The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

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Bovine embryos ) collected from superovulated cows were exposed to open pulled straw (OPS) vitrification before in vitro culture for 72 hours. The aim of this cryopreservation study applied to embryos of different developmental stages and morphological quality grades was to assess embryo survival and process of embryo handling during commercial embryo transfer (ET) procedures using the conventional freezing method as a control.During the culture of thawed quality grade 1 embryos, the hatched blastocyst stage was reached by 38.6% (27/70) of the vitrified and by 34.5% (29/84) of the conventionally frozen (control) embryos (p > 0.05). The corresponding proportions for quality grade 2 and 3 embryos were 18.5% (10/54) vs. 5.4% (2/37) and 11.8% (8/68) vs. 2% (1/50), p > 0.05, respectively.Hatching rates of embryos vitrified or conventionally frozen at the morula stage were 35.7% (15/42) and 30.3% (17/56), p > 0.05, respectively. No significant difference was found between hatching rates of embryos vitrified or conventionally frozen at the stage of early blastocysts (29.3%; 12/41 vs. 34%; 18/54). The hatching rates of embryos vitrified or conventionally frozen at the blastocyst and expanded blastocyst stages were 30.8% (12/39) vs. 21.6% (11/51), p > 0.05 and 29.3% (11/37) vs.11.4% (5/44), p < 0.05, respectively.The study demonstrated about the same survival rates for vitrified and conventionally cryopreserved embryos of all quality grades and developmental stages during in vitro embryo culture. Expanded blastocysts survived better vitrification than conventional freezing (p < 0.05). OPS vitrification is an effective and rapid method of cryopreservation of bovine embryos.

To increase the developmental competence of bovine oocytes isolated from small, medium, and large follicles (2-3, 3-4, and 4-6 mm in diameter, respectively), we tried to modify the conditions for their in vitro culture. The first step involved conditions maintaining at least for 48 hr a reversible inhibition of the germinal vesicle breakdown (GVBD) and the second step stimulated the resumption of meiosis and completion of nuclear and cytoplasmic maturation during the subsequent 20-22 hr of culture. The effectiveness of this model depended mainly on the medium composition (reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity by agar, the reduction of oxygen concentration (within 6%-8%), the reduction of the proportion between the number of cumulus-oocyte complexes (COCs), and the reduction of the amount of a medium (within 6-7 mul per COC) to amplify the GVBD-inhibitory effect of oocyte surrounding granulosa cells. The COCs were incubated in clumps of 6-7 COCs. The effectiveness and reversibility of GVBD inhibition depended also on the duration of COCs isolation. The full reversibility of the GV block was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 hr) maturation technique was evaluated by the percentage of total and hatched blastocysts at day 9. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from growing follicles, mainly from the smallest category (13.9% vs. 7.1% and 9.2% vs. 3.3% for total blastocysts and hatched, respectively). However, the two-step culture of oocytes from large regressing follicles substantially reduced the blastocyst yield (9.7% vs. 39.1% and 4.9% vs. 26.7% for total blastocysts and hatched, respectively). The transfer of ten blastocysts (developed after two-step culture) to ten recipients resulted in seven pregnancies.

The aim of this study was to evaluate the applicability of ovum pick-up equipment for follicular fluid collection from various follicular structures (experiment 1) and for recovery of follicular fluid for acid-base balance analysis (experiment 2). An ultrasound scanner equipped with a 5-MHz convex transducer was used for transvaginal ultrasound-guided follicular aspiration. A 17-gauge, 60-cm aspiration needle was connected with a shortened aspiration line. The fluid was aspirated manually into a 2 ml plastic syringe at a speed of approximately 0.2 ml/s. The success of aspiration was higher in ovarian cysts (100%) and single follicles larger than 13 mm (76.7%) compared to single follicles smaller than 12 mm (20%, p < 0.001). The success of aspiration of multiple follicles on day 4 (diameter of 7-9 mm) was higher (90.9%) compared to follicles on day 2 (diameter of 4-6 mm) (66.7%, p < 0.05) in experiment 1. The fluid from ovarian cysts > 25 mm in diameter was aspirated in a two-step procedure (samples 1 and 2) for the determination of pH, HCO 3 , BE, pCO 2 and pO 2 (experiment 2). The indicators were compared between samples 1 and 2. Higher pO 2 as well as pH and lower pCO 2 in sample 1 compared to sample 2 showed insufficient anaerobic conditions during the first phase of the puncture in experiment 2. Our study brings for the first time the finding that the ovum pick-up equipment used in our experiments is suitable for the collection of follicular fluid only from larger follicular structures. The sampling of follicular fluid for acid-base balance assays requires the development of a special new device to prevent samples from coming into contact with air during aspiration.