Viability Of Preantral Follicles Research Articles

Effects of bovine ovarian storage conditions and vitrification on isolated preantral follicle viability.

The decreasing rate of successful pregnancies, both naturally and through assisted conception, has led to innovations in the way eggs, sperm, and embryos are stored. Despite these advances, the use of assisted reproductive techniques to preserve endangered or rare species remains unexplored. Since the location where samples are collected and facilities are often far apart, we aim to address part of this challenge by comparing different methods to store and handle ovarian tissue before freezing. This may pave the way for further research in preserving endangered species, despite the challenges posed by the distance between sample collection sites and suitable facilities.

Post-thaw viability of mouse preantral follicles after cryopreservation with cryotube freezing and OPS vitrification procedures.

In the field of reproductive science, there is an increased interest in the application of ovarian preantral follicles. Since the ovary contains a great amount of preantral follicles (PAF), the cryopreservation and in vitro culture of such follicles support the fertility preservation of domestic animals with high genetic value, endangered or zoo animals, and women before anticancer therapy. To date, no standard freezing or vitrification protocol is available in human or animals. The aim of the present study was to examine the viability of preantral follicles cryopreserved using freezing or vitrification protocols: cryotube freezing or OPS vitrification.

Microencapsulation and nanowarming enables vitrification cryopreservation of mouse preantral follicles

Preantral follicles are often used as models for cryopreservation and in vitro culture due to their easy availability. As a promising approach for mammalian fertility preservation, vitrification of preantral follicles requires high concentrations of highly toxic penetrating cryoprotective agents (up to 6 M). Here, we accomplish low-concentration-penetrating cryoprotective agent (1.5 M) vitrification of mouse preantral follicles encapsulated in hydrogel by nanowarming. We find that compared with conventional water bath warming, the viability of preantral follicles is increased by 33%. Moreover, the cavity formation rate of preantral follicles after in vitro culture is comparable to the control group without vitrification. Furthermore, the percentage of MII oocytes developed from the vitrified follicles, and the birth rate of offspring following in vitro fertilization and embryo transfer are also similar to the control group. Our results provide a step towards nontoxic vitrification by utilizing the synergistic cryoprotection effect of microencapsulation and nanowarming.

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200–300 μm) were randomly assigned into four groups. Group1 – Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2– FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2– FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (−196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Heterotopic ovarian allotransplantation in goats: Preantral follicle viability and tissue remodeling

An appropriate implantation site favors angiogenesis and avoids ovarian tissue damage after tissue grafting. The objective of this study was to evaluate the effects of intramuscular (IM) and subcutaneous (SC) sites for ovarian grafts in goats by evaluating follicular morphology and activation, preantral follicle and stromal cell densities, tissue DNA fragmentation, collagen types I and III depositions, and graft revascularizations. Ovarian cortical tissue was transplanted in IM or SC sites and recovered 7 or 15 days post-transplantation. There was a greater percentage of developing follicles and lesser follicular and stromal cell densities in all grafted tissues as compared to ovarian tissues of the control group. The stromal cell density and percentage of normal follicles were positively associated. At 15 days post-transplantation, tissues at the SC and IM sites had similar amounts of DNA fragmentation and type III collagen content. In contrast, tissues at the SC, as compared with IM site, had greater abundances of collagen type I. Furthermore, there was a positive association between collagen type I and percentage of morphologically normal follicles post-transplantation. In addition to a marked decrease in follicular density 15 days post-transplantation in ovarian grafts at the SC and IM sites, low percentages of normal follicles and follicular activation were observed similarly in both transplantation sites. There were also positive associations of stromal cell density and abundance of type I collagen fibers with the percentage of intact follicles in grafted ovarian tissues.

Effect of cryoprotectant type and concentration on the vitrification of collared peccary (Pecari tajacu) ovarian tissue

The aim of the present study was to establish a protocol for solid surface vitrification of peccary ovarian tissue by using different cryoprotectants. Ovarian pairs from five adult females were fragmented and two fragments (fresh control group) were immediately subjected to morphological evaluation using classical histology, transmission electron microscopy, and viability analysis using fluorescent probes. The remaining fragments (n = 18) were vitrified using a solid surface method with different concentrations (3 or 6 M) of ethylene glycol (EG), dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). After 2 weeks, samples were re-warmed and evaluated. A decrease in the percentage of morphologically normal preantral follicles (PFs) was verified for all the groups in comparison to the fresh control (92.0 ± 2.8%); however, if only the primordial follicles are considered, the most effective preservation (P < 0.05) was achieved with the use of EG at 3 M (74.2±7.3%) or DMSO at 6 M (75.0 ± 4.2%). Ultrastructural analysis indicated there were well-preserved PFs in all the groups evaluated, having well-defined membranes, a few vacuoles, and organelles that were uniformly distributed throughout the cytoplasm, mainly round and elongated mitochondria in close association with lipid droplets. Viability was preserved (P < 0.05) with the use of EG at 3 (97%) or 6 (97%) M, DMSO at 3 (100%), and DMF at 6 (97%) M. Solid surface vitrification, therefore, is an effective method for conservation of peccary female germplasm, especially with the use of EG at 3 M, which was highly effective for preservation of both the morphology and viability of PFs.

