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BOR - Papers in Press, published online ahead of print September 3, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016774
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BIOLOGY OF REPRODUCTION 70, 12–17 (2004)
DOI: 10.1095/biolreprod.103.016774
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

In Vitro Growth of Preantral Follicles Isolated from Cryopreserved Ovine Ovarian Tissue

Sandra Cecconi, Giulia Capacchietti, Valentina Russo, Paolo Berardinelli, Mauro Mattioli, and Barbara Barboni1

Dipartimento di Scienze e Tecnologie Biomediche,2 Università degli Studi dell'Aquila, 67100, L'Aquila, Italy Dipartimento di Scienze Biomediche Comparate,3 Università degli Studi di Teramo, 64100, Teramo, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.

estradiol, follicle, follicular development, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cryopreservation is widely applied in reproductive biology to preserve the reproductive potential of excess embryos and, therefore, to reduce the need for repeated gonadotropin-stimulation protocols. The storage of unfertilized eggs, either immature or mature, has been proposed as a strategy to preserve fertility, especially in young patients with cancer or in women at risk for premature ovarian failure. Despite many attempts, few results have been reported regarding the possibility of fertilizing thawed human oocytes to obtain newborn babies [1–3]. The low efficiency of this procedure originates from different forms of cell damage that occur during freezing/thawing, which dramatically affects further embryo survival [2, 4, 5]. Indeed, most of the thawed human oocytes show clear signs of degeneration, as evidenced by zona pellucida hardening and abnormal cytoplasmic morphology [6]. Oocyte cryopreservation has provided good results in the mouse, because the high survival rate of thawed oocytes has allowed the production of embryos with an uncompromised ability to develop to term [7–10].

The possibility of restoring fertility and achieving pregnancies after transplantation of cryopreserved ovarian cortical strips has been reported for large mammals [11], rodents [12–15], and recently, humans [16]. In effect, primordial/primary follicles survive the cryopreservation and grafting procedures in high numbers and can undergo subsequent development. However, the applicability of this procedure is affected by two main problems: first, restoration of fertility is impaired by postgrafting ischemia, which determines a dramatic loss of potentially developing follicles [17, 18], and second, reimplantation of stored ovarian cortex after chemotherapy does not exclude an eventual relapse of cancer [19].

Thus, an alternative option to preserve fertility relies on the possibility of obtaining mature oocytes from in vitro-cultured small follicles isolated from frozen/thawed tissue. This, in turn, implies the identification of culture conditions capable of successfully supporting full follicular development. As far as the culture of freshly collected preantral follicles, it is possible in rodents [20–23] and large mammals [24] to obtain large numbers of in vitro-grown (IVG) oocytes capable of being successfully fertilized. To date, the birth of offspring derived from oocytes of primordial follicles cultured entirely in vitro has been reported only for the mouse [25]. Concerning the in vitro culture of small follicles from cryopreserved ovarian cortex, the production of fertilizable mouse oocytes has been reported by many authors [10, 26, 27]. In large mammals, Newton et al. [28] analyzed the capacity of sheep granulosa cell-oocyte complexes, enzymatically isolated from ovarian cortical strips stored with dimethyl sulfoxide (DMSO), to develop in vitro after thawing. In their study, however, data regarding antral formation and estradiol release were reported, but little information was provided about oocyte quality.

The aim of the present study was to compare the in vitro development of intact sheep preantral follicles isolated from cryopreserved ovarian cortical strips stored with two widely used cryoprotectants (CPAs), DMSO and ethylene glycol (EG) [11, 12, 14, 16, 27, 28]. To provide information about the degree of cell damage induced by cryopreservation, at the end of culture the IVG antral follicles and related oocytes (obtained from tissue unfrozen, frozen, or exposed to CPAs) were analyzed with respect to their morphology as well as some qualitative parameters, such as estradiol release, metabolic cooperativity, and oocyte chromatin organization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

All chemicals used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.

