Slow Freezing,but Not Vitrification Supports Complete Spermatogenesis in Cryopreserved,Neonatal Sheep Testicular Xenografts

The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly. The purpose of this study was to determine, for the first time, the ability of two substantially different cryopreservation approaches, slow freezing versus vitrification, to preserve testicular tissue of the neonatal sheep and subsequently allow initiation of spermatogenesis post-xenografting. Testis tissue from four lambs (3-5 wk old) was processed and then untreated or subjected to slow freezing or vitrification. Tissue pieces (fresh, n = 214; slow freezing, then thawing, n = 196; vitrification, then warming, n = 139) were placed subcutaneously under the dorsal skin of SCID mice and then grafts recovered and evaluated 17 wk later. Grafts from fresh and slow frozen tissue contained the most advanced stages of spermatogenesis, including normal tubule architecture with elongating spermatids in ~1% (fresh) and ~10% (slow frozen) of tubules. Fewer than 2% of seminiferous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue. Results demonstrate that slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ~10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa. Although a first for any ruminant species, findings also illustrate the importance of preemptive studies that examine cryo-sensitivity of testicular tissue before attempting this type of male fertility preservation on a large scale.     

Effects of Vitamin A on In Vitro Maturation of Pre-Pubertal Mouse Spermatogonial Stem Cells

Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7), 9 (D9) and 11 (D11) days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re) or retinoic acid (RA) alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type), intra-tubular cell death and proliferation (PCNA antibody) and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7°C, -8°C or -9°C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10^-6M and retinol at 3.3.10^-7M, as well as retinol 10^-6M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8°C, after 9 days of organotypic culture using 10^-6M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10^-6M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8°C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular tissue.     

Over expression of interleukin-1alpha,interleukin-1beta and interleukin-1 receptor antagonist in testicular tissues from sexually immature mice as compared to adult mice

The levels of IL-1alpha, IL-1beta and IL-1Ra were higher in homogenates of testicular tissue from sexually immature than those from mature mice. Immunohistochemical staining of testicular tissues from sexually immature and adult mice show that differentiated germ cells express higher levels of IL-1alpha compared to Sertoli cells and Leydig cells/interstitial cells. Peritubular cells of sexually immature and adult mice did not express IL-1alpha. Testicular tissue cells of adult mice showed high levels of expression of IL-1beta, mainly in the cytoplasm and nucleus of the spermatogonia and in spermatocytes. Sertoli cells and Leydig/interstitial cells were also highly stained for IL-1beta. However, peritubular cells did not express IL-1beta. On the other hand, testicular tissue cells from sexually immature mice, showed high levels of IL-1beta, mainly in spermatocytes. Spermatogonia showed low levels of IL-1beta expression. Also, high levels of IL-1beta expression were detected in Leydig/interstitial cells. Peritubular cells clearly showed IL-1beta expression. Testicular tissue cells from adult mice, showed IL-1Ra expression in spermatogonia, Sertoli and Leydig/interstitial cells. IL-1Ra expression was clearly present in the Golgi apparatus of spermatogonia and Sertoli cells. However, peritubular cells did not show IL-1Ra expression. Testicular tissue cells from sexually immature mice, also showed high levels of IL-1Ra expression mainly in the cytoplasm and nucleus of the spermatogonia and Sertoli cells. In addition, Leydig/interstitial cells and peritubular cells also expressed IL-1Ra. Our results demonstrate, for the first time, the expression of IL-1beta in germ and Sertoli cells, and IL-1Ra in Leydig/interstitial cells of testicular tissues from adult and sexually immature mice, under in vivo conditions. In addition, the relative elevated levels of the IL-1 system in the testis of immature mice compared to mature mice may indicate its involvement in the spermatogenesis.     

Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing

Vitrification is a novel cryopreservation method for mammalian blastocysts. This study was designed to compare different vitrification methods and slow freezing for their effects on survival rate and DNA integrity in mouse and human blastocysts. In Experiment 1, embryo survival and DNA integrity were compared between mouse blastocysts with collapsed and non‐collapsed blastoceles. In Experiment 2, embryo survival and DNA integrity were compared between vitrified and slow‐frozen mouse blastocysts. In Experiment 3, embryo survival and DNA integrity were compared between vitrified and slow‐frozen human blastocysts. Fresh blastocysts were used as controls in all experiments. Higher (P < 0.05) blastocyst survival rates were obtained in mouse blastocysts vitrified with collapsed versus intact blastoceles, although DNA‐integrity indices in the surviving blastocysts were the same among vitrified and fresh blastocysts. More mouse blastocysts (P < 0.05) survived after vitrification (100%) as compared to slow freezing (82.5%). DNA‐integrity indices examined in the surviving blastocysts were also higher (P < 0.001) in fresh (93.6%) and vitrified/warmed (93.7%) blastocysts than in slow‐frozen/thawed (75.8%) ones. More human blastocysts survived with a higher DNA‐integrity index after vitrification/warming than after slow freezing/thawing. These results indicate that higher survival rates can be obtained by vitrification of blastocele‐collapsed blastocysts, and that vitrification causes less cell apoptosis in both mouse and human blastocysts compared to slow freezing. Vitrification of blastocysts after blastocele collapse by single laser pulse supports a higher survival rate and less DNA apoptosis, suggesting that laser blastocele collapse is a safe procedure for blastocyst vitrification. Mol. Reprod. Dev. 79: 229–236, 2012. © 2011 Wiley Periodicals, Inc.     