44 Proliferation and Antral Formation of Preantral Follicle Within Cryopreserved Cat Ovarian Tissue Transplanted into Nude Mice

Transplantation of cryopreserved ovarian tissue is a fertility preservation technique that results in live births in patients who have undergone pelvic chemo- or radiotherapy. It can be used for conservation purposes in endangered animals by transplanting the cryopreserved ovarian tissue of animals with highly valuable genetics into the immunodeficient animal to grow the follicles. This study aimed to examine the preantral follicular viability and K i-67 proliferation index of the preantral follicles within fresh and cryopreserved cat ovarian tissue transplanted into nude mice as a model for endangered felids. Adult female nude mice (n = 9) were chosen to be the hosts of ovarian tissues from 2-year-old domestic cat. Ovarian cortical tissues were cut into 66 small pieces (1 × 1 × 0.2 mm3). Half of the fragments were cryopreserved using slow-freezing method with 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose and thawed using thawing medium with 0.75 M DMSO and 0.5 M sucrose. Three pieces of fresh and cryopreserved tissues were transplanted into the subcutaneous pocket of the left and right side of the back of the nude mouse, respectively. Follicular viability and proliferation index were investigated after graft retrieval on Day 15 post-transplantation. Evaluation of preantral follicle viability was based on the integrity of the basement membrane, pyknotic bodies, and oocyte integrity. Proliferation index was determined by percentage of preantral follicle that had K i-67 immunopositive granulosa cells. Preantral follicle viability data was analysed using Kruskal-Wallis test and proliferation index data were analysed using general linear model test of least squares means. The percentages of viable follicles of all stages were decreased at Day 15 (Table 1). The percentage of proliferating preantral follicles in the fresh ovarian fragments was higher after transplantation (11 ± 2% and 46 ± 24% before and after transplantation, respectively; P &lt; 0.05). However, the percentages of proliferating follicles were not different between before and after cryopreserved tissue transplantation (35 ± 8% and 45 ± 33%, respectively). In conclusion, our findings showed the possibility of domestic cat ovarian tissue transplantation into the nude mouse. although cryopreserved ovarian tissue did not tolerate the short-term transplantation as the fresh tissue. This approach needs further investigation to optimize the transplantation technique in terms of ischaemic reperfusion and neovascularization. Table 1.Percentage of viable follicles per area of 0.0625 mm2 within fresh and cryopreserved cat ovarian fragments at Day 0 and Day 15 of the study

Mesenchymal Stem Cells Facilitate In Vitro Development of Human Preantral Follicle.

Biological folliculogenesis is a lengthy and complicated process, and follicle growth microenvironment is poorly understood. Mesenchymal stem cells (MSCs) have been shown to establish a supportive microenvironment for wound repair, autoimmune diseases amelioration, and tumor development. Therefore, this study is aimed to investigate whether MSCs could help to reconstruct a microenvironment to facilitate the in vitro follicle development. Here we show human MSCs significantly promote the survival rates, increase the growth velocity, and improve the viability of preantral follicles in a dose-dependent manner. Further analyses reveal that growth differentiation factor 9 and bone morphogenetic protein 15 in oocytes and inhibin βA and transforming growth factor β1 in granulose cells within the follicles cocultured with MSCs express notably higher than those in the follicles cultured without MSCs. In summary, our findings demonstrate a previously unrecognized function of MSCs in promoting preantral follicle development and provide a useful strategy to optimize fertility preservation and restoration by facilitating in vitro follicle growth.

