Conjugated linoleic acid improves oocyte cryosurvival through modulation of the cryoprotectants influx rate.

BACKGROUND: In cryopreservation, oocytes are subjected to extreme hyperosmotic conditions, inducing large volume changes that, along with an abrupt temperature drop, interfere with their developmental competence. Our objectives in this work were to find conditions enabling an increase in oocyte cryosurvival and subsequent development. METHODS: Abattoir-derived bovine oocytes were cultured without (Control group) or with trans-10,cis-12 conjugated linoleic acid isomer (CLA group). Comparative observations were made for 1) the oocyte developmental competence after exposure to cryoprotectants followed or not by vitrification/warming, 2) the oocyte membrane permeability to water (using the non-permeant cryoprotectant sucrose) and 3) the oocyte membrane permeability to two cryoprotectants (ethylene glycol, EG, and dimethyl sulfoxide, DMSO). Mature oocytes cultured with or without CLA and vitrified/warmed or only exposed to cryoprotectants without vitrification were subjected to in vitro fertilization; embryo culture proceeded until the blastocyst stage. The oocyte membrane permeabilities to water and cryoprotectants were estimated using mature oocytes subjected to hyperosmotic challenges. For water permeability, 200 mM sucrose was used, whereas for the cryoprotectant permeability, a 10 % solution of both EG and DMSO was used. The data were analyzed using the MIXED procedure and Student's T-test. RESULTS: CLA supplementation improves the developmental competence of vitrified/warmed and cryoprotectants exposed oocytes (p < 0.01) and reduces their membrane permeability to water (37 %, p < 0.001) and to cryoprotectants (42 %, p < 0.001). CONCLUSIONS: By slowing the fluxes of water and of permeant cryoprotectants, CLA contributed to improved oocyte cryosurvival and post-thawed viability. This isomer supplementation to the maturation media should be considered when designing new protocols for oocyte cryopreservation.

Inhibition of ovine in vitro fertilization by anti-Prt antibody: hypothetical model for Prt/ZP interaction.

BACKGROUND: The impact of prion proteins in the rules that dictate biological reproduction is still poorly understood. Likewise, the role of prnt gene, encoding the prion-like protein testis specific (Prt), in ram reproductive physiology remains largely unknown. In this study, we assessed the effect of Prt in ovine fertilization by using an anti-Prt antibody (APPA) in fertilization medium incubated with spermatozoa and oocytes. Moreover, a computational model was constructed to infer how the results obtained could be related to a hypothetical role for Prt in sperm-zona pellucida (ZP) binding. METHODS: Mature ovine oocytes were transferred to fertilization medium alone (control) or supplemented with APPA, or pre-immune serum (CSerum). Oocytes were inseminated with ovine spermatozoa and after 18 h, presumptive zygotes (n=142) were fixed to evaluate fertilization rates or transferred (n=374) for embryo culture until D6-7. Predicted ovine Prt tertiary structure was compared with data obtained by circular dichroism spectroscopy (CD) and a protein-protein computational docking model was estimated for a hypothetical Prt/ZP interaction. RESULTS: The fertilizing rate was lower (P=0.006) in APPA group (46.0+/-6.79%) when compared to control (78.5+/-7.47%) and CSerum (64.5+/-6.65%) groups. In addition, the cleavage rate was higher (P<0.0001) in control (44.1+/-4.15%) than in APPA group (19.7+/-4.22%). Prt CD spectroscopy showed a 22% alpha-helical structure in 30% (m/v) aqueous trifluoroethanol (TFE) and 17% alpha in 0.6% (m/v) TFE. The predominant alpha-helical secondary structure detected correlates with the predicted three dimensional structure for ovine Prt, which was subsequently used to test Prt/ZP docking. Computational analyses predicted a favorable Prt-binding activity towards ZP domains. CONCLUSIONS: Our data indicates that the presence of APPA reduces the number of fertilized oocytes and of cleaved embryos. Moreover, the CD analysis data reinforces the predicted ovine Prt trend towards an alpha-helical structure. Predicted protein-protein docking suggests a possible interaction between Prt and ZP, thus supporting an important role for Prt in ovine fertilization.

Is prnt a pseudogene? Identification of ram Prt in testis and ejaculated spermatozoa.

A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP(C)), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP(Sc)) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific--Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.