Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation

Background

The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep.

Methods

Mature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries).

Results

Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries.

Conclusion

These data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

Bovine oocyte vitrification using the Cryotop method: Effect of cumulus cells and vitrification protocol on survival and subsequent development

The ability to successfully cryopreserve mammalian oocytes has numerous practical, economical and ethical benefits, which may positively impact animal breeding programs and assisted conception in humans. However, oocyte survival and development following vitrification remains poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) bovine oocytes, (2) to compare empirical and theoretical vitrification protocols, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. In Experiment 1, cumulus-enclosed and partially-denuded GV and MII oocytes were vitrified in 15% EG + 15% Me₂SO + 0.5 M sucrose in two steps. In Experiment 2, GV oocytes were vitrified either as above or using theoretical modeling based on permeability and osmotic tolerance characteristics in 30% EG + 11.4% trehalose in three steps or 40% EG + 11.4% trehalose in four steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA) 1% X-1000, 1% Z-1000 or both in three steps. In Experiment 1, the survival, cleavage and blastocyst rate of cumulus-enclosed oocytes was significantly higher than those of partially-denuded oocytes when vitrified at the GV stage (93.8% vs. 81.3%, 65.8% vs. 47.3%, 11.3% vs. 4.0%, respectively, P < 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% vs. 91.8%, 35.2% vs. 36.8%, 5.0% vs. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited significantly higher developmental competence than those vitrified at the MII stage (P < 0.05). In Experiment 2, there were no significant differences in the survival, cleavage and blastocyst rate among three protocols (86.0% vs. 92.8% vs. 91.2%, 44.8% vs. 54.4% vs. 45.6%, 5.0% vs. 5.4% vs. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P < 0.05) than non-vitrified control oocytes. In Experiment 3, the presence of ice blockers did not alter the cleavage rate or blastocyst development (P > 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes using Cryotop method and conventional IVF procedure. Theoretical analysis of permeability characteristics and tolerance limits could not explain the low developmental competence of vitrified oocytes.     

Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl-pyrrolidone and 0.4% trehalose and they were warmed in a 0.3M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly (P<0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P<0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified-warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.     

Vitrification of oocytes from endangered Mexican gray wolves (Canis lupus baileyi)

Careful genetic management, including cryopreservation of genetic material, is central to conservation of the endangered Mexican gray wolf. We tested a technique, previously used to vitrify human and domestic animal oocytes, on oocytes from domestic dogs as a model and from the endangered Mexican wolf. This method provided a way to conserve oocytes from genetically valuable older female Mexican wolves as an alternative to embryos for preserving female genes. Oocytes were aspirated from ovaries of 36 female dogs in December and March (0 to 65 oocytes per female) and from six female wolves (4 to 73 per female) during their physiologic breeding season, or following stimulation with the GnRH agonist deslorelin. Oocytes from dogs were pooled; half were immediately tested for viability and the remainder vitrified, then warmed and tested for viability. All oocytes were vitrified by being moved through media of increasing cryoprotectant concentration, placed on Cryotops, and plunged into liquid nitrogen. There was no difference in viability (propidium iodide staining) between fresh and vitrified, warmed dog oocytes (65.7 and 61.0%, respectively, P = 0.27). Oocyte viability after warming was similarly assessed in a subset of wolves (4 to 15 oocytes from each of three females; total 29 oocytes). Of these, 57.1% of the post-thaw intact oocytes were viable, which was 41.4% of all oocytes warmed. These were the first oocytes from a canid or an endangered species demonstrated to have maintained viability after vitrification and warming. Furthermore, our results demonstrated that vitrification of oocytes with the Cryotop technique was an option for preserving female gametes from Mexican wolves for future use in captive breeding programs, although in vitro embryo production techniques must first be developed in canids for this technique to be used.     

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.     

Specific activation requirements of in vitro-matured sheep oocytes following vitrification-warming

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase‐II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified‐warmed, and unvitrified‐activated oocytes, the present study investigated how vitrification‐warming process may affect developmental competence of in vitro‐matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified‐warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified‐activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using the maximum ionomycin concentration (5 µM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified‐warmed oocytes (28.4 ± 1.4%) was achieved with a minimal duration of ionomycin treatment (1 min), and further extending the duration dramatically reduced developmental potential of vitrified‐warmed oocytes. These results suggested that vitrified‐warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture. Mol. Reprod. Dev. 79:434–444, 2012. © Wiley Periodicals, Inc.     

Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods

This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P<0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P>0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.     

Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.     

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer

We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients.     

Gene expression profiles of vitrified in vivo derived 8-cell stage mouse embryos detected by high density oligonucleotide microarrays

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to compare the gene expression patterns, and identify those most affected. For this, 8-cell stage embryos were collected from ICR mice and vitrified with solid surface vitrification technique, while maintaining equal numbers of embryos as control. Total RNAs were extracted and two rounds of amplification were employed. Finally three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22 K oligonucleotide slides and the results were analyzed with subsequent verification by independent real-time PCR analyses. The two rounds of amplification with 5 ng tRNA input have yielded 15-16 microg of cRNA. The analyses of repeated hybridizations showed 20,183 genes/ESTs as common signatures, and unsupervised analysis identified 628 differentially expressed (P < 0.01) genes. However, with at least 1.5-fold change considerations, 183 genes were differentially expressed (P < 0.01) out of which 107 were upregulated. The independent analysis with real-time PCR and unamplified samples fully confirmed the results of microarray, indicating the linearity of amplification. Furthermore, this novel gene expression study for vitrified embryos identified many new candidate genes with overrepresentation in some important biological processes. Thus, it is possible to conclude that the expression pattern reflected a broad spectrum of consequences of vitrification on embryos, with most effects on metabolism, regulatory role and stress response genes and allowed the identification of new candidate marker genes for cryosurvival.     