Collection of Cortical Tissue

Prepubertal sheep ovaries were obtained from the local abattoir and transported to the laboratory in a thermostatic container at 30°C within 1 h of collection. The ovaries were freed from ligaments and rinsed several times in PBS supplemented with antibiotics (75 mg/L of penicillin-G, 50 mg/L of streptomycin sulfate). Then, the ovaries were placed into Hepes-buffered medium 199 and cut into fragments of approximately 0.5 x 0.5 x 0.5 cm with a surgical blade. These pieces were used to isolate unfrozen preantral follicles, to cryopreserve ovarian strips, to carry out the CPA toxicity test, and to isolate unfrozen preantral follicles.

Freezing and Thawing

Dimethyl sulfoxide and EG were used at the final concentration of 1.5 M in M2 medium supplemented with 10% fetal calf serum, antibiotics (75 mg/L of penicillin-G, 50 mg/L of streptomycin sulfate), and 0.1 M sucrose. Ovarian pieces were first aspirated into plastic insemination straws (Cryo Biosystem, Group IMV Technologies, Paris, France), held on ice for 15 min, and transferred to a programmable freezer (Cryo-Logic; IMV). The straws were cryopreserved by using a slow-cooling protocol as described by Gosden et al. [11]. Briefly, straws were cooled from 0°C to -9°C at 2°C/min, seeded manually, cooled again to -40°C at 0.3°C/min, cooled to -140°C at 10°C/min, and finally, plunged into liquid nitrogen and stored for as long as 3 mo. Thawing was carried out by rapidly warming the straws at 38°C; dilution of the cryoprotectant was performed at room temperature with Hepes-buffered TCM199 supplemented with decreasing concentrations of DMSO or EG and 0.1 M sucrose (three steps, 15 min each). Tissue strips were finally transferred into prewarmed isolation medium.

Follicle Culture

Preantral follicles were mechanically isolated from either frozen or unfrozen (fresh) ovarian fragments in Hepes-buffered TCM199. Preantral follicles were then measured by using an ocular micrometer (40x magnification) inserted into an inverted-phase microscope. Selected follicles with a mean diameter of 170 ± 20 µm were individually transferred to 96-V-well microtiter plates (Greiner Labortechnuk, Frickenhausen, Germany) and cultured in 25 µl of {alpha}-MEM supplemented with 2% fetal calf serum, 1% insulin-transferrine-selenium, antibiotics (75 mg/L of penicillin-G, 50 mg/L of streptomycin sulfate), and 1 µg/ml ovine FSH. Culture was carried out for 10 days at 38°C and 5% O2 (gas mixture: O2, 5%; CO2, 5%; N2, 90%). Culture media were changed every other day. For each well, 15 µl of conditioned media were stored at -80°C for estradiol determination. At the end of culture, follicles were measured and analyzed to record the presence of an antral-like cavity. Oocyte-cumulus cell complexes (OCCs) were then isolated from IVG antral follicles and analyzed for their morphological aspect. The OCCs presenting continuous and compact layers of cumulus cells were classified as healthy complexes and used either to determine metabolic coupling between the germinal and somatic compartment or to evaluate oocyte chromatin configurations. As a control, in vivo-grown OCCs were collected from antral ovarian follicles.

Histology

Follicles were fixed in 10% neutral buffered formalin for 1 h. Samples were dehydrated through increasing concentrations of ethanol, cleared in xylene, and embedded into Paraplast (plastic paraffin; Sherwood Medical Co., St. Louis, MO) for 24 h. Serial sections (thickness, 5 µm) were cut, transferred to glass slides, and stained with hematoxylin and eosin.

Evaluation of Oocyte Chromatin Organization

To evaluate germinal vesicle (GV) chromatin configurations, oocytes from OCCs recovered from control and IVG antral follicles, either unfrozen or frozen, and devoid of surrounding cumulus cells were fixed in 3.7% paraformaldehyde. After a 15-min incubation in 0.1 mg/ml of Hoechst 33342 in PBS, oocytes were then mounted in 50% PBS-50% glycerol and observed with a fluorescence microscope (40x magnification; Axioplan 2; Zeiss, Göttingen, Germany). The GV conformations were classified as unrimmed (GVu) or rimmed (GVr) based on the degree of heterochromatin associated with the nucleolus [29–31]. Oocytes presenting different chromatin arrangements or dispersed chromosomes were also recorded and classified as abnormal configurations.

