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 INHßA and INHßB proteins in porcine oocytes cultured in vitro is dependent on the follicle size.

The current study aimed to investigate differential expression of inhibin ßA (INHßA) and inhibin ßB (INHßB) 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 INHßA and INHßB protein expression pattern using western blot analysis before or after 44 h of oocyte IVM. An increased expression of INHßA 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 INHßB levels were observed in oocytes recovered from large follicles compared with small (P < 0.01). As INHßA and INHßB are expressed in both porcine follicular somatic cells and oocytes, it can be assumed that these transforming growth factor beta (TGFß) 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.

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.

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.

Differential expression of GDF9, TGFB1, TGFB2 and TGFB3 in porcine oocytes isolated from follicles of different size before and after culture in vitro.

The TGFB superfamily genes are involved in several important cell functions, including proliferation and differentiation, and the role of the expression of these genes in growth and development of theca and granulosa cells is well recognised. However, the dependence between the stage of oocyte maturation or follicular size and the expression of these genes in pigs is still not entirely known. This study was aimed at investigating the expression pattern of GDF9, TGFB1, TGFB2 and TGFB3 in porcine oocytes before and after in vitro maturation (IVM) as well as in oocytes collected from follicles of different sizes. RQ-PCR was performed to analyse the expression of GDF9, TGFB1, TGFB2 and TGFB3 in oocytes before and after IVM (oocytes cultured for 44 h in TCM-199), isolated from large (> 5 mm), medium (3-5 mm) and small (< 3 mm) follicles collected from ovaries of 28 puberal crossbred Landrace gilts after slaughter. We found an increased expression of both TGFB1 and TGFB2 in oocytes before IVM collected from large as compared to medium and small follicles (P < 0.05, P < 0.001, P < 0.01, P < 0.05, respectively). In these groups of oocytes we did not observe differences in GDF9 and TGFB3 mRNA levels. However, after IVM, GDF9 protein distribution in oocytes was significantly higher in large and medium follicles as compared to small ones (P < 0.01, P < 0.001, respectively). Moreover, an increased TGFB1, TGFB2 and TGFB3 proteins pattern was observed in oocytes of large compared to small follicles. The highest GDF9 and TGFB1 mRNA levels were found in oocytes after IVM compared to those before IVM. Based on our study we can suppose that the distribution pattern of TGFB superfamily genes is associated with the stage of maturation of porcine oocytes and the follicle size. Furthermore, GDF9 and TGFB1 may serve as molecular markers of the develop-mental potential of porcine oocytes. The confocal microscopic observation revealed that TGFB1 and TGFB3 were translocated between the zona and the cytoplasm of oocytes, depending on the stage of maturation and follicle size.

Expression and cellular distribution of INHA and INHB before and after in vitro cultivation of porcine oocytes isolated from follicles of different size.

Cumulus-oocyte-complexes (COCs) were collected from small (<3 mm), medium (3-5 mm), and large (>5 mm) porcine follicles, and the INHA and INHB expression and cellular localization were studied. Developmentally competent (BCB+) COCs were cultured for 44 h. Samples of mRNA were isolated before and after in vitro maturation (IVM) from oocytes collected from follicles of different size for RQ-PCR assay. The INHA and INHB protein distribution within the oocytes was observed by confocal microscopy. INHA mRNA expression was increased in oocytes from large compared to medium and small follicles before IVM (P < 0.001), and to oocytes of small follicles after IVM (P < 0.001). The INHB expression was not different before IVM, but the IHNB mRNA level was gradually higher in oocytes from large follicles after IVM (P < 0.01). INHA was not differently expressed before IVM; however, in large follicle oocytes the protein was distributed in the peripheral area of the cytoplasm; in oocytes from small follicles it was in the entire cytoplasm. After IVM, INHA was strongly expressed in oocytes from small follicles and distributed particularly in the zona pellucida (ZP). Similarly and both before and after IVM, INHB protein was highly expressed in small follicle oocytes and within the cytoplasm. In summary, INHs can be recognized as a marker of porcine oocyte quality.

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.