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a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
b Monsanto, St. Louis, Missouri 63198
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, follicular development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Developing a follicular culture system that will result in fertilizable oocytes would be advantageous not only for understanding folliculogenesis but also for the preservation and long-term storage of female germ cells. Transplantation of cryopreserved primordial follicles to sterilized animals can restore fertility [10], and trials have been carried out with human ovarian tissue [11]. Recent research has focused on developing a preantral follicular culture system because preantral follicles are a large potential source of oocytes that could be used in vitro for studying early folliculogenesis and embryo production.
Various methods have been developed to isolate and culture preantral follicles from mouse [12], rat [13], hamster [14], pig [15], bovine [16, 17], and human [6, 18] ovaries. Furthermore, both complete media (murine [12, 19–22], porcine [15], bovine [16, 17, 23], human [6, 18, 24]) and simple medium (porcine [25]) have been used for the culture of preantral follicles. As medium supplementation, fetal calf serum (FCS) (bovine [16, 26], porcine [15, 27]) and prepubertal gilt serum (PGS) (porcine [25]) were employed. So far, fertilizable oocytes have been obtained from in vitro culture of mouse primordial follicles [28] and early preantral follicles [29]. However, conditions for complete development of preantral follicles in vitro have not been established in the human or in domestic animals. This difficulty is likely due to the length of folliculogenesis and a lack of information on the regulation of preantral follicular and oocyte growth.
To develop a culture system that will result in porcine oocytes capable of undergoing in vitro maturation, fertilization, and embryonic development, we conducted a preliminary experiment to determine the follicular diameter at which antrum formation was initiated in vivo in prepubertal gilts and subsequently designed a series of experiments to 1) compare the effects of complete medium versus simple medium and FCS versus PGS on follicular growth rate, antrum formation, and recovery rate of cumulus cell-oocyte complexes (COCs) in vitro; 2) determine the importance of FSH in stimulating preantral follicular growth and antrum formation and in suppressing apoptosis of granulosa cells in cultured follicles determined by in situ terminal deoxynucleotidyl transferase dUTP nick end-label (TUNEL) staining; 3) determine the effect of serum type and various concentrations of FSH on porcine preantral follicular growth and development in vitro; and 4) reevaluate the culture system developed by Wu et al. [25] by assessing the meiotic competence of oocytes retrieved from cultured porcine preantral follicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries from prepubertal gilts were collected at a local abattoir, transported to the laboratory within 3 h at 25–28°C, and rinsed three times in 0.9% NaCl supplemented with 75 µg/ml penicillin G and 50 µg/ml streptomycin. Blood was collected and allowed to clot at room temperature from prepubertal gilts near the expected age of puberty from the University of Missouri-Columbia animal herd. The procedure for blood-sample collection was approved by the University of Missouri Animal Care and Use Committee. After centrifugation, prepubertal gilt serum (PGS) was harvested, aliquoted, and stored at -20°C until used for follicular culture. All chemicals were obtained from Sigma (Sigma Chemicals, St. Louis, MO) unless otherwise indicated.
Follicular Histology
A preliminary experiment was conducted to determine the follicular diameter at which antrum formation was initiated in prepubertal gilts. Ovaries collected from seven prepubertal gilts (6–7 mo of age and >100 kg body weight) at a local abattoir were sliced (<1 mm thick) with a hand microtome (Stadie-Riggs tissue slicer) and fixed in 10% (v/v) neutral buffered formalin for >48 h. Fixed tissues were washed in distilled water with several changes over 48 h, dehydrated through a graded series of ethanol (70–100%), cleared in xylene, and embedded in paraffin, and serial sections (6 µm thick) were prepared. The sections were deparaffinized in xylene, rehydrated through a graded series of ethanol (100–50%), and stained with hematoxylin/eosin (Fisher Scientific, Pittsburgh, PA). Follicular diameter at the widest point was determined with an ocular micrometer. Follicles were classified into groups by diameter (<200, 201–250, 251–300, 301–350, and >350 µm), and the proportion of follicles that had initiated antrum formation within each size group was recorded.
