure was also found. Additionally, the amount of accumulated mRNAs was reduced (ap-proximately 75-80%, as compared to normal mouse oocytes), and protein synthesis in transgenic oocytes was much lower. Taking into account these and our own observations, it may be presumed that mature and/or immature, developmentally competent and/or incompetent oocytes manifest distinct activity of the mRNA synthesis machinery.
Regarding the BCB staining test, it was shown (19, 34) that only double staining with the BCB test may have a toxic effect on oocytes and down-regulates their fertilization potential, inhibiting the expression of ZP glycoproteins and their cellular distribution. A similar mRNA expression pattern, as demonstrated in our study, may suggest that the BCB staining test is a good predictor of the developmental competence of oocytes, but it is not a sufficient procedure for determining the fertilization potential of porcine oocytes. However, the much lower expression of mRNAs encoding ITGB2, ZP3, and ZP3α in BCB– oocytes reveals that these probably developmentally incompetent cells have a lower fertilization potential. In addition, the expres-sion of ZP3α mRNA reduced to approximately 5% suggests that this gene and its encoded protein have a less pronounced role in the achievement of fertiliza-tion potential by porcine oocytes.
Although there are several reports indicating a role of ZP glycoproteins in mammalian fertilization, there are still only few data describing the role of ITGB2 in this process (7, 26). Kempisty et al. (17) investigated the expression of mRNAs encoding integrins (alphaL, alphaM, beta1 and beta6), CD9, and CD18 antigens, as well as zona pellucida glycoproteins (pZP1, pZP2, pZP3 and pZP3alpha) in oocytes collected from pu-beral gilts and multiparous sows. They found a sig-nificantly increased expression of alphaL, alphaM,
Fig. 1. Relative abundance of ITGB2, ZP3 and ZP3α tran-scripts in competent and incompetent porcine oocytes. The porcine oocytes (before IVM, BCB+ and BCB–) isolated from pubertal gilts were immediately used to isolate RNA, which was reverse-transcribed into cDNA. The presence of ITGB2 (A), ZP3 (B) and ZP3α (C) transcripts was evaluated by RQ-PCR analysis. Results are presented as mean ± SEM with the level of significance, * P < 0.05, ** P < 0.01, *** P < 0.001
Before IVM BCB+ BCB– Relative abundance of mRNA 0 0,5 1 1,5 2 2,5 ITGB2 A Before IVM BCB+ BCB– Relative abundance of mRNA 0 2 1 4 3 6 5 8 7 10 9 Zp3 B Before IVM BCB+ BCB– Relative abundance of mRNA 0 0,1 0,05 0,2 0,15 0,3 0,25 0,4 0,35 0,5 0,45 Zp3α C
beta1, and beta6 integrins, CD9 antigen, and of pZP2 and pZP3 mRNAs in oocytes isolated from puberal gilts compared to oocytes isolated from multiparous sows. The higher level of transcripts may be related to the age of sows. On the other hand, it may also be hypothesized that the higher expression of these mRNAs may be associated with a higher mRNA ac-cumulation (maternal transcripts) in competent oocytes isolated from younger compared to older sow donors, in which maternal templates may be degraded follow-ing maturation and the attainment of the MII stage. In a previous study (19), ITGB2 and ZP3 mRNAs, as well as the respective proteins, were analyzed after a single and double exposure to BCB. Contrary to that study, we did not find an increase in ITGB2 mRNA levels after a single exposure to BCB. However, we obtained the same results regarding ZP3 mRNA expression. Taking into account both of these observations, it can be presumed that a single BCB staining may have a stimulatory effect or exert no effect on the expression of ITGB2 and ZP3 mRNAs.
In conclusion, the differential mRNAs expression pattern of genes encoding ITGB2, ZP3, and ZP3α is related to the stage of the developmental compe-tence of oocytes and is down- and/or up-regulated in a maturation-stage-dependent manner.
References
1. Antosik P., Kempisty B., Bukowska D., Jackowska M., Wlodarczyk R., Budna J., Brüssow K. P., Lianeri M., Jagodzinski P. P., Jaskowski J. M.: Follicular size is associated with the levels of transcripts and proteins of selected molecules responsible for the fertilization ability of oocytes of puberal gilts. J. Reprod. Dev. 2009, 55, 588-593.