Ultrastructural changes of sheep cumulus-oocyte complexes following different methods of vitrification

To determine the ultrastructural changes of sheep cumulus-oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified-warmed sheep COCs.     

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P < 0.05) than that of the liquid nitrogen group (81.1%). When the vitrified–thawed oocytes were matured in vitro for 24 h, the maturation rate in liquid helium group (50.6%) was higher (P < 0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P < 0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified–thawed oocytes were matured 24 h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.     

Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes,WNT signaling pathway and apoptotic genes expression

This study compared slow freezing and vitrification of ovarian tissue by evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Ovarian tissue was obtained from women aging 27–38 years old. Ovarian cortex from each patient was divided into three pieces and randomly grouped as slow freezing, vitrification and control groups for investigation of WNT signaling gene expression and β-CATENIN presence as well as histological studies. The stromal structure of all ovaries were preserved. The number of secondary follicles decreased in vitrified group (P < 0.05). WNT-3, β-CATENIN, FZD-2 and GSK-3β expressions were significantly higher in slow frozen and vitrified groups, compared to control group (P < 0.05). On the contrary, AXIN1 expression in slow frozen samples were significantly lower than that of the vitrified and control group. The expression of apoptotic genes, excluding CASP3, was significantly decreased in slow-frozen samples (P < 0.05). Conversely, BAX:BCL-2 percentage significantly increased in vitrification versus slow freezing and control(P < 0.05). Follicles in slow frozen samples displayed nuclear and cytoplasmic β-CATENIN staining, while control and vitrification groups only showed β-CATENIN protein in the cytoplasm. The presented data show that slow freezing results in a better preservation regardless of the type of follicle. Therefore, it is concluded that slow freezing is still an ideal method for ovary cryopreservation.     

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.     

Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries

The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.     

Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification

We cryopreserved infantile rat ovaries by vitrification and assessed their viability by autotransplantation. Hemilateral ovarian transplantation was performed on rats on postnatal Days 10 to 12. The left ovary of each rat was dissected out, cryopreserved by vitrification using a modified vitrification solution (VS1), and then autotransplanted under the capsule of the right kidney. The right ovary of each rat was removed. For the control, the left ovary was dissected out from each rat and was immediately transplanted by the same procedure, without cryopreservation. Rats were nursed until weaning, and then the day of vaginal opening, estrous cyclicity from the day of vaginal opening until postnatal Day 84, and histology of ovarian grafts at postnatal Day 84 were examined. The time course of development of endocrine function of cryopreserved grafts was similar to that of fresh grafts. In ovarian transplants recovered on postnatal Day 84, antral follicles and corpora lutea (CL) were observed in addition to small follicles, although the number of antral follicles in cryopreserved grafts was smaller than in the fresh grafts. These results indicate that cryopreservation of ovarian tissue by vitrification can be used for the preservation of fertility and endocrine function of ovaries.     

Vitrification of in vitro cultured porcine two-to-four cell embryos

The objective of this experiment was to evaluate the effect of a 5-day period of in vitro culture of two-to-four cell porcine embryos up to the blastocyst stage on their ability to survive vitrification and warming. In order to increase the cooling rate, superfine open pulled straws and Vit-Master((R)) technology were used for vitrification. Two-to-four cell embryos were collected from weaned sows (n=11) on day 2 (D0=onset of estrus). Some embryos (N=63) were vitrified within 3h after collection, warmed and cultured for 120h (Group V2). Additionally, 81 two-to-four cell embryos were cultured for 96h in order to obtain blastocysts; these were then vitrified, warmed and cultured for 24h (Group VB; N=65). The remaining two-to-four cell embryos were used as controls and thus not vitrified (control embryos; N=70) but were cultured in vitro for 120h. The V2, VB and control embryos were evaluated for their developmental progression and morphology during culture. All embryos (V2, VB and controls) were fixed on the same day of development in order to assess the total number of blastomeres. The survival and blastocyst formation rates obtained from V2 embryos were very poor (9.6+/-0.7% and 3.2+/-0.5%, respectively). The survival and hatching rates of VB embryos (75.0+/-0.69% and 33.6+/-0.13%) were lower (p<0.001) than those obtained with control embryos (89.1+/-0.8% and 47.5+/-0.12%). Hatched VB embryos had a lower (p<0.01) total cell number than hatched control embryos (70.3+/-4.5 versus 90.6+/-3.2, respectively). There was no difference between expanded VB and control blastocysts. In conclusion, blastocysts derived from in vitro culture of two-to-four cell pig embryos could be successfully vitrified using SOPS straws and Vit-Master.