Estradiol Determination

The levels of estradiol-17ß released by follicles during the 10-day culture period were determined using an Estradiol EIA kit (582251; Cayman Chemical Company, Ann Arbor, MI). The sensitivity of the assay, defined as the amount of steroid giving a 10% drop with binding of the enzyme-conjugated estradiol, was 9 pg/ml. Intra- and interassay precision, expressed as the coefficients of variations for replicate determination of the sample, was 3.8% (10 replicates) and 5.5% (eight replicates), respectively. The levels of estradiol in samples of conditioned media were expressed as pg/follicle.

Determination of Intercellular Cooperativity

Metabolic cooperativity was measured as previously described by Mattioli et al. [32]. The OCCs obtained from control and IVG antral follicles were incubated, in part, as naked oocytes after stripping the surrounding cumulus cells, and, in part, as OCCs. Both groups of germ cells were then incubated for 2 h in culture medium containing 100 µCi/ml of [3H]uridine (28 Ci/mmol; Amersham, Little Chalfont, U.K.). Afterward, cumulus cells were removed from the OCCs, and all naked oocytes were individually transferred into 20 µl of SDS buffer for radioactivity determination. Coupling index (CI) values were determined using the formula A - B/B, where A and B indicate the uptake (expressed in cpm) of [3H]uridine in the oocytes incubated with cumulus cells or without cumulus cells, respectively.

Toxicity Test

To evaluate CPA toxicity, ovarian pieces were aspirated into straws containing 1.5 M EG or DMSO and then incubated at room temperature for 15 min. Afterward, the CPAs were removed as described for thawed sample, and preantral follicles were placed in culture for 10 days. In vitro follicle growth, estradiol levels, and oocyte-cumulus cell intercellular cooperativity were determined as previously described.

Statistical Analysis

Data are presented as the percentage or mean ± SEM. Differences in estradiol production were evaluated by ANOVA followed by the Tukey-Kramer test for comparison of multiple groups. The CI values were compared by chi-square test; all other data were analyzed by Student t-test. Values with P < 0.05 were considered to be statistically different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Follicular Development and Morphological Evaluation

The first series of experiments was performed to compare the morphological architecture of preantral follicles isolated from unfrozen or frozen ovarian cortex. Although thawed preantral follicles appeared morphologically similar to those isolated from unfrozen tissue, histological analysis performed after 1 day of culture revealed that follicle organization was dramatically affected by cryopreservation. The majority (94%; n = 33) of preantral follicles isolated from unfrozen cortex (Fig. 1a) showed oocytes with agranular cytoplasm and a central GV surrounded by several layers of granulosa cells and a well-organized thecal layer. By contrast, 50% of EG (n = 15) and 34% of DMSO (n = 15; EG vs. DMSO, P < 0.01) cryopreserved follicles revealed a damaged structure, frequently characterized by loss of intercellular communications between the oocyte and the surrounding granulosa cells, and signs of pyknosis in the GV as well as in some granulosa cells (Fig. 1b). The remaining follicles (Fig. 1c) showed a normal morphological architecture of the oocyte and neighboring granulosa cells. However, some signs of fractures in the stromal tissue and within the thecal layer were evident, independent of the CPA that was used.



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  		FIG. 1. Photomicrographs of sheep preantral follicles cultured for 1 day. a) Follicles isolated from unfrozen tissue. The germinal vesicle-stage oocyte is surrounded by several layers of granulosa cells. b) Frozen, damaged follicle. Irrespective of the CPA used, disruption of intercellular contacts among granulosa cells and between the innermost granulosa layer and the oocyte is recognizable. c) Frozen follicle. Minor freezing damage is evident, including areas of detachment of theca cells from the basal membrane (arrowheads). Bar = 50 µm

