Search

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP. Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230-1300 cm(-1) amide III region and the 1020-1140 cm(-1) spectral range in RMS spectra, respectively. The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in β-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram (Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 +/- 4.6% versus 56.0 +/- 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 +/- 4.6%) than in ram semen (62.7 +/- 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.

The objective of the present study was to compare the developmental capacity of sheep oocytes obtained by OPU after two different ovarian stimulations, and cryotolerance to vitrification procedures of in vitro derived embryos after in vitro maturation, fertilisation and culture of these oocytes. Sheep were divided into three groups: (A) no treatment (control); (B) constant doses of FSH (FSH-c); (C) decreasing doses of FSH (FSH-d). Ovine groups FSH-c and FSH-d were synchronised by the insertion of intravaginal sponges left in situ for 7 days; FSH (total dose: 96IU) was administered in four doses given every 12h starting on Day 5. Twelve hours after the last FSH administration oocytes were collected by OPU technique. The control group showed a significantly lower number ( P<0.05 ) of follicles (166) than FSH-c (294) and FSH-d (317) groups, while the number of follicles >5mm was significantly higher ( P<0.01 ) in FSH-d group, showing that this protocol stimulates the growth of a different follicle population compared to FSH-c group. The control group showed a higher number of <2mm follicles ( P<0.01 ). We did not find any difference in oocyte quality between the three groups and therefore the percentage of discarded oocytes was similar. No significant differences were found between control, FSH-c and FSH-d groups in terms of maturation (90.9, 85.7 and 87.7%, respectively) and fertilisation rates (75.2, 80.9 and 83.7%, respectively) while a significantly higher ( P<0.01 ) blastocyst rate was observed in the FSH-c group than in the FSH-d and control groups (20.4% versus 11.8 and 13.7%, respectively). After vitrification, warming and 72 h in vitro culture, the hatching rate was significantly higher ( P<0.01 ) in the control (87.5%) and FSH-c (90.5%) groups than in the FSH-d group (66.7%). Control and FSH-c groups showed a significantly higher ( P<0.001 ) number of total cells than FSH-d group ( 217.6+/-26.5 and 203.0+/-33.2 versus 147.5+/-20.2 ), while no differences were observed in ICM cell rates in the control ( 35.6+/-3.8 ), FSH-c ( 37.1+/-4.6 ) and FSH-d ( 36.6+/-6.7 ) groups. These results indicate that donor sheep stimulated with FSH-c produced better quality oocytes and blastocysts showing better cryotolerance than ewes given the decreasing doses treatment.

To evaluate resumption of metabolic activity of vitrified ovine embryos during a short time immediately after warming, blastocysts collected from superovulated Sarda ewes were incubated with (35)S-methionine. In vitrified/warmed embryo groups the protein secretion significantly (P < 0.05) increased from 0 to 24 hr of culture, reaching significantly (P < 0.01) higher activity at 18-24 hr and dropping to values similar to the control nonvitrified embryo group at 29-35 hr. Within the control group at 29-35 hr there was a significantly (P < 0.01) higher level of protein secretion compared to the other interval times. The electrophoretic pattern showed a 48-50 kDa secreted protein identified as urokinase-type plasminogen activator (PA). The caseinolytic assay of PA activity showed a course similar to protein secretion in both vitrified and control groups. During 29-35 hr of culture, we did not observe any improvement in PA activity as seen for secreted proteins. At this time, we observed the secretion of a new 20 kDa protein that was not present in vitrified/warmed embryos. Analysis of BrDU incorporation in newly synthesised DNA showed a significant (P < 0.01) improvement in positive cell number from 3 to 9 hr after warming, reaching a value similar to that of the control group at 12 hr of culture. Our results suggest that vitrified/warmed embryos require 9-12 hr of culture to complete resumption of DNA synthesis and 29-35 hr to re-acquire the full capacity of protein secretion but not the qualitative secretion pattern., (Copyright 2003 Wiley-Liss, Inc.)

The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.