Isolation of Preantral Follicles and Assessment of Follicular Antrum Formation
Ovarian cortical tissue (<1 mm thick) was sliced from the ovarian surface. Based on the results of the preliminary experiment, preantral follicles with a diameter of 250–300 µm were visualized under the dissecting microscope and manually dissected with a 28-gauge needle and surgical blade. Hepes-buffered Tyrode medium containing 0.01% polyvinyl alcohol (w/v) was used for collection and holding of preantral follicles. Isolated follicles from different ovaries were pooled and processed for culture. This size class of follicles would be expected to form an antrum along with a 10% to 20% increase in follicular diameter in vivo.
After isolation, good-quality follicles were selected based on their morphology as described by Gutierrez et al. [30]. Briefly, follicles whose oocyte and granulosa cells were completely surrounded by the basement membrane, thecal cells, and stromal tissue were classified as good. As demonstrated in cattle [31], follicles selected in this way showed no signs of atresia. To assess antrum formation following culture, three follicles from each treatment group in experiment 1, as described below, were fixed with 10% (v/v) paraformaldehyde in Dulbecco PBS for 30 min and stained with 5 mg/ml ethidium bromide in Dulbecco PBS for 15 min at room temperature. After staining, the follicles were examined by confocal microscopy (Bio-Rad MRC-600, Richmond, CA) to confirm the presence of an antrum.
Culture of Follicles
The basal culture medium was North Carolina State University medium 23 (NCSU23) or TCM199 (Life Technologies, Gaithersburg, MD), both supplemented with 3.5 µg/ml insulin, 10 µg/ml transferrin, 100µg/ml L-ascorbic acid, 7.5% (v/v) PGS or fetal calf serum (FCS, Life Technologies, Gaithersburg, MD), and various concentrations of FSH (ICN Biomedicals, CA) according to the experimental design. Three follicles were placed per well in four-well plates (NUNC International, Naperville, IL) containing 500 µl medium and were cultured for 4 days. The plates were incubated at 39°C under 5% CO2 in air. To avoid rapid changes in the constituents and pH of the culture medium, half of the medium (250 µl) was replaced with freshly prepared medium every other day.
Experimental Design
Experiment 1 To determine the effects of different culture media (simple medium, NCSU23 versus complete medium, TCM199) and serum type (PGS versus FCS) on preantral follicular growth, antrum formation, and cumulus cell-oocyte complex recovery rate, a 2 x 2 factorial experimental design was used. Based on the culture system described by Wu et al. [25], the concentration of FSH used in the culture medium was 6 mIU/ml in this experiment. Follicular diameter was measured with an ocular micrometer daily and antrum formation was determined. At the end of the 4-day culture period, follicles were carefully opened with two fine needles and the COCs were morphologically evaluated. A cumulus cell-oocyte complex with continuous and compact layers of cumulus cells was considered to be an intact COC (refer to Fig. 1D). The recovery rate of intact COCs was calculated based on the number of follicles cultured in each treatment group. Oocyte diameter, without zona pellucida, was recorded as well. This experiment was replicated nine times with a total of 27 follicles cultured per treatment group.
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Experiment 2 This experiment studied the importance of FSH in stimulating follicular growth, antrum formation, and regulation of granulosa cell apoptosis in cultured follicles. The preliminary study of different concentrations of FSH in the culture medium (1, 2, 3, and 6 mIU/ml) showed that there was no difference among 2, 3, and 6 mIU/ml FSH concentrations in stimulating follicular growth and antrum formation. Thus, 2 mIU/ml FSH was used in this experiment. As described above, a total of 36 preantral follicles were cultured in NCSU23 medium with 2 mIU/ml FSH (n = 18) or without FSH (control; n = 18). Fetal calf serum was used as serum supplementation. At the end of the 4-day culture, all follicles were fixed in 10% (v/v) neutral buffered formalin overnight and apoptotic granulosa cells were detected using TUNEL staining as described below.
Experiment 3 A 2 x 3 factorial experimental design was used to determine the effects of serum type (FCS and PGS) and various concentrations of FSH (0.5, 1, and 2 mIU/ml) on preantral follicular growth and on the recovery rate of intact COCs from cultured follicles. Follicles were cultured for 4 days in NCSU23 medium as described in experiment 2. Follicular diameter and antrum formation were monitored daily. The recovery rate of intact COCs was recorded using the same criteria as described in experiment 1. A total of 126 follicles were studied in the 2 x 3 factorial experiment, with 21 follicles cultured in each treatment group.