Freshly isolated and frozen preantral follicles grew in vitro from a mean diameter of approximately 170 µm to a final diameter of approximately 300–310 µm after 10 days of culture, without significant differences among the experimental groups tested (Table 1). Extrusion of oocytes during culture, which represents a sign of follicle degeneration, was infrequent in all groups analyzed (<5%). The presence of a translucent, antral-like cavity was recognizable starting from Day 6, and by the end of culture, the proportion of follicles forming an antral-like cavity was similar in unfrozen and frozen follicles. This result was confirmed by histological analysis, which evidenced a similar morphological aspect between IVG antral follicles derived from unfrozen (Fig. 2a) or cryopreserved (Fig. 2b) tissue. In fact, in both groups of follicles, two distinct granulosa cell populations were clearly recognizable, with the OCC being clearly distinguishable from mural granulosa cells. The IVG follicles obtained from fresh tissue (Fig. 2a) showed a well-organized thecal layer. Cryopreserved follicles showed a certain degree of stromal and thecal disorganization while maintaining an intact lamina, as revealed by the columnar aspect of the mural granulosa adjacent to the basal membrane (Fig. 2b).


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  		TABLE 1. In vitro development of sheep preantral follicles obtained from frozen or unfrozen ovarian cortex and cultured for 10 days.a



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  		FIG. 2. Histological analysis of IVG antral follicles from (a) unfrozen or (b) cryopreserved ovarian tissue. Both IVG follicles show a comparable morphological aspect. Bar = 50 µm

Estradiol Determination

The levels of estradiol released during the culture period were determined only for follicles forming antral cavities. As reported in Figure 3, unfrozen follicles released increasing amounts of estradiol, with a sharp rise from Day 6 to Day 10 (P < 0.01). A similar final level of this steroid was determined for EG follicles, although this value was lower than that for control up to Day 6 (P < 0.05). Production of estradiol by DMSO cryopreserved follicles was comparable to that of EG cryopreserved cells up to Day 6, but from this day onward, it remained significantly lower compared with the unfrozen and EG groups (P < 0.01).



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  		FIG. 3. Estradiol secretion by IVG antral follicles obtained from fresh or cryopreserved ovarian tissue frozen with DMSO or EG. Values are expressed as the mean ± SEM of at least 20 follicles/treatment. *P < 0.01

Assessment of Oocyte Development and Metabolic Cooperativity

On Day 10, OCCs were recovered from IVG antral follicles and their morphological aspect assessed. As shown in Table 1, the percentage of healthy OCCs derived from IVG unfrozen follicles was significantly higher compared to those from cryopreserved follicles, even if EG appeared to protect OCC quality more efficiently (Table 1) (EG vs. DMSO, P < 0.01). The extent of metabolic cooperativity between the somatic and germinal compartment was measured by comparing CI values between the different experimental groups. The CI values were arbitrarily sorted into two classes: "high" (range, 10–30) and "low" (<10). As shown in Figure 4, 100% and 80% ± 10%, of OCCs from control and IVG unfrozen follicles, respectively, displayed high CI values, in comparison with 51% ± 6% of EG and 35 ± 7% of DMSO groups.



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  		FIG. 4. Mean percentage of oocytes with high CI values (10 <= CI < 30). Data are expressed as the mean percentage ± SEM of oocytes that show a high CI in seven independent experiments. The number of oocytes evaluated is at least 30 for each group. Values with different superscripts are significantly different (P < 0.01)

To assess oocyte growth, only the diameters of oocytes isolated from healthy OCCs were recorded at the end of culture. Starting from a mean diameter of 75 ± 5 µm, oocytes collected from unfrozen and EG cryopreserved complexes reached a final diameter that was slightly higher than, although not significantly different than, that of DMSO oocytes (unfrozen, 125 ± 2 µm; EG, 120 ± 3 µm; DMSO, 116 ± 10 µm; P > 0.05) . In all the experimental groups tested, however, the final mean size was significantly lower than that recorded for fully grown oocytes (140 ± 6 µm; P < 0.01). The analysis of GV chromatin organization showed that the rimmed configuration (GVr) was recognizable in 64% and 42% (n = 25; P < 0.05) of the oocytes obtained from complexes retrieved in vivo or from unfrozen IVG follicles, respectively. Cryopreservation negatively affected the acquisition of such a configuration, which was shown by only 25% of EG and 19% of DMSO oocytes (EG vs. DMSO, P < 0.05; unfrozen vs. frozen, P < 0.01). The remaining oocytes displayed the unrimmed configuration (GVu), with the exception of the cryopreserved groups, in which approximately 20% of oocytes presented abnormal chromatin configurations.