2. Bansal F., Gupta S. K.: Binding characteristics of sperm with recombinant human zona pellucida glycoprotein-3 coated beads. Indian J. Med. Res. 2009, 130, 37-43.
3. Blanco M. R., Demyda S., Moreno Millán M., Genero E.: Developmental com-petence of in vivo and in vitro matured oocytes: A review. Biotechnol. Mol. Biol. Rev. 2011, 6, 155-165.
4. Bromfield E. G., Nixon B.: The function of chaperone proteins in the assem-blage of protein complexes involved in gamete adhesion and fusion processes. Reproduction 2013, 145, R31-42.
5. Depa-Martynow M., Kempisty B., Lianeri M., Jagodzinski P. P., Jedrzejczak P.: Association between fertilin beta, protamines 1 and 2 and spermatid-specific linker histone H1-like protein mRNA levels, fertilization ability of human sper-matozoa, and quality of preimplantation embryos. Folia Histochem. Cytobiol. 2007, 45, 79-85.
6. Fabryova K., Simon M.: Function of the cell surface molecules (CD molecules) in the reproduction processes. Gen. Physiol. Biophys. 2009, 28, 1-7. 7. Fusi F., Bronson R. A.: Failure to detect integrins of the beta-2 subclass on the
human sperm surface. Am. J. Reprod. Immunol. 1991, 25, 143-145.
8. Gadella B. M.: Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation. Reprod. Fertil. Dev. 2012, 25, 26-37.
9. Gupta S. K., Bhandari B., Shrestha A., Biswal B. K., Palaniappan C., Malhotra S. S., Gupta N.: Mammalian zona pellucida glycoproteins: structure and function during fertilization. Cell Tissue Res. 2012, 349, 665-678.
10. Gupta S. K., Chakravarty S., Suraj K., Bansal P., Ganguly A., Jain M. K., Bhandari B.: Structural and functional attributes of zona pellucida glycoproteins. Soc. Reprod. Fertil. Suppl. 2007, 63, 203-216.
11. Howes E., Pascall J. C., Engel W., Jones R.: Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization. J. Cell. Sci. 2001, 114, 4127-4136. 12. Hynes R. O.: Integrins: bidirectional, allosteric signaling machines. Cell 2002,
110, 673-687.
13. Jackowska M., Kempisty B., Antosik P., Bukowska D., Budna J., Lianeri M., Rosinska E., Wozna M., Jagodzinski P. P., Jaskowski J. M.: The morphology of porcine oocytes is associated with zona pellucida glycoprotein transcript contents. Reprod. Biol. 2009, 9, 79-85.
14. Jansen S., Ekhlasi-Hundrieser M., Töpfer-Petersen E.: Sperm adhesion mol-ecules: structure and function. Cells Tissues Organs 2001, 168, 82-92.
15. Jaslow C. R., Patterson K. S., Cholera S., Jennings L. K., Ke R. W., Kutteh W. H.: CD9 expression by human granulosa cells and platelets as a predictor of fertiliza-tion success during IVF. Obstet. Gynecol. Int. 2010, doi: 10.1155/2010/192461. 16. Jovine L., Qi H., Williams Z., Litscher E., Wassarman P. M.: The ZP domain is a conserved module for polymerization of extracellular proteins. Nat. Cell Biol. 2002, 4, 457-461.
17. Kempisty B., Antosik P., Bukowska D., Jackowska M., Lianeri M., Jaskowski J. M., Jagodzinski P. P.: Assessment of zona pellucida glycoprotein and integrin transcript contents in porcine oocytes. Reprod. Biol. 2009, 9, 71-78. 18. Kempisty B., Depa-Martynow M., Lianeri M., Jedrzejczak P., Darul-Wasowicz A.,
Jagodzinski P. P.: Evaluation of protamines 1 and 2 transcript contents in sper-matozoa from asthenozoospermic men. Folia Histochem. Cytobiol. 2007, 45, 109-113.
19. Kempisty B., Jackowska M., Piotrowska H., Antosik P., Wozna M., Bukowska D., Brüssow K. P., Jaskowski J. M.: Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test. Theriogenology 2011, 75, 1525-1535.