Experiment 4 To evaluate the culture system described by Wu et al. [25], the same method as presented in that article was used to culture preantral follicles in NCSU23 medium.
In Vitro Maturation of Cumulus Cell-Oocyte Complexes and Assessment of Meiotic Competence
An in vitro maturation/in vitro fertilization (IVM/IVF) system has been established in our laboratory for porcine oocytes. Utilizing this system, approximately 37% to 41% of porcine oocytes collected develop to blastocysts [32, 33]. Using the same IVM system, oocyte meiotic competence was determined in the present study. Briefly, COCs recovered from each experiment were transferred to drops of TCM199 supplemented with 1 µg/ml FSH, 1 µg/ml porcine LH, and 10 ng/ml epidermal growth factor (EGF) under oil and cultured for 44 h. At the end of the maturation period, oocytes were fixed in acetic acid:ethanol (1:3, v/v) for 48–72 h at room temperature, stained with 1% (w/v) orcein in 45% acetic acid (v/v), and the meiotic stage of oocytes was assessed as described by Motlik and Fulka [34].
Detection of Apoptosis by TUNEL Staining
Histological tissue sections of cultured follicles were stained for apoptotic DNA using the in situ terminal deoxynucleotidyl transferase (TdT) TUNEL staining (Intergen, Purchase, NY). At the end of the 4-day culture in experiment 2, all follicles were fixed in 10% (v/v) neutral buffered formaldehyde overnight, dehydrated, and embedded in paraffin. The follicles embedded in paraffin were cut into 5-µm sections and mounted on silane-coated glass slides. Each eighth and ninth sections were used for TUNEL staining. After deparaffinization in xylene, tissue sections were incubated with proteinase K (20 µg/ml) and treated with 3% (v/v) hydrogen peroxide (H2O2) in PBS to block endogenous peroxidase activity. The slides were subsequently pretreated with TdT buffer, incubated with anti-digoxigenin antibody conjugated to peroxidase, stained in a solution of diaminobenzidine (the substrate for peroxidase), and counterstained in hematoxylin (BioGenex, San Ramon, CA). After counterstaining, the slides were mounted with supermount permanent aqueous mounting medium (BioGenex) and the number of apoptotic and total granulosa cells was immediately determined under a microscope (Olympus AX70) at 200x magnification. Negative controls were processed in an identical manner in each experiment. For negative controls, the TdT buffer was substituted with the same volume of PBS. Biological positive control slides were included with each assay. These control slides contained atretic follicles based on morphology isolated from a prepubertal gilt ovary [35].
Statistical Analysis
All dependent variables were analyzed for normality using the Wilk-Shapiro test [36]. The effects of medium, serum type, and FSH on follicular diameter on each day of culture were analyzed with the repeated measures GLM procedure of SAS [36], and follicular growth rate was analyzed using SAS GLM procedure [36] with initial follicular size as a covariate. Effects on antrum formation and COC recovery rate were analyzed using SAS GLM procedure [36] by including medium, serum, and the medium x serum interaction (experiment 1) or serum, FSH, and the serum x FSH interaction (experiment 3) in the model. In experiment 2, SAS ANOVA and GLM procedures were used to analyze the effect of FSH on antrum formation and apoptosis of granulosa cells in cultured follicles, respectively [36]. Significant differences among treatment groups in follicular growth rate, antrum formation, and COC recovery rate were determined using culture well as the error term. A 95% confidence level was used to indicate significance. Data are reported as least-squares means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two hundred and six preantral follicles were examined to determine the follicular diameter at which antrum formation was initiated in prepubertal gilts. Based on follicular diameter, these follicles were classified into five groups (<200, 201–250, 251–300, 301–350, and >350 µm) and the proportion of follicles that had an antrum was 0%, 6.7%, 23.1%, 46.2%, and 94.1%, respectively. Based on these results, follicles ranging from 250 to 300 µm in diameter without an obvious antrum were used for all experiments reported here.