Toxicity Test

Evaluation of the toxic effects of CPAs on in vitro follicular development showed that preantral follicles obtained from cortical tissues exposed to 1.5 M EG (n = 55) or DMSO (n = 60) grew in vitro (unfrozen, 310 ± 8 µm; EG, 318 ± 10 µm; DMSO, 315 ± 9 µm; P > 0.05) and formed, by the end of culture, antral-like cavities with percentages comparable to those of fresh controls (unfrozen, 78% ± 3%; EG, 70% ± 6%; DMSO, 73% ± 9%; P > 0.05). Estradiol levels, as detected during the whole culture period, evidenced a similar steroidogenic activity among the different experimental groups. In fact, starting from Day 6 of culture, the levels of estradiol released from follicles exposed either to DMSO or EG increased significantly, reaching a mean value on Day 10 of 520 ± 25 pg/follicle, which was similar to that detected for the unfrozen control (504 ± 17 pg/follicle; P > 0.05). In addition, CI determinations performed on healthy OCCs (unfrozen, 56% ± 3%; EG, 53% ± 8%; DMSO, 57% ± 9%; P > 0.05) revealed that exposure to CPAs did not affect the metabolic cooperativity between the oocyte and the surrounding cumulus cells, because the majority of oocytes presented high levels of CI (unfrozen, 80% ± 10%; EG, 71% ± 9%; DMSO, 77% ± 9%; P > 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that the cryopreservation of sheep cortical tissue allows the recovery of viable preantral follicles, which can be grown in vitro with different efficiencies depending on the CPA used. Although the toxicity test reveals similar functional performance in preantral follicles exposed to EG and DMSO, the freezing/thawing procedures show that EG more efficiently preserves ovine follicle survival. The in vitro development demonstrated, in fact, that EG cryopreserved follicles showed a higher percentage of healthy, metabolically coupled OCCs, and an estradiol secretion significantly higher than that recorded in DMSO frozen follicles.

The culture system used in the present study was defined in our previous work [33]. This system is based on the mechanical isolation of small follicles from ovarian cortical tissue. Although it is a time-consuming procedure, it allows the recovery of theca-intact follicles, which maintain their three-dimensional structure throughout the whole culture period, efficiently form antral cavities, and produce fertilizable oocytes [21–24]. Also, in humans, mechanical isolation of preantral follicles results in the formation of steroid-secreting antral follicles [34]. Indeed, physiological follicular development requires the presence of both granulosa and thecal/stromal cells, which play a fundamental role in the modulation/coordination of gonadotropin stimulation and steroid secretion [35]. Several reports, however, have described in vitro follicular development and antral cavity formation with reference to enzymatically isolated preantral follicles, either from unfrozen [28, 36–39] or cryopreserved tissue [28, 40]. In these experiments, theca cell removal does not impair antral cavity formation [28, 36–40], and estradiol release occurs following androstenedione supplementation in culture media [28].