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

64 CRYOPRESERVATION OF CAT OVARIAN TISSUE BY VITRIFICATION

The cryopreservation of ovarian tissue is linked to a wide range of possible applications, from oocyte harvesting to allo- and xenotransplantation. These procedures have significant potential for the preservation of valuable genetic material and endangered-species conservation. The objectives of the present study were to (1) compare viability of preantral follicles obtained from fresh v. vitrified feline ovarian cortex, (2) evaluate the effect of apoptotic inhibitors (ROCK inhibitor v. glutathione) on viability of follicles from vitrified samples, and (3) determine the optimal inhibitor concentration for follicle viability. In Experiment 1, 5 × 5 × 1 mm cortical tissue samples were obtained from excised cat ovaries and assigned to either the fresh control or vitrification group. Fresh samples were processed through a 230-micron-pore dissection strainer to collect preantral follicles. Follicles were then stained in Trypan blue to determine membrane integrity and survival rates. Vitrification samples were first equilibrated in 7.5% dimethyl sulfoxide and 7.5% ethylene glycol at ~22°C and then in vitrification solution consisting of 20% dimethyl sulfoxide, 20% ethylene glycol, and 0.5 M sucrose. They were then vitrified on a thin, perforated, metal strip (Cryotissue, Kitazato Biopharma, Fujinomiya, Japan). Samples were later warmed in 1.0 M sucrose at 38°C. Follicles were then collected and assessed for survival. In Experiment 2, follicles were collected from samples vitrified/warmed in cryo-media supplemented with either 3 × 104 nM ROCK inhibitor or 6 nM glutathione. Follicles from samples vitrified/warmed without inhibitor treatment were used as controls. In Experiment 3, tissue samples were vitrified/warmed in cryo-media supplemented with 0, 2, 6, or 10 nM glutathione before follicle viability was determined. Data were evaluated by chi square analysis. In Experiment 1, 637 and 340 follicles were collected from fresh and vitrified samples, respectively. Overall, survival was higher in freshly collected follicles when compared to those from the vitrified group (67 v. 18%, respectively; P &lt; 0.05). Evaluation of apoptotic inhibitors was determined through collection of 314, 354, and 506 follicles from inhibitor-free, ROCK inhibitor, and glutathione-treated media, respectively. Follicles from samples vitrified in inhibitor-free media and in ROCK inhibitor survived at a lower rate than those from glutathione-treated samples (10 and 13% v. 18%, respectively; P &lt; 0.05). In Experiment 3, a total of 539, 641, 625, and 632 follicles were collected from samples treated in 0, 2, 6, and 10 nM glutathione, respectively. There were no statistical differences in follicle survival among the 0, 2, and 6 nM groups. However, follicles treated in 10 nM glutathione survived at a higher rate than those vitrified/warmed in the absence of glutathione (20 v. 14%; P &lt; 0.05). In summary, viability of preantral follicles from ovarian cortical tissue was significantly reduced by vitrification. Despite this, tolerance of such follicles to cryopreservation was improved by vitrifying and warming in cryo-media containing 10 nM glutathione. Partially funded by the LSU/ACRES Collaborative Project.

Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method

A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production.

In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF‐1

Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 ± 22.1 μm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 ± 14.4 μm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 ± 15.9 μm, 63.6%, respectively) than that cultured without IGF-1 (26.7 ± 14.4 μm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase.

VIABILIDADE DE FOLÍCULOS PRÉ-ANTRAIS BOVINOS ISOLADOS DE OVÁRIOS APÓS VITRIFICAÇÃO

The aim of this study was to evaluate isolated bovine preantral follicles after vitrification procedure. Ovaries were obtained at a local slaughterhouse and submitted to follicular isolation. From the obtained suspension, one part was destined to control group. The remaining suspension was divided into two parts, one to the toxicity test and the other one to the vitrification procedure. For vitrification, the follicles were exposed for 1 min to vitrification solution-1, composed of 10% ethylene glycol, 10% DMSO in saline solution (NaCl 0.9%), and then 1 min to vitrification solution-2, composed of 20% ethylene glycol, 20% DMSO in the same saline solution supplemented with 0.5 M sucrose. The follicles were vitrified by use of glass capillaries and remained vitrified for 24 h. After warming, they were transferred to a 0.25 M sucrose solution for 5 min and then transferred to a 0.15 M sucrose solution for another 5 min. For the toxicity test, the follicles were only exposed to cryoprotectants and analised. The preantral follicles were classified as non-viable orviable when they were stained or not stained by trypan blue, respectively. Differences (P&lt;0.05) in follicular viability were observed among the control, non-vitrified group (93.6%), and exposed (64.1%) or vitrified follicles (66%). The percentages of normal follicles found after exposure to cryoprotectant did not differ significantly (P&gt;0.05) from that found after vitrification, indicating that the reduction of percentage of viable preantral follicles was due to a toxic effect of cryoprotectants and not to the vitrification procedure itself.KEYWORDS: bovine; cryoprotectants; oocytes; vitrification.

