Jędrzej M. Jaśkowski

The morphology of porcine oocytes is associated with zona pellucida glycoprotein transcript contents

We hypothesized that oocyte morphology may be associated with the accumulation of specific mRNAs encoding proteins responsible for the gamete fertilization ability. Therefore, the aim of the study was to evaluate the transcript levels of porcine zona pellucida (pZP1, pZP2, pZP3 and pZP4) glycoproteins in oocytes classified by a four-grade morphological scale (I-IV) accounting for either a homogeneous cytoplasm and a complete cumulus oophorus (grade I) or a heterogenous cytoplasm and decreased number of cumulus layers in the other grades (II, III and IV). We observed a significant increase of all investigated pZP glycoprotein mRNAs in grade I oocytes as compared to other grades (p<0.05). Our observations suggest that porcine oocyte morphology is associated with pZP transcript contents and may be related to an increased fertilization ability of higher quality oocytes.

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

Brilliant cresyl blues (BCB) staining test is a useful tool in assessing the competence of cumulus-oocyte-complexes (COCs) in several mammalian species. It is mostly used to select gametes after they are recovered from the ovary or before and after IVM to isolate those oocytes that reach developmental competency. However, there is evidence that double exposure to BCB test may lead to impaired fertilization or even have a toxic effect on cells. The aim of the present study was to investigate the expression pattern of sperm-egg interaction molecules in oocytes after single and double exposure to BCB test. Follicles were dissected from porcine ovaries after slaughter and aspirated COCs were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The BCB test was applied to COCs before and after IVM. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH; BCB test), real-time quantitative PCR reaction methods, western blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoprotein 3 (pZP3), and integrin beta 2 (ITGB2), as well as the levels of pZP3 and ITGB2 proteins. In the control group, assessment of the expression of the investigated genes was performed before and after IVM without BCB test. We observed a significantly higher level of pZP3 mRNA in oocytes after single exposure to BCB test compared to control before and after IVM (P < 0.001), and to double staining (P < 0.05). The level of ITGB2 mRNA was also increased in gametes after single exposure to BCB test as compared to control before and after IVM (P < 0.001, P < 0.01, respectively), and double staining (P < 0.05). Western blot analysis demonstrated a higher level of pZP3 protein in oocytes after single staining with BCB as compared to control both before and after IVM (P < 0.001, P < 0.05, respectively) and double staining (P < 0.05). Confocal microscopic observations have revealed the same pattern of increased level of pZP3 and ITGB2 expression after single exposure to BCB test. In both cases we detected specific cytoplasmic localization of both proteins. The ITGB2 protein has zona pellucida and membrane localization in control oocytes before IVM. After IVM and after single exposure to BCB, ITGB2 was also strongly detected in the cytoplasm. In both cases, after double exposure to BCB both proteins were detected only partially in the cytoplasm. Our results suggest that (i) single exposure to BCB increased the expression of sperm-oocyte interaction genes, (ii) double exposure to BCB leads to only partial expression of pZP3 and ITGB2 in oocyte cytoplasm, (iii) the BCB staining test itself may be a cause of specific pZP3 translocation from the zona pellucida to the cytoplasm, and that (iv) in vitro maturation of oocytes may increase ITGB2 expression and translocation from the zona pellucida to the cytoplasm.

Assessment of zona pellucida glycoprotein and integrin transcript contents in porcine oocytes

Using reverse transcription and real-time quantitative PCR analysis we evaluated the transcript levels of integrins (alphaL, alphaM, beta1, and beta6), CD9 and CD18 antigens as well as zona pellucida glycoproteins (pZP1, pZP2, pZP3 and pZP3alpha) in oocytes isolated from puberal gilts (n=20) and multiparous sows (n=20). We found significantly (p<0.05) higher transcript contents of alphaL, alphaM, beta1, and beta integrins, CD9 antigen, and pZP2 and pZP3 in puberal gilt oocytes compared to multiparous sow oocytes. Our results suggest that a decrease in the level of oocyte transcripts encoding essential proteins involved in oocyte fertilization may be associated with increased porcine female age.

The expression of genes encoding zona pellucida glycoproteins in canine cumulus-oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol

The role of progesterone (P4) and estradiol-17beta (E2) on the efficiency of canine oocyte maturation in vitro is recognized, but little is known about the influence of both steroids on the expression of zona pellucida (ZP) glycoproteins. It has been shown that E2 and P4 used in the IVC significantly influenced canine oocytes meiotic competence, although the effect is specifically related to the combination of hormones used in the experiment. Because both of these steroids may stimulate or inhibit maturation competence of oocytes in a dose-dependent manner, there is a high possibility that they also influence the fertilization ability of canine oocytes. Our study was aimed to analyze whether genes, encoding ZP glycoproteins, are regulated by P4 or E2. Canine cumulus oocyte complexes (COCs) were recovered from anestrous mongrel bitches after ovariohysterectomy and cultured in serum-free tissue culture medium 199. The expression pattern of ZP glycoproteins 2 and 3 (ZP2 and ZP3) mRNAs, using quantitative real-time polymerase chain reaction (RQ-PCR), and of ZP3 and ZP4 proteins, using Western blot analyses, was examined in oocytes after the supplementation of the culture medium with (1) 0.5 μg/mL, 1.0 μg/mL, and 2.0 μg/mL of P4 (experiment 1), or with (2) 2.0 μg/mL E2, and with (3) a combination of E2 (2.0 μg/mL) and P4 (0.5, 1.0, or 2.0 μg/mL, respectively; experiment 2). The analysis revealed an inhibited expression of ZP2 mRNA in oocytes after in vitro maturation (IVM) with different P4 supplementations as compared with oocytes before IVM. The expression of ZP3 mRNA was stimulated (P < 0.01) by the supplementation of 1.0 μg/mL P4. The expression of both ZP3 and ZP4 proteins was also stimulated after the treatment with 1.0 μg/mL P4. On the other hand, the level of ZP2 mRNA was inhibited (P < 0.01) after the supplementation with E2 or with combinations of E2 and P4 as compared with control oocytes. The expression of ZP3 mRNA was significantly higher after the supplementation with E2 and 0.5 μg/mL P4. Similarly, ZP3 and ZP4 proteins were highly expressed (P < 0.01) after such hormone supplementation. The results clearly show that in vitro, P4 regulates the expression of ZP glycoproteins in a dose-dependent manner. We demonstrated that E2 used alone and in combination with P4 upregulates the expression of ZP3 mRNA as well as ZP3 and ZP4 protein in canine oocytes. ZP2 mRNA is downregulated by E2 alone and in combination with E2 and P4. Furthermore, ZP glycoproteins expression is regulated by E2 alone or in combination with P4, and such synergistic or adverse effect is P4 concentration-dependent.

Real-time proliferation of porcine cumulus cells is related to the protein levels and cellular distribution of Cdk4 and Cx43

The proper maturation of cumulus somatic cells depends on bidirectional communication between the oocyte and the surrounding cumulus cells (CCs). The aim of this study was (i) to investigate maturation markers, such as Cx43 and Cdk4 protein levels, and (ii) to analyze the distribution of these two proteins in CCs cultured for 44, 88, 132, and 164 hours in both separated and cumulus-enclosed oocyte cultures. CCs were isolated from porcine ovarian follicles after the treatment of the recovered COCs with collagenase. Then, the separated CCs were cultured in TCM-199 for 0 to 164 hours, using a real-time cellular analyzer; however, the immunostaining was performed only after 44, 88, and 132 hours. The protein levels and distribution were analyzed using confocal microscopy. After the CCs underwent in vitro cultivation (IVC) for 25 hours, a logarithmically increasing normalized proliferation index was found throughout the entire 164 hours cultivation time. The Cx43 and Cdk4 proteins were observed at higher levels after 44 hours of culture than before IVC. After 88 and 132 hours of IVC, no significant alterations in either mRNA or protein levels of Cx43 and Cdk4 were found. Cx43 and Cdk4 were localized in the cell nucleus before IVC, whereas after 44, 88, and 132 hours of IVC, both proteins translocated to the cytoplasm. In cumulus-enclosed oocyte cultures, Cdk4 was localized both in the nucleus and cytoplasm, whereas Cx43 was only in the cytoplasm. Additionally, only low levels of the cumulus expansion markers MIS and SNAT3 were observed. In summary, we could demonstrate that the in vitro cultivation of CCs was associated with cell proliferation and that Cx43 and Cdk4 gene expression was upregulated after IVC, resulting in significantly higher protein levels. Moreover, the two proteins translocated from the nucleus to the cytoplasm of the CCs during IVC. The protein distribution is presumably related to different protein functions during bidirectional communication via gap junction communication.

Are the levels of Cdk4 and Cx43 proteins of porcine oocytes associated with follicular size

Gap junction connections are formed by proteins which play an important role in oocyte developmental competency but there is little information on the relationship between follicle size and the expression of genes encoding these proteins. The aim of this study was to investigate the potential association between follicle size and the levels of Cdk4 and Cx43 proteins using western blot analysis and confocal microscopic observations. Cumulus-oocyte complexes (COCs) were collected from puberal gilts (n = 20) of large (>5 mm), medium (3-5 mm), and small (<3 mm) follicles, and stained with BCB. BCB+ COCs, which had finished their growth phase, were cultured in TCM 199 for 44 h. Western blot analysis revealed an increased level of Cdk4 protein in oocytes isolated from large follicles as compared to medium (P < 0.05) and small (P < 0.01) ones. We did not detect differences in Cx43 protein levels in oocytes collected from any follicle class. Confocal microscopic observation revealed a specific membrane and zona pellucida localization of Cdk4 protein in oocytes isolated from large follicles, but an exclusively cytoplasmatic distribution of Cdk4 in oocytes from smaller follicle categories. The effect of follicular size on Cdk4 is indicated by the higher level of Cdk4 protein in oocytes isolated from large follicles and its variable distribution – perhaps resulting from a specific translocation mechanism – in the membrane, zona pellucida, and cytoplasm. IVM may also have a significant effect on Cdk4, as seen from the considerable difference in the expression and localization of Cdk4 protein in oocytes after IVM.

