Abstract. The maturation and developmental competence of the oocyte is acquired during folliculogenesis. It is still unclear whether follicle size is associated with the levels of transcript and protein encoding molecules contributing to the fertilization ability of the porcine oocyte. Follicles were dissected from porcine ovaries after slaughter and classified as small (< 3 mm), medium (3-5 mm) or large (>5 mm), aspirated cumulus-oocyte complexes were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH) using a brilliant cresyl blue (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 glycoproteins pZP1, pZP2, pZP3, pZP3 alpha and integrins beta 1 and beta 2, as well as the levels of pZP3 and integrin beta 2 proteins. We observed significantly higher levels of pZP1, pZP3 and integrin beta1 and beta2 transcripts in oocytes collected from medium follicles as compared with small follicles (P<0.001). Moreover, we found an increased content of all investigated mRNAs in oocytes isolated from large follicles as compared with small follicles (P<0.001). Western-blot analysis demonstrated a higher level of pZP3 protein in oocytes isolated from large and medium follicles as compared with small follicles (P<0.001). Our results suggest that the levels of transcripts and proteins for selected molecules contributing to the fertilization ability of oocytes are associated with follicular size in puberal gilts. Key words: Follicular size, Integrins, Oocyte, Pig, ZPs glycoproteins (J. Reprod. Dev. 55: [588][589][590][591][592][593] 2009) ocyte quality has a significant influence on early embryonic development and survival, establishment and maintenance of pregnancy, fetal development and even the health of offspring in postnatal life [1][2][3][4][5]. The developmental competence of an oocyte may be affected by follicle size [6][7][8]. The growth of the oocyte within the follicle is accompanied by expression of a series of genes [9][10][11]. Some of these genes are involved in cell cycle control and meiotic progression, resulting in full meiotic competence of the oocyte [12,13]. Several other proteins are involved in cellular progress as characterized by complete developmental competence of the gamete prior to fertilization [14,15]. During the growth phase, several messenger RNA (mRNA) and their respective proteins are accumulated and stored long-term to be used after fertilization and subsequent proper embryo development [1,16]. The abilities of fertilization and embryo development are acquired by an oocyte only after a long period of growth and development. Specific follicular growth and somatic follicular cell-oocyte interactions lead to the acquisition of high developmental potential for the oocyte.The porcine zona pellucida is composed of four major glycoproteins: ZP1...

Cat oocytes have the unique ability to mature in vitro after temporary storage at 4°C which can provide opportunities to rescue oocytes from the ovaries of endangered felids after sudden death or medical ovariohysterectomy. It has been demonstrated that factors such as season, culture conditions and morphological quality of oocytes influence the meiotic competence of domestic cat oocytes. In the present study we determined the meiotic maturation rate and incidence of apoptosis or necrosis in domestic cat oocytes collected from ovaries stored at different temperatures. Nuclear status and the presence of the first polar body were evaluated by fluorescence DAPI staining. Cell death was detected using Annexin-V, a phospholipid-binding protein that detects translocation of phosphatidyl-serine to the outer cytoplasmic membrane. Most oocytes (77.5%) collected from ovaries immediately after ovariohysterectomy (control group) resumed meiosis and reached metaphase II. A similarly high percentage of oocytes underwent nuclear maturation after recovery from ovaries stored for 6 h at 4°C (68.6%) or at room temperature (55.5%), but the rate of maturation after recovery from ovaries stored for 24 h at 4°C was greatly reduced (15.3%) Not surprisingly, the highest percentage of apoptotic oocytes were seen in Group 3, and the lowest frequency of apoptotic oocytes were observed in Group 1. Correspondingly, Group 1 had the highest percentage of necrotic oocytes. Thus, our results indicate that storage of domestic cat ovaries at room temperature, even for a short time, can negatively influence the competence of oocytes to undergo nuclear maturation in vitro.

The influence of passage time of the transfer gun through the uterine cervix and body to the embryo insertion site on pregnancy rate was analysed in 248 recipient heifers (mean age: 15-17 months). Embryos (90 fresh and/or 88 and 70 frozen in glycerol and ethylene glycol, respectively, grades 4 and 5, stage 1 or 2) were transferred to the ipsilateral uterine horn on day 7. Two different transfer guns were used in this experiment: a sterilisable steel transfer instrument to be used without sheaths with a removable tip made of gold-plated stainless steel (Wörrlein Minitüb) or a transfer stylet with sheaths with a metal tip and a side opening (Cassou gun, IMV Technologies). The time of passage of the instruments through the uterine cervix and body to the site of embryo deposition in the uterine horn was measured in the study. In order to randomise the risk of errors, all manipulations were carried out by the same experienced operator. The average time needed for the insertion of embryos into the uterus was 50.6 seconds (s) and it was longer for the transfer gun with sheaths than for the metal-tipped transfer gun (60.1 and 40.8 s, respectively) (P < 0.001). The average conception rate was 45.6%. If the time needed to insert embryos into the uterus was 10-60 s, the conception rate was 53.4% (up to 20, 21-30, 31-40, 41-50 and 51-60 s - 57.7, 52.5, 50, 51.5 and 50%, respectively). In contrast, if the time needed to insert the embryo in the uterine horn was longer than 60 s, the conception rate was 20.4% (61-80, 80.1-120 and > 120 s - 28.0, 6.0 and 24.9%, respectively). Thus, it cannot be excluded that the type of the applied transfer gun may influence pregnancy rate in recipient cows due to its effect on cervical passage time.

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.