Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.     

Towards whole sheep ovary cryopreservation

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 °C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12–16 °C/min from −196 to −133 °C) in liquid nitrogen vapour, followed by rapid thawing in a 45 °C water bath at about 200 °C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4 ± 2.2% of small follicles were viable while histological analysis showed that 48 ± 3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.     

Effect of ovarian tissue vitrification method on mice preantral follicular development and gene expression

Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group.     

Towards whole sheep ovary cryopreservation

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 °C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12–16 °C/min from −196 to −133 °C) in liquid nitrogen vapour, followed by rapid thawing in a 45 °C water bath at about 200 °C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4 ± 2.2% of small follicles were viable while histological analysis showed that 48 ± 3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.     

Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development

The cryopreservation of ovarian tissue has been reported to affect the development of preantral follicles. However, the effect of cryopreservation of ovarian tissue on the development of primordial follicles remains to be elucidated. This study was conducted to evaluate the effect of cryopreservation on the development of frozen-thawed mouse primordial follicles. One-day-old mouse ovaries were cryopreserved by either slow-freezing or a vitrification method. The development of primordial follicles was evaluated histologically and also with markers for follicle development such as: GDF-9, inhibin-alpha subunit and ZP3 in fresh and frozen-thawed ovaries cultured for five days. The proportion of apoptotic and necrotic areas was analyzed in fresh and frozen-thawed ovaries at one and five days after culture, in order to examine the viability of ovarian cells that influence primordial follicle development. The development rate of primordial follicles was significantly lower in slow-frozen and vitrified ovaries than the fresh controls after five days of in vitro culture (P<0.05). The mRNA expression for all developmental markers was slightly decreased in the frozen-thawed ovaries; this difference was not significant. The proportion of apoptosis was significantly increased in the slow-frozen and vitrified ovaries compared to the fresh ovaries at one day (P<0.05); however, there was no difference at five days after culture. The proportion of the area of necrosis was significantly higher in slow-frozen and vitrified ovaries compared to the fresh ovaries at one and five days after culture (P<0.05). Our preliminary data suggest that ovarian tissue cryopreservation using slow-freezing and vitrification methods inhibits development of primordial follicles. This may be caused by the death of ovarian cells through apoptosis and necrosis after cryopreservation.     

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.     

Zinc supplementation of vitrification medium improves in vitro maturation and fertilization of oocytes derived from vitrified-warmed mouse ovaries

Oocyte cryopreservation is an approach for fertility preservation for normal women and cancer patients facing chemo and radiotherapy. The present study evaluated the effect of adding zinc chloride to the vitrification medium used for whole mouse ovaries and then assessing the in vitro maturation and fertilization of oocytes when they were subsequently extracted from these vitrified ovarian tissues. Four vitrification solutions with 0, 100,150 and 200 μg/dl zinc (V0, V1, V2 and V3 respectively) were compared. The viability of oocytes isolated from ovaries vitrified-warmed in the highest concentration of zinc (V3) was significantly higher after 24 than in the control V0 group (72.99 vs 85.97). Progression to the MII stage, fertilization and cleavage by 48 h was also higher in the V3 than V0 control group (35.55 vs 44.73), (47.67 vs 63.74), (28.72 vs 43.03) (P < 0.05) respectively. These results indicate that supplementation of vitrification medium for intact ovaries with zinc can improve the oocyte viability and in vitro maturation-fertilization rate.     

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.     

Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension

The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O₂) or high (20% O₂) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO₂, and 5% or 20% O₂. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 μm (5% O₂) and 11.78 ± 1.06 μm (20% O₂) on Day 4 and 20.94 ± 1.24 μm (5% O₂) and 19.12 ± 1.36 μm (20% O₂) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O₂ (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.     

Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method

Background

Cryopreservation of preantral follicles or ovarian tissues would enable the storage of large numbers of primordial follicles or preantral follicles and preserves the structural integrity of somatic and reproductive cells. In the present study, we compared the developmental potential of cryopreserved two-week-old mouse preantral follicles, ovarian tissue slices, two-week-old mouse ovaries and newborn mouse ovaries using a metal plate with a high cooling rate for cooling the droplet of vitrification solution.

Methods

Groups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries) were exposed to 4% ethylene glycol (EG) in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20–30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180°C in the volume of 2 μl and 6 μl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-μl droplets of α-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs) were collected for IVM and IVF. Fertilization and embryo cleavage were scored.

Results

After the vitrification of 14-day-old preantral follicles using 2 μl or 6 μl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 μl and 6 μl droplet groups. As comparing 14-day old ovarian tissue (ovarian tissue slices and whole ovaries) and whole newborn ovaries vitrified in 6 μl droplet, lower success rates of antral-like cavity formation and COCs collection were found in the whole ovaries group.

Conclusion

Our results suggest that the metal plate surface vitrification method is an appropriate and convenient method for cryopreservation of mouse ovaries and preantral follicles. The droplet volume of vitrification solution in 2 μl and 6 μl can be an option.     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

Cryopreservation of mouse ovarian tissue following prolonged exposure to an Ischemic environment.