Our results confirm previous observations indicating that the freezing/thawing process severely damages preantral follicles, which are considered to be less resistant than primordial ones to the injuries caused by cryopreservation [40]. This can be explained by the increased complexity of the growing follicles, which makes them more sensitive to cryodamage. The physiological differentiation of mammalian ovarian follicles is strictly dependent on the existence of a bidirectional, regulative dialogue between the germinal and somatic cell compartments. This dialogue is exerted either through the production of paracrine/autocrine factors or through the presence of functional gap junctions [41]. The important role of this specialized intercellular contact is confirmed by the finding that follicular development in mice lacking Cx37 (i.e., the connexin forming heterologous gap junctions) is irreversibly arrested at early stages of folliculogenesis [42, 43]. From our results, the evaluation of postthaw follicle viability by morphological analysis shows that cryopreservation can cause disruption of gap junctional communication, thus impairing follicle survival. The extent of such damage is dependent on the CPA used, as demonstrated by the finding that follicles survive at a higher percentage when frozen in EG than when frozen in DMSO. Moreover, cryopreservation by itself can induce variable degrees of junctional damage, as demonstrated by the lower percentage of IVG oocytes retrieved from frozen antral follicles showing high metabolic cooperativity values. Indeed, this result shows the limits of morphological analysis, which provides similar percentages of antral-like cavity formation and the absence of apparent morphological differences between groups. The different CI values determined can be explained only in part by a negative effect of culture conditions. In fact, a decrease (~20%) in the percentage of oocytes belonging to the class with high cooperativity values has been recorded for IVG unfrozen oocytes. However, the observation that the majority of EG follicles contain healthy-looking OCCs, the CI values and estradiol release of which are significantly higher in respect to the DMSO groups, further confirms that different CPAs can differentially affect overall postthaw viability.

The quality of IVG oocytes has also been evaluated by analyzing chromatin configuration at the GV stage. Data in the literature have demonstrated that in the mouse, the transition from a decondensed (GVu) to a condensed (GVr) perinuclear configuration occurs in vivo at the time of antrum formation [29, 30] and is coincident with transcriptional inactivation characterizing the oocytes approaching the end of their growth phase [30, 44–46]. In our experiments, we have observed that oocytes collected in vivo from antral follicles display a significantly higher percentage of GVr configuration with respect to IVG unfrozen oocytes. Because IVG oocytes reach a lower final diameter, this result is in agreement with previous observations conducted on bovine [47] and human oocytes [31], demonstrating a relationship between oocyte size and GV remodeling. However, the finding that a very low number of IVG cryopreserved oocytes display the rimmed organization strongly suggests that freezing/thawing also dramatically affects the occurrence of processes involved in the acquisition of a chromatin conformation necessary for sustaining complete nuclear maturation and successful embryonic development [48, 49].

Finally, our results also demonstrate that the in vitro culture of sheep preantral follicles is a useful test for evaluating damage induced by CPA and/or freezing procedures, as previously proposed for the mouse [27] and the sheep [50]. However, a comprehensive evaluation of cryodamage cannot be based exclusively on morphological analysis; rather, it needs to be assessed by more detailed biochemical investigations.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Delia Nardinocchi for her precious technical assistance and Dr. Giovanni Coticchio for critical reading of the manuscript.


   FOOTNOTES
 
1 Correspondence: Barbara Barboni, Dipartimento di Scienze Biomediche Comparate, Università degli Studi di Teramo, 64100, Teramo, Italy. FAX: 39 086 141 1285; barboni{at}ifv.vet.unite.it Back

Received: 28 February 2003.

First decision: 27 March 2003.

