Gene Expression of Monocarboxylate Transporters and Oocyte-secreted Factors in Bovine Cumulus-oocyte Complexes Selected by Brilliant Cresyl Blue.

Oocyte selection based on the brilliant cresyl blue (BCB) staining test has been successfully used to differentiate between competent and incompetent bovine oocytes. Here, the expression of genes involved in transport of monocarboxylates (Mct1-4) and oogenesis specific genes (Bmp15, Gdf9 and Has2) in BCB+ and BCB- selected immature and mature bovine cumulus-oocyte complexes (COC) was evaluated. In order to find specific molecular markers to characterize successful oocyte maturation, our study was also aimed at identifying the expression of Mcts and oogenesis specific genes in denuded oocytes and cumulus cells. Immature COCs morphological appropriate were (i) stained with 26 mm BCB for 90 min before IVM, (ii) exposed to same incubation conditions as stained COCs, but without BCB (holding group) or (iii) transferred into a maturation medium immediately after morphological selection (control group). mRNA expression was investigated by RT-PCR in COCs before and after IVM. No relationship was observed in the relative expression of Has2, Gdf9, Bmp15 or Mct1, 2 and 4 transcripts between BCB+ and BCB- COCs. Transcripts analysis showed that Gdf9 and Bmp15 in BCB+, BCB- and holding groups were up-regulated (p < 0.05) before IVM, while Has2 was up-regulated (p < 0.01) after IVM in the control group. Other genes remained stable during maturation (Mct1, 2 and 4). Our results showed, for the first time, Mct1, 2 and 4 expression in bovine COCs. Mct1 and Mct4 transcripts were present in denuded oocytes and cumulus cell, while Mct2 was detected only in cumulus cells. These differences between the three isoforms in localization suggest unique roles for each in monocarboxylate transport during maturation.

Vitrification of Rattus norvegicus immature cumulus-oocyte complexes using hyaluronic acid.

The aim of this study was to assess in vitro meiosis resumption and nuclear maturation of Rattus norvegicus oocytes after vitrification with different cryoprotective solutions. Cumulus-oocyte complexes (COCs) were exposed to an equilibration solution for 4 min placed in cryoprotective solutions for 1 min and vitrified in open pulled straws. Cryoprotective solutions were prepared with 15% ethylene glycol + 15% dimethyl sulfoxide + 0.5 M sucrose and different supplements, to form the following groups: G1, 20% fetal bovine serum in modified phosphate-buffered saline (mPBS); G2, 0.4% bovine serum albumine in mPBS; G3, 1% hyaluronic acid in mPBS; and G4, 0.4% polyvinyl alcohol in mPBS. Seven days after vitrification, the COCs from G1 to G4 were warmed and in vitro matured for 30 h along with the control group. Hoechst staining was performed to assess meiosis resumption and nuclear maturation rates. Control group showed higher meiosis resumption (77.88%) and nuclear maturation rates (55.75%) compared to all vitrified groups. Among the vitrified COCs, G3 showed the highest meiosis resumption and nuclear maturation rates (G1, 26.5 and 15.38%; G2, 22.12 and 11.54%; G3, 34.55 and 20%; G4, 20.17 and 9.24%). Supplementation of the vitrification solution with 1% hyaluronic acid provided better results, compared to the other supplements. Hyaluronic acid can be useful to vitrify rat COCs associated with other cryoprotectant agents.

Gene expression and developmental competence of bovine embryos produced in vitro with different serum concentrations.

The objective of this study was to determine the developmental rates and relative abundance of Hsp 70.1 and Glut-1 transcripts in in vivo- and in vitro-produced (IVP) bovine embryos in media supplemented with bovine serum albumin (BSA) or different oestrous cow serum concentrations. In experiment 1, in vitro maturation and culture media were supplemented with 0.4% BSA or 1, 5, 10 or 20% of oestrous cow serum (ECS). The analysis of the expression of Hsp 70.1 and Glut-1 was carried out in individual days 7 and 8 embryos by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. In experiment 2, in vivo-produced morulae were collected on day 7 of the oestrous cycle and employed for the comparison of the relative abundances of Hsp 70.1 and Glut-1 transcripts with IVP morulae produced using two protein sources (10% ECS or 0.4% BSA). No differences were observed in cleavage rate among groups, but blastocyst formation (27%) and hatching rates (78%) were significantly higher in IVP embryos produced with 20% ECS than the other groups (p<0.05). No significant differences were observed in the relative abundances of Hsp 70.1 and Glut-1 mRNA in days 7 and 8 blastocysts expanded blastocysts between groups. The abundances of mRNA for those genes were similar between IVP and in vivo-produced morulae. In spite of the alterations observed in embryonic development, the presence of serum at distinct concentrations did not appear to alter the relative abundance profiles of Hsp 70.1 and Glut-1 compared with controls or the BSA supplementation to the IVP media.

Current status of sperm cryopreservation: why isn't it better?

Cryopreservation extends the availability of sperm for fertilization; however, the fertilizing potential of the frozen-thawed sperm is compromised because of alterations in the structure and physiology of the sperm cell. These alterations, characteristics of sperm capacitation, are present in the motile population and decrease sperm life-span, ability to interact with female tract, and fertilizing ability. The etiology of such alterations may represent a combination of factors, such as inherited fragility of the sperm cell to withstand the cryopreservation process and the semen dilution. Although the former is difficult to address, approaches that make-up for the dilution of seminal fluid may be sought. The aim of this work is to review aspects of sperm cryopreservation paralleled by events of capacitation and evaluate the possible roles of sperm membrane cholesterol, reactive oxygen species, and seminal plasma as mediators of cryopreservation effects on sperm function.