20. Kim M. K., Fibrianto Y. H., Oh H. J., Jang G., Kim H. J., Lee K. S., Kang S. K., Lee B. C., Hwang W. S.: Effects of estradiol-17beta and progesterone supple-mentation on in vitro nuclear maturation of canine oocytes. Theriogenology 2005, 63, 1342-1353.
21. Kölle S., Dubois C. S., Caillaud M., Lahuec C., Sinowatz F., Goudet G.: Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte matura-tion, and embryogenesis. Mol. Reprod. Dev. 2007, 74, 851-859.
22. Larson J. E., Krisher R. L., Lamb G. C.: Effects of supplemental progesterone on the development, metabolism and blastocyst cell number of bovine embryos produced in vitro. Reprod. Fertil. Dev. 2011, 23, 311-318.
23. Lay K. M., Ashizawa K., Nakada T., Tatemoto H.: N-glycosylation of zona glycoproteins during meiotic maturation is involved in sperm-zona pellucida interactions of porcine oocytes. Theriogenology 2011a, 75, 1146-1152. 24. Lay K. M., Oshiro R., Arasaki C., Ashizawa K., Tatemoto H.: Role of
acidifica-tion elicited by sialylaacidifica-tion and sulfaacidifica-tion of zona glycoproteins during oocyte maturation in porcine sperm-zona pellucida interactions. J. Reprod. Dev. 2011b, 57, 744-751.
25. Opiela J., Kątska-Książkiewicz L.: The utility of Brilliant Cresyl Blue (BCB) staining of mammalian oocytes used for in vitro embryo production (IVP). Reprod. Biol. 2013, 177-183.
26. Orvieto R., Ben-Rafael Z., Abir R., Bar-Hava I., Fisch B., Molad Y.: Controlled ovarian hyperstimulation: a state of neutrophil activation. Am. J. Reprod. Immunol. 1999, 42, 288-291.
27. Pawlak P., Pers-Kamczyc E., Renska N., Kubickova S., Lechniak D.: Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts. Theriogenology 2011, 832-840.
28. Rath D., Töpfer-Petersen E., Michelmann H. W., Schwartz P., von Witzendorff D., Ebeling S., Ekhlasi-Hundrieser M., Piehler E., Petrunkina A., Romar R.: Structural, biochemical and functional aspects of sperm-oocyte interactions in pigs. Soc. Reprod. Fertil. Suppl. 2006, 62, 317-330.
29. Redgrove K. A., Anderson A. L., McLaughlin E. A., O’Bryan M. K., Aitken R. J., Nixon B.: Investigation of the mechanisms by which the molecular chaperone HSPA2 regulates the expression of sperm surface receptors involved in human sperm-oocyte recognition. Mol. Hum. Reprod. 2013, 19, 120-135.
30. Somfai T., Ozawa M., Noguchi J., Kaneko H., Karja N. W., Fahrudin M., Nakai M., Maedomari N., Dinnyés A., Nagai T., Kikuchi K.: In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa. Anim. Reprod. Sci. 2008, 107, 131-147.
31. Thellin O., Zorzi W., Lakaye B., De Borman B., Coumans B., Hennen G., Grisar T., Igout A., Heinen E.: Housekeeping genes as internal standards: use and limits. J. Biotechnol. 1999, 75, 291-295.
32. Wang W. H., Abeydeera L. R., Prather R. S., Day B. N.: Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization. Mol. Reprod. Dev. 1998, 49, 308-316. 33. Willingham-Rocky L. A., Hinrichs K., Westhusin M. E., Kraemer D. C.: Effects
of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro. Reproduction 2003, 126, 501-508. 34. Wongsrikeao P., Otoi T., Yamasaki H., Agung B., Taniguchi M., Naoi H.,
Shimizu R., Nagai T.: Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos. Theriogenology 2006, 66, 366-372.
35. Yonezawa N., Kanai-Kitayama S., Kitayama T., Hamano A., Nakano M.: Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex. Zygote 2012, 20, 389-397.
36. Yu J., Deng M., Medvedev S., Yang J., Hecht N. B., Schultz R. M.: Transgenic RNAi-mediated reduction of MSY2 in mouse oocytes results in reduced fertility. Dev. Biol. 2004, 268, 195-206.
Corresponding author: DVM Bartosz Kempisty, PhD, Department of Histology and Embryology, Department of Anatomy, Poznan University of Medical Sciences, Poznan, Poland; e-mail: etok@op.pl