Accepted: 26 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997 68:724-726[CrossRef][Medline]
  2. Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human egg cryopreservation. Hum Reprod 1998 13:3156-3159[Abstract/Free Full Text]
  3. Porcu E, Fabbri R, Petracchi S, Ciotti PM, Flamigni C. Ongoing pregnancy after intracytoplasmic injection of testicular spermatozoa into cryopreserved human oocytes. Am J Obstet Gynecol 1999 180:1044-1045[CrossRef][Medline]
  4. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986 i 884-886
  5. Al-Hasani S, Diedrich K, Van Der Ven H, Reinicke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod 1987 2:695-700[Abstract/Free Full Text]
  6. Pickering SJ, Braude PR, Johnson MH. Cryoprotection of human oocytes: inappropriate exposure to DMSO reduces fertilization rates. Hum Reprod 1991 6:142-143[Abstract/Free Full Text]
  7. Levron J, Willadsen SM, Shimmel T, Cohen J. Cryopreservation of activated mouse oocytes and zygote reconstruction after thaw. Hum Reprod 1998 13:109-116
  8. Sztein JM, O'Brien MJ, Farley JS, Mobraaten LE, Eppig JJ. Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term. Hum Reprod 2000 15:567-571[Abstract/Free Full Text]
  9. Liu J, Van der Elst J, Van den Broecke R, Dhont M. Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation. Biol Reprod 2001 64:171-178[Abstract/Free Full Text]
  10. De la Pena EC, Takahashi Y, Katagiri S, Atabay EC, Nagano M. Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles. Reproduction 2002 123:593-600[Abstract]
  11. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum Reprod 1994 9:597-603[Abstract/Free Full Text]
  12. Cox SL, Shaw J, Jenkin GJ. Transplantation of cryopreserved fetal ovarian tissue to adult recipients in mice. J Reprod Fertil 1996 107:315-322[Abstract/Free Full Text]
  13. Gunasena KT, Villines PM, Crister ES, Critser JK. Live births after autologous transplant of cryopreserved mouse ovaries. Hum Reprod 1997 12:101-106
  14. Candy CJ, Wood MJ, Whittingham DG. Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries. J Reprod Fertil 1997 110:11-19[Abstract/Free Full Text]
  15. Sztein J, Sweet H, Farley J, Mobraaten L. Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking. Biol Reprod 1998 58:1071-1074[Abstract/Free Full Text]
  16. Oktay K. Ovarian tissue cryopreservation and transplantation: preliminary findings and implications for cancer patients. Hum Reprod Update 2001 7:526-534[Abstract/Free Full Text]
  17. Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil 1998 114:341-346[Abstract/Free Full Text]
  18. Aubard Y, Piver P, Cogni Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999 14:2149-2154[Abstract/Free Full Text]
  19. Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Unfrozen and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 1996 11:1668-1673[Abstract/Free Full Text]
  20. Eppig JJ, Schroeder AC. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol Reprod 1989 41:268-276[Abstract]
  21. Spears N. In vitro growth of oocytes. Hum Reprod 1994 9:969-970[Free Full Text]
  22. Cortvrindt R, Smitz J, Van Steirteghem AC. In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system. Hum Reprod 1996 11:2656-2666[Abstract/Free Full Text]
  23. Cortvrindt R, Smitz J. Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa cell proliferation. Theriogenology 1998 49:845-859[CrossRef][Medline]
  24. Wu J, Carrell DT, Wilcox AL. Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection. Biol Reprod 2001 65:1579-1585[Abstract/Free Full Text]
  25. O'Brien MJ, Pendola JK, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod 2002 68:1682-1686
  26. Cortvrindt R, Smitz J, Van Steirteghem AC. A morphological and functional study of the effect of slow freezing followed by complete in vitro maturation of primary mouse ovarian follicles. Hum Reprod 1996 11:2648-2655[Abstract/Free Full Text]
  27. Newton H, Illingworth P. In vitro growth of murine preantral follicles after isolation from cryopreserved ovarian tissue. Hum Reprod 2001 16:423-429[Abstract/Free Full Text]
  28. Newton H, Picton H, Gosden RG. In vitro growth of oocytes-granulosa cell complexes isolated from cryopreserved ovine tissue. J Reprod Fertil 1999 115:141-150[Abstract/Free Full Text]
  29. Mattson BA, Albertini DF. Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol Reprod Dev 1990 25:374-383[CrossRef][Medline]