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.

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.

Isolation of pre-antral follicles from human ovarian medulla tissue

Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre-antral follicles from human ovarian medulla and evaluating its follicular content. Medulla was collected from 40 girls/women aged 3-35 years undergoing cryopreservation of the ovarian cortex. Follicle density was assessed for all patients and pre-antral follicles were isolated from 22 patients. On the basis of the neutral red (NR) staining of follicles and enzymatic digestion with a mixture of Collagenase IV and Liberase Thermolysin Medium, viable pre-antral follicles were isolated. NR accumulated in follicles resulting in a distinct red staining within the medulla. Follicle density of the medulla varied from 0 to 9824 follicles/gram of medulla and was significantly higher (P< 0.001) in the 3-9-year age group when compared with older groups (10-35 years). Enzymatic digestion combined with follicle identification by NR yielded a high output of isolated and viable pre-antral follicles from medulla, of which, 3607 follicles were collected and classified. The percentage of primordial and growing follicles decreased and increased, respectively, with age (P< 0.0001 and <0.0007). Discarded medulla contained a considerable pool of pre-antral follicles, especially in young girls. Our new method allowed the isolation of viable pre-antral follicles from human ovarian medulla and provides a unique opportunity for basic scientific studies and for culture and grafting purposes.

202 EFFECTS OF ASCORBIC ACID ON IN VITRO CULTURE OF CATTLE PREANTRAL FOLLICLES

The mechanisms that regulate the gradual exit of ovarian follicles from the nongrowing, primordial pool are poorly understood. The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine the effects of this addition on the growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; 1 fragment was immediately fixed in Bouin’s solution (control). The other fragments were cultured for 2, 4, 6, or 8 days on culture plates in minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, BSA, and antibiotics (MEM+) or in MEM+ plus ascorbic acid (5, 25, 50, 100, or 200 μg mL-1). Ovarian tissue was processed for classical histology, TEM, and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Preantral follicles were classified according to their development stage (primordial, intermediate, primary, and secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare percentages of follicles with PCNA-positive granulosa cells. All analyses were done with Statistical Analysis System (SAS Institute, Cary, NC, USA); P ≤ 0.05 was considered significant. Compared with control fragments, the percentage of primordial follicles was reduced (P ≤ 0.05) and the percentage of growing follicles was increased (P ≤ 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (P ≤0.05), but not when cultures were supplemented with 25, 50, and 100 μg mL-1 of ascorbic acid. Ultrastructural and immunohistochemical analysis of ovarian cortical fragments cultured for 8 days, however, showed the integrity and viability of follicles only when fragments were cultured in the presence of 50 μg mL-1 of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg mL-1 not only stimulates the activation and subsequent growth of cattle primordial follicles that are cultured in vitro for 8 days but also safeguards the viability of these preantral follicles. E. R. Andrade and A. A. Alfieri are recipients of the PRODOC/CAPES fellowship.

Effects of storing dromedary camel ovaries at 4 or 20°C for different periods of time on the morphology and viability of pre-antral follicles

The purpose of this study was to evaluate the effect of cooling ovarian tissue on camel pre-antral follicles. Ovaries were maintained in saline solution (0.9%) at 4 or 20°C for 6, 12 18 or 24 h. After storage, COCs were recovered from dromedary ovaries and were morphologically and viability evaluated by trypan blue exclusion test. The percentage of morphologically normal and live growing follicles was significantly reduced in ovarian tissue stored at 20°C for 12, 18 or 24 h. To assessment the viability of stored follicles, morphologically normal and live COCs isolated from ovaries stored at 4°C for 24 h and at 20°C for 6 h were in vitro matured (IVM) in TCM 199 + 10 μg/ml FSH + 10 IU hCG/ml + 10% FCS + 50 μg/ml gentamycin. COCs were incubated for 30 h at 38.5°C under 5% CO2 in humidified air. Follicles stored in either condition presented the same growth pattern in vitro as fresh follicles. We conclude that storage of camel ovaries at 4°C for up to 24 h or at 20°C for up to 6 h did not affect the morphology of growing follicles or their ability to mature in vitro.

Quantification of dimethyl sulfoxide perfusion in sheep ovarian tissue: a predictive parameter for follicular survival to cryopreservation.

The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ∼0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.

Quantification of Dimethyl Sulfoxide Perfusion in Sheep Ovarian Tissue: A Predictive Parameter for Follicular Survival to Cryopreservation

The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ∼0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.