Experiment 1: Comparison of Effects of Simple Medium (NCSU23) Versus Complete Medium (TCM199) and Prepubertal Gilt Serum Versus Fetal Calf Serum on Follicular Growth, Antrum Formation, and COC Recovery
All follicles used for experiment were manually isolated, and a representative follicle is shown in Figure 1A. The initial follicular diameter was not different among treatment groups (mean diameter = 265.8 ± 8.3 µm; P > 0.05). Serum type had no effect on growth rate (28.2 versus 32.6 µm/day for FCS and PGS, respectively, P > 0.05). Culture medium had significant effects on follicular growth (Fig. 2). Follicles cultured in TCM199 grew faster than in NCSU23 (35.9 versus 24.8 µm/day, P < 0.02), and there was no interaction between medium and serum on follicular growth rate (P > 0.05). The comparison of follicular growth rate between culture days showed that the growth rate during the first 2 days of culture was higher than during the last 2 days in both culture-medium groups (mean growth rate 101.1 versus 8.8 µm/2 days for the first and last 2 days of culture, respectively, P < 0.01; Fig. 2).
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Antrum formation was detected between Days 2 and 3 of culture (Fig. 1B) and was confirmed by confocal microscopy (Fig. 1C). At the end of culture, a higher cumulative percentage of antral follicles was obtained from follicles cultured in NCSU23 than in TCM199 (92.6% ± 4.7% versus 72.3% ± 4.8%, P < 0.01). There was also a significant interaction between medium type and serum type on the cumulative percentage of antral follicles (P < 0.02), indicating that the effect of medium on follicular antrum formation depended on the serum type supplemented.
At the end of follicular culture, follicles were carefully cut open and cumulus cell-oocyte complexes were recovered. A representative intact COC is shown in Figure 1D. An effect of serum type was detected on the COC recovery rate (P < 0.03), with PGS-supplemented groups higher. Further analysis of the data showed that COC recovery rate from follicles cultured in TCM199 supplemented with FCS was significantly lower than that from follicles cultured in TCM199 supplemented with PGS (P < 0.04). No difference was found between the two serum groups cultured in NCSU23 (Table 1). There was no interaction between medium type and serum type on COC recovery rate (P > 0.05). Oocyte diameter did not differ among groups (P > 0.05) and the mean diameter of all oocytes collected was 100.7 ± 11.4 µm. Because a primary objective for developing a preantral follicular culture system was to increase the percentage of preantral follicles that formed an antrum and to recover more COCs, NCSU23 medium was chosen for the following experiments.
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In order to evaluate oocyte meiotic competence, 43 oocytes were isolated from cultured follicles, matured for 44 h, and examined for nuclear maturation. No oocytes progressed beyond the germinal vesicle stage after in vitro maturation.
Experiment 2: Importance of FSH in Stimulating Follicular Growth, Antrum Formation, and Suppressing Apoptosis of Granulosa Cells
A follicular growth curve during the 4-day culture period is presented in Figure 3. Compared with the control group (0 mIU/ml FSH), follicles cultured in 2 mIU/ml FSH grew larger (P < 0.01) from Days 2 to 4 and a higher proportion (83.3%) formed an antrum. Follicles cultured without FSH did not increase in diameter and no antrum formation was observed after 4 days of culture. At the beginning of culture, the percentage of apoptotic granulosa cells in the freshly isolated follicles was 0.1% ± 0.04%. After the 4-day culture, the percentage of apoptotic granulosa cells in cultured follicles was 3.4% ± 0.7% and 1.3% ± 0.2% for the control and 2 mIU/ml FSH groups, respectively (P < 0.01). A cross-section of a cultured follicle with apoptotic granulosa cells is presented in Figure 4.
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Experiment 3: Effect of Various Concentrations of FSH and Serum Type on Follicular Growth, Antrum Formation, and COC Recovery
Follicular diameter at the beginning of culture (Day 0), follicular growth rate, cumulative percentage of antral follicles, and intact COC recovery rate are summarized in Table 2. There was no difference in follicular diameter on Day 0 (mean diameter = 303.9 ± 4.2 µm; P > 0.05). Over the entire range of concentrations studied, the best results in terms of follicular growth rate were obtained with 2 mIU/ml FSH (P < 0.01). The cumulative percentage of antral follicles at the end of culture was greater in follicles cultured in FCS-supplemented medium than those cultured in PGS-supplemented medium (97.2% ± 4.3% versus 72.7% ± 4.3%, P < 0.02). In addition, the recovery rate of intact COCs was higher from follicles cultured in FCS-supplemented medium compared with PGS-supplemented groups (69.5% ± 9.1% versus 37.0% ± 9.1%, P < 0.04). No interaction between FSH and serum type was found on follicular growth rate or cumulative percentage of antral follicles (P > 0.05).