  30. Johnson LD, Albertini DF, McGinnis LK, Biggers JD. Chromatin organization, meiotic status and meiotic competence acquisition in mouse oocytes from cultured ovarian follicles. J Reprod Fertil 1995 104:277-284[Abstract/Free Full Text]
  31. Combelles CM, Cekleniak NA, Racowsky C, Albertini DF. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod 2002 17:1006-1016[Abstract/Free Full Text]
  32. Mattioli M, Galeati G, Bacci ML, Seren E. Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocytes. Gamete Res 1988 21:223-232[CrossRef][Medline]
  33. Cecconi S, Barboni B, Coccia M, Mattioli M. In vitro development of sheep preantral follicles. Biol Reprod 1999 60:594-601[Abstract/Free Full Text]
  34. Abir R, Franks S, Mobberley MA, Moore PA, Margara RA, Winston RML. Mechanical isolation and in vitro growth of preantral and small antral human follicles. Fertil Steril 1997 68:682-688[CrossRef][Medline]
  35. Greenwald GS, Roy SK. Follicular development and its control. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994:629–723
  36. Figureido JR, Hulshof SCJ, Van den Hurk R, Ectors FJ, Fontes RS, Nusgens B, Bevers MM, Beckers JF. Development of a new mechanical and enzymatical method for the isolation of intact preantral follicles from fetal, calf and adult bovine ovaries. Theriogenology 1993 40:789-799
  37. Wandji SA, Eppig JJ, Fortune JE. FSH and growth factors affect the growth and endocrine functions in vitro of granulosa cells of bovine preantral follicles. Theriogenology 1996 45:817-832
  38. Roy SK, Treacy BJ. Isolation and long-term culture of human preantral follicles. Fertil Steril 1993 59:783-790[Medline]
  39. Hirao Y, Nagai T, Kubo M, Miyano T, Miyake M, Kato S. In vitro growth and maturation of pig oocytes. J Reprod Fertil 1994 100:333-339[Abstract/Free Full Text]
  40. Gosden RG, Mullan J, Picton HM, Yin H, Tan SL. Current perspective on primordial follicle cryopreservation and culture for reproductive medicine. Hum Reprod Update 2002 8:105-110[Abstract/Free Full Text]
  41. Cecconi S. Growth and differentiation of small ovarian follicles in mammals: problems and perspective. J Reprod Develop 2002 48:431-445[CrossRef]
  42. Simon AM, Goodenough DA, Li E, Paul DL. Female infertility in mice lacking connexin 37. Nature 1997 385:525-529[CrossRef][Medline]
  43. Carabatsos MJ, Sellitto C, Goodenough DA, Albertini DF. Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence. Dev Biol 2000 226:167-179[CrossRef][Medline]
  44. Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev 1995 42:437-442[CrossRef][Medline]
  45. Bouniol-Baly C, Hamraoui L, Guibert J, Beaujean N, Szollosi MS, Debey P. Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes. Biol Reprod 1999 60:580-587[Abstract/Free Full Text]
  46. De la Fuente R, Eppig JJ. Transcriptional activity of the mouse oocyte genome: companion granulosa cells modulate transcription and chromatin remodeling. Dev Biol 2001 229:224-236[CrossRef][Medline]
  47. Fair T, Hyttel P, Greve T, Boland M. Nucleus structure and transcriptional activity in relation to oocyte diameter in cattle. Mol Reprod Dev 1996 43:503-512[CrossRef][Medline]
  48. Christians E, Boiani M, Garagna S, Dessy C, Redi CA, Renard JP, Zuccotti M. Gene expression and chromatin organization during mouse oocyte growth. Dev Biol 1999 207:76-85[CrossRef][Medline]
  49. Zuccotti M, Ponce RH, Boiani M, Guizzardi S, Govoni P, Scandroglio R, Garagna S, Redi CA. The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst. Zygote 2002 10:73-78[CrossRef][Medline]
  50. Amorim CA, Rondina D, Rodrigues APR, Costa SHF, Goncalves PBD, de Figueiredo JR, Giorgetti A. Isolated ovine primordial follicles cryopreserved in different concentrations of ethylene glycol. Theriogenology 2003 60:735-742[CrossRef][Medline]



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S. Cecconi, A. Mauro, G. Capacchietti, P. Berardinelli, N. Bernabo, A. R. Di Vincenzo, M. Mattioli, and B. Barboni
Meiotic Maturation of Incompetent Prepubertal Sheep Oocytes Is Induced by Paracrine Factor(s) Released by Gonadotropin-Stimulated Oocyte-Cumulus Cell Complexes and Involves Mitogen-Activated Protein Kinase Activation
Endocrinology, January 1, 2008; 149(1): 100 - 107.
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