Combination of intracellular cryoprotectants preserves the structure and the cells proliferative capacity potential of adult collared peccary testicular tissue subjected to solid surface vitrification

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.     

Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing

Grafting of cryopreserved testicular tissue is a promising tool for fertility and testicular function preservation in endangered species, mutant animals, or cancer patients for future use. In this study, we aimed to improve the whole neonatal mouse testicular tissue cryopreservation protocols by comparing cryosurvival, spermatogenesis, and androgen production of grafted testicular tissue after cryopreservation with three different vitrification protocols and an automated computed controlled-rate freezing. Whole neonatal mouse testes were vitrified with various vitrification solutions (V1) 40% EG + 18% Ficoll + 0.35 M Sucrose, (V2) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M PG), or (V3) 15% EG + 15% PG + 0.5 M Sucrose (total solute concentration V1:74.34%, V2:44.0%, and V3:49.22% wt/vol). Alternatively, neonatal testicular tissue was also frozen in 0.7 M DMSO +5% fetal bovine serum using controlled-rate freezing and compared to fresh grafted testicular tissue, sham grafted controls, and the vitrification protocol groups. Fresh (n = 4) and frozen-thawed (n = 4) testes tissues were grafted onto the flank of castrated male NCr Nude recipient mouse. The grafts were harvested after three months. Fresh or frozen-thawed grafts with controlled-rate freezing had the highest rate of tissue survival compared to other vitrified protocols after harvesting (p < 0.05). Both controlled-rate freezing and V1 protocol groups displayed the most advanced stages of spermatogenesis with elongated spermatids and spermatozoa in 17.6 ± 1.3% and 16.3 ± 1.9% of seminiferous tubules based on histopathological evaluation, respectively. Hosts of the testicular graft from controlled-rate freezing had higher levels of serum testosterone compared to all other vitrified-thawed graft groups (p < 0.05). This study shows that completed spermatogenesis from whole neonatal mouse testes were obtained when frozen with controlled-rate freezing and V1 vitrification solution and that testicular cryopreservation efficacy vary with the protocol and vitrification technique.     

Calves born after open pulled straw vitrification of immature bovine oocytes

The aim of this study was to evaluate the developmental capacity of immature bovine oocytes after vitrification with 20% ethylene glycol (EG)+20% dimethyl sulfoxide (Me(2)SO) and 0.5M sucrose (SUC), by open pulled straw (OPS) technology. The effect of treatment with cytochalasin D before vitrification was also examined. No differences were observed in cleavage and blastocyst rates among the group vitrified without cytochalasin D treatment (Vitri) (49.0% and 6.1%) and that with cytochalasin D treatment before vitrification (CDVitri) (46.4% and 3.6%), but both were lower (P<0.05) than the unvitrified control group (85.1 and 45.9%). Calves were obtained after transfer of fresh and vitrified blastocysts from the Vitri group and after transfer of vitrified blastocysts from the CDVitri group. Cytochalasin D treatment does not improve the development of immature bovine vitrified oocytes. The results show that a small proportion of immature oocytes vitrified with this technology are fully competent to produce blastocysts, which may be transferred immediately or vitrified before transfer, and go on to develop healthy offspring.     

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.     

Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.     

Vitrification of caprine secondary and early antral follicles as a perspective to preserve fertility function

The present study aimed to evaluate the structure, survival and development of isolated caprine (secondary-SEC and early antral-EANT) follicles, after vitrification in the presence of synthetic polymers and in vitro culture. Additionally, transzonal projections (TZPs) and p450 aromatase enzyme were evaluated. After isolation, SEC and EANT follicles were in vitro cultured for six days or vitrified. After one week, SEC and EANT follicles were warmed and also in vitro cultured for six days. Data revealed that the percentage of morphologically normal follicles was similar between fresh and vitrified follicles in both follicular categories and antrum formation rate was similar between fresh and vitrified SEC follicles. Fluorescence by calcein-AM did not show difference between fresh and vitrified (SEC and EANT) follicles, however, the trypan blue test showed low viability for vitrified follicles. The integrity of TZPs was not affected between fresh and vitrified SEC follicles, however, in vitrified EANT follicles, there were signs of TZPs loss. Regarding steroidogenic function, it was observed a positive staining for p450 aromatase enzyme in fresh and vitrified SEC and EANT follicles. It was concluded that SEC follicles seem to be more resistant to vitrification than EANT follicles, as shown by the trypan blue test and TZPs assay. Future studies may confirm this hypothesis, in order to consolidate the use of SEC and EANT follicles as an alternative to ovary cryopreservation.     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.     

Vitrification of collared peccary ovarian tissue using open or closed systems and different intracellular cryoprotectants

This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions – NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ± 8.6%); however, the SSV was only efficient with DMSO alone (63.9 ± 7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ± 2.9% viable cells with 2.0 ± 0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.     