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Sixty-five recovered intact COCs were placed into IVM medium and were examined for nuclear maturation after 44 h of culture. Again, none of the oocytes matured beyond the germinal vesicle stage.
Experiment 4: Reevaluating the Culture System Described by Wu et al. [25]
In total, 981 preantral follicles were isolated and cultured using the method as described by Wu et al. [25]. Three hundred forty-three COCs were recovered from cultured follicles at the end of the 4-day culture and 181 COCs were cultured in IVM medium. However, no oocytes developed to the metaphase II stage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A variety of complete media including TCM199 have been used in attempts to develop culture systems for preantral follicles in a number of species. Alternatively, NCSU23, a simple medium, was used by Wu et al. [25] in a porcine preantral follicular culture system and oocytes recovered from these follicles could be fertilized after in vitro maturation. To our knowledge, no report has been published comparing the effects of a simple medium versus a complete medium on preantral follicular growth and development in vitro in any species. TCM199 is a complex medium containing amino acids, vitamins, ribonucleosides, and deoxyribonucleosides in addition to the usual inorganic salts and energy sources (glucose) of a simple medium such as NCSU23. However, when follicles were cultured in NCSU23, despite the lower growth rate of cultured follicles, antrum formation was improved. More work is needed to optimize culture conditions employing TCM199. Unlike the murine species [37], in which the oocyte is almost fully developed in the preantral follicle, the porcine oocyte continues to grow after antrum formation. Therefore, it is speculated that porcine preantral follicles require all the ingredients found in the complete medium for oocyte RNA and protein synthesis and for oocyte growth and maturation.
When serum is a component of culture medium, it provides growth factors and improves preantral follicular growth and survival (human [24], pig [27]). Fetal calf serum is often used in the culture of bovine [16, 26] and porcine [15, 27] preantral follicles; however, Wu et al. [25] supplemented prepubertal gilt serum in a porcine preantral follicular culture system. We compared prepubertal gilt serum with fetal calf serum in the current study. Our results demonstrated that fetal calf serum provided the superior serum supplement for preantral follicular culture, and a higher number of intact cumulus cell-oocyte complexes could be recovered. Therefore, fetal calf serum, not prepubertal gilt serum, was found to provide a superior source of serum for developing a preantral follicular culture system.
Folliculogenesis in the pig from the primordial to the preovulatory stage is a lengthy process [38] and is regulated by an interaction among endocrine, paracrine, and autocrine factors in the ovary [2]. Although very little is known about the regulation of early development at the preantral follicular stage, the role of FSH in regulating folliculogenesis has been well recognized. Driancourt et al. [39] studied the role of gonadotropins in regulating porcine folliculogenesis in vivo by using hypophysectomy or treatment with a GnRH antagonist (Antarelix) that blocks pulsatile LH secretion but does not affect FSH. They demonstrated that the growth of 0.19- to 1.1-mm size follicles is gonadotropin independent, whereas growth from 1.1 to 2 mm is FSH dependent and growth of follicles larger than 2 mm is LH dependent. From a biochemical perspective, as follicles develop 2–3 layers of granulosa cells and the theca interna and externa cells begin to differentiate, FSH receptors appear on granulosa cells of preantral follicles in pigs [40, 41], and follicles become gonadotropin dependent. The current experiments demonstrated that preantral follicles could respond to increasing amounts of FSH, implying that culture of preantral follicles provides a powerful and simple approach to study early follicular regulation by FSH.
FSH plays a critical role in early follicular development in vitro. In the mouse, omission of FSH from the culture medium resulted in follicular cell death and the vitality of granulosa cells was compromised [42]. Hsueh et al. [5] and Chun et al. [8] suggested that the diffusion of several essential chemical and physical factors through the basal membrane could be compromised in the absence of FSH. The cultures without FSH more frequently resulted in extrusion of the oocyte from its original follicular structure [42], which may be caused by an impairment in gap junctions or reduced number of gap junctions [5, 43]. Addition of FSH to culture media promotes preantral follicular growth and survival and antrum formation in the mouse [42] and rat [44]. In the human, FSH promotes antrum formation and estrogen production in vitro [18]. Collectively, these data support the vital role of FSH in maintaining healthy oocyte growth and follicular structure. FSH appears to play an important role