In cases in which ovarian tissue is to be cryopreserved for tissue or gene banking it is important to maintain its integrity and viability. This study examined how delays between the death of an animal and the collection/cryopreservation of its ovarian tissue influenced follicle viability. Mouse ovaries were placed in PBS+antibiotic (in vitro) or left within the body (in situ) at room temperature for 0, 3, 6, 12, or 24 h following the death of the donor. These ovaries were cryopreserved at 1 degrees C/min on dry ice or in a -84 degrees C freezer using a passive cooling device or by conventional slow cooling (0.3 degrees C/min). The ovaries were grafted under the kidney capsule of ovariectomized recipient mice and collected 2 weeks later, and the size and number of follicles were determined. Cryopreserved ovarian tissue grafted immediately after the death of the donor contained numerous viable and healthy follicles independent of the cooling procedure (dry ice, 134 +/- 32; -84 degrees C, 165 +/- 54; slow, 214 +/- 55 follicles per half ovary). Tissues stored in vitro before cryopreservation retained viable follicles up to 12 h after death (dry ice, 30 +/- 15; -84 degrees C, 86 +/- 45; slow, 93 +/- 33), whereas tissue left in situ had significantly reduced follicle numbers within 3 h of death (dry ice, 36 +/- 12; -84 degrees C, 19 +/- 6; slow, 28 +/- 7). No significant difference was found between the cooling rates tested, indicating that a passive cooling container which cools at 1 degrees C/min is a suitable alternative to conventional slow cooling. We conclude that ovarian tissues for cryobanking should be cryopreserved as soon as possible after collection or death of the animal to ensure maximal follicular survival.     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage

The cryopreservation of immature oocytes would generate a readily available, non-seasonal source of female gametes for research and reproduction. In domestic animals, the most promising results on oocyte cryopreservation have been reported in cattle, few studies have been conducted on buffalo. The aim of the present study was to compare the use of different vitrification solutions and various cryodevices on viability and developmental competence of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cumulus oocyte-complexes (COCs) obtained at slaughterhouse from mature buffalo ovaries were randomly divided into three main groups and vitrified by using either straw or open pulled-straw (OPS) or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol (GLY); VS1 or 20% EG + 20% dimethylsulfoxide (DMSO); VS2, respectively. Following vitrification and warming, viable COCs were matured in vitro for 22 h. Some COCs were denuded and stained with 1.0% aceto-orcein to evaluate nuclear maturation, whereas the others were fertilized and cultured in vitro for 7 days to determine the developmental competence. Although the recovery rate (64.9%) was the lowest in the oocytes vitrified by SSV using 20% EG + 20% DMSO as compared to the other groups, the best survival rate of the COCs was achieved in the same treatment (96.7%), which was significantly higher (P < 0.05) than those vitrified using traditional straws (71.8% in VS1 and 73.6% in VS2) or those vitrified using OPS and VS1 (73.9%). Furthermore, in the nuclear maturation test, the highest maturation rate (75.5%) was achieved in SSV vitrified COCs using 20% EG + 20% DMSO (VS2), which was similar to the controls (77.1%). Post IVF and embryo culture, the highest cleavage and blastocyst development rates were obtained in COCs vitrified in 20% EG + 20% DMSO using SSV (47.1% and 24.0%, respectively), which showed no difference from the controls (61.2% and 46.9%, respectively). Our results clearly show that the combination of SSV and 20% EG + 20% DMSO could be used effectively to vitrify GV stage buffalo COCs.     

Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods

Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in “warming solution” consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM⁺) followed by washes in MEM⁺ with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM⁺ plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM⁺ (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as “live” and “dead” markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.CAPES/Brazil supported this work. Regiane Rodrigues dos Santos is a recipient of a grant from CAPES/Brazil.     

Survival rate of preantral follicles derived from vitrified neonate mouse ovarian tissue by Cryotop and conventional methods

The aim of this study was to investigate the growth and survival rate of preantral follicles isolated from vitrified ovarian tissue by Cryotop and conventional methods. The ovaries of 14-day-old mice were separated and divided into four groups as following: Cryotop group, vitrified by Cryotop; CV (Conventional; CV) group, vitrified by conventional straw; toxicity test group and control group. After warming the vitrified ovaries, isolated preantral follicles from four groups were cultured for 4 days to compare survival rate and follicular growth between above-mentioned groups. Survival rate (97.3%) in toxicity test group alike the control group (98.7%) were significantly higher (P<0.05) than the Cryotop (92.7%) and CV (47.7%) groups. Increase in follicle diameters after 4 days in Cryotop and CV groups was significantly lower (P<0.05) than the control and toxicity test groups, but growth and survival rate of follicles in Cryotop group was significantly higher (P<0.05) than the CV group. These results demonstrated that ovarian tissue vitrification by Cryotop highly preserves the viability rate of preantral follicles.     

Effectiveness of slow freezing and vitrification for long-term preservation of mouse ovarian tissue

This study was conducted to evaluate the interaction between cryo-damage and ART outcome after cryopreservation of mouse ovarian tissues with different methods. Either a vitrification or a slow freezing was employed for the cryopreservation of B6CBAF1 mouse ovaries and follicle growth and the preimplantation development of intrafollicular oocytes following parthenogenesis or IVF were monitored. Both cryopreservation protocols caused significant damage to follicle components, including vacuole formation and mitochondrial deformities. Regardless of the cryopreservation protocols employed, a sharp (P < 0.0001) decrease in follicle viability and post-thaw growth was detected. When IVF program was employed, significant (P < 0.05) decrease in cleavage and blastocyst formation was notable in both modes of cryopreservation. However, such retardation was not found when oocytes were parthenogenetically activated. In the IVF oocytes, slow freezing led to better development than vitrification. In conclusion, a close relationship between cryopreservation and ART methods should be considered for the selection of cryopreservation program.     

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n?=?16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n?=?16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

     

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n=16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.     

A novel strategy for conservation of European eel (Anguilla anguilla) genetic resources: Cryopreservation of ovarian stem cells

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me₂SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN₂ at −80 °C displayed the highest OSC viability. Different cooling rates ranging from −1 to −40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me₂SO) with VS3 (3 M PG and 3 M Me₂SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species.