Seasonal changes in the seminiferous epithelium of rhesus and bonnet monkeys

With a view to elucidate seasonal variations in testicular spermatogenesis, quantitative analysis of spermatogenic cells was carried out in non-human primate species viz. rhesus (Macaca mulatta) and bonnet (M. radiata) monkeys during breeding (October-December) and non-breeding (May-June) seasons. The results revealed significant inhibition of testicular germ cell population during non-breeding compared with the breeding period in both the species. Quantitative determination of Sertoli cell-germ cell ratio showed a marked decrease in the number of type A-spermatogonia, spermatocytes (non-pachytene and pachytene) and spermatids (in steps 1-12 of spermiogenesis) in rhesus monkey during the non-breeding period. Bonnet monkeys exhibited the significant decline in the number of primary spermatocytes and spermatids during the non-breeding phase. In addition, average diameter of round seminiferous tubules and nuclear diameter of Leydig cells also decreased significantly in rhesus monkeys. However, bonnet monkeys did not show any significant change in nuclear diameter/morphology of Leydig cells, testicular tubular diameter and number of type A-spermatogoniae. Sertoli cell number did not show any significant change during both breeding and non-breeding periods in both the species. The results of this study indicate a prominent seasonal variation in testicular spermatogenic/Leydig cells in rhesus monkeys than those observed in bonnet monkeys.     

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.     

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.     

Viability, maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices

This study was designed to evaluate the effects of vitrification on immature porcine and ovine oocytes, collected at a slaughterhouse, by performing vitrification in devices with different volumes. Viability was evaluated both before and after vitrification and maturation. Immediately after warming, the percentage of viable pig oocytes was 81% regardless the type of device, while in the control (after oocyte selection) was 95%. The viability of matured pig oocytes after warming, vitrified in beveled edge open straws (BES) was 6%, in small-open-pulled-straw (SOPS) was 17% and in cryotop was 4%, while the viability of the control group was 86%. The viability and maturation results were similar with all devices. Embryo development (ED) was observed in fresh porcine oocytes with 15% 2-8 cell embryos, 7% morulae and 3% blastocysts, and non-embryo cleavage was observed in warmed oocytes. The viability of sheep oocytes immediately after warming averaged 90% in all devices, while that of the control (after oocyte selection) averaged 95%. The viability of warmed oocytes after maturation was: BES 21%, SOPS 30%, cryotop 21% and control group 86%; while maturation values were 11, 21, 34 and 70%, respectively. After vitrification, the highest ED was achieved with ovine oocytes vitrified in SOPS, with 17% morulae development and it was the only device in which blastocysts developed. A direct relationship was observed between viability and actin filament integrity in both species.     

Influence of gonadotropins on adrenalectomy induced changes in the testis of albino mouse

Effects of adrenalectomy and administration of gonadotropins on cell counts of different cell types of spermatogenesis and morphology of the Leydig cells were studied in 30 day old mice. Adrenalectomy (duration, 12 days; age at autopsy 42 days) caused a significant decrease in the diameters of seminiferous tubules and Leydig cell nucleus and, cell counts of intermediate spermatogonia, round and elongated spermatids. Administration of FSH (75 micrograms/0.1 ml saline) + LH (25 micrograms/0.1 ml saline) everyday for 12 days to adrenalectomized mice restored testicular activity as revealed by significant increases in mean diameter of the Leydig cell nuclei and cell counts of intermediate spermatogonia and elongated spermatids over those of adrenalectomized mice. The results indicate that (i) testis of adrenalectomized mouse responds to gonadotropin treatment and (ii) impairment in gonadotropin secretion is possibly a major factor in inducing testicular regression following adrenalectomy.     

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer

We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blastocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated.     

Evaluation of sheep ovarian tissue cryopreservation with slow freezing or vitrification after chick embryo chorioallantoic membrane transplantation

The aim of our investigations was to compare the effectiveness of two methods for cryopreservation of sheep ovarian tissue, slow freezing and vitrification. The quality of cryopreserved tissues was evaluated after 5 days of thawing and chorioallantoic membrane (CAM) transplantation. Follicular structure, stromal integrity and neovascularization were assessed. The areas of fibrosis and necrosis were measured using MICROVISIBLE software, and proliferation was assessed with Ki-67 immunostaning. After 5 days of culture, the proportion of primordial follicles decreased, whereas the primary and intermediary follicles increased insignificantly (p > .05). Only necrosis in the vitrified culture group increased significantly (p < .05). It was established also that 5 days CAM culture was not suitable methodology for detection of folliculogenesis. Follicular quality decreased after culture, but was better in fresh and slow frozen tissues than after vitrification (p < .05). Cellular proliferative activity fell, but it preserved to some extent in all groups. In conclusion, follicles was preserved better in grafted tissue after slow freezing than vitrification and stroma was more susceptible to ischemia in vitrified rather than conventional freezing in this view. Vitrification may not be a suitable alternative to the slow freezing.     

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, −10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.