Short-term Cultivation of Porcine Cumulus Cells Influences the Cyclin-dependent Kinase 4 (Cdk4) and Connexin 43 (Cx43) Protein Expression—A Real-time Cell Proliferation Approach

Abstract The CC (cumulus cell) proliferation index in relation to the expression and distribution of Cdk4 and Cx43 proteins, which are crucial factors for oocyte maturation, was investigated. Cumulus-oocyte complexes (COCs) were recovered from pubertal crossbred Landrace gilts and treated with collagenase, and separated CCs were cultured in standard TCM199 medium for 44 h. At each step of in vitro cultivation (IVC) of CCs (0, 12, 24 and 44 h), a normalized proliferation index was assessed. Cdk4 and Cx43 protein expression and the CC-specific cellular distribution were analyzed by confocal microscopic observation. The normalized proliferation index (number of cells attached, measured by impedance) was increased in the first 12 h of IVC (P<0.01) and differed between 12 h and 24 h of cultivation (P<0.001). Later, between 24 h–44 h of IVC, the CC proliferation rate was stable, and no significant differences were observed. Based on the confocal microscopic observation, increased expression of both Cdk4 and Cx43 was found after 44 h of IVC compared with the expression of these proteins before IVC. Moreover, after IVC, a substantial translocation of Cdk4 and Cx43 was noted from the nucleus to the cytoplasm of CCs. In conclusion, it was demonstrated for the first time that CCs can be cultured in vitro separately without oocytes and that the proliferation index was significantly increased in the first 12 h of IVC, which may reflect the process of ordinary cumulus cell expansion. Furthermore, the expression of both Cdk4 and Cx43 in CCs suggested that these proteins may be regarded as markers not only of proper oocyte maturation but also of CC differentiation. Translocation of these proteins into the cytoplasm of CCs after 44 h of IVC may be related to the expansion process.

Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation

This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus-oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.

Expression of INHβA and INHβB proteins in porcine oocytes cultured in vitro is dependent on the follicle size

Summary The current study aimed to investigate differential expression of inhibin βA (INHβA) and inhibin βB (INHβB) in porcine oocytes before or after in vitro maturation (IVM) isolated from follicles of various sizes. Porcine oocytes isolated from large, medium and small follicles (40 from each) were used to study the INHβA and INHβB protein expression pattern using western blot analysis before or after 44 h of oocyte IVM. An increased expression of INHβA was found in oocytes collected from large and medium follicles compared with small follicles before or after IVM (P < 0.001, P < 0.05, respectively). Similarly, higher INHβB levels were observed in oocytes recovered from large follicles compared with small (P < 0.01). As INHβA and INHβB are expressed in both porcine follicular somatic cells and oocytes, it can be assumed that these transforming growth factor beta (TGFβ) superfamily factors are involved in the regulation of molecular bi-directional pathways during follicle and oocyte development, and can be recognized as markers of follicle and oocyte maturation. Moreover, the current study clearly demonstrated that inhibin expression is substantially associated with porcine follicle growth and development.

Microfluidic method of pig oocyte quality assessment in relation to different follicular size based on lab-on-chip technology.

Since microfollicular environment and the size of the follicle are important markers influencing oocyte quality, the aim of this study is to present the spectral characterization of oocytes isolated from follicles of various sizes using lab-on-chip (LOC) technology and to demonstrate how follicle size may affect oocyte quality. Porcine oocytes (each, n = 100) recovered from follicles of different sizes, for example, from large (>5 mm), medium (3–5 mm), and small (<3 mm), were analyzed after preceding in vitro maturation (IVM). The LOC analysis was performed using a silicon-glass sandwich with two glass optical fibers positioned “face-to-face.” Oocytes collected from follicles of different size classes revealed specific and distinguishable spectral characteristics. The absorbance spectra (microspectrometric specificity) for oocytes isolated from large, medium, and small follicles differ significantly (P < 0.05) and the absorbance wavelengths were between 626 and 628 nm, between 618 and 620 nm, and less than 618 nm, respectively. The present study offers a parametric and objective method of porcine oocyte assessment. However, up to now this study has been used to evidence spectral markers associated with follicular size in pigs, only. Further investigations with functional-biological assays and comparing LOC analyses with fertilization and pregnancy success and the outcome of healthy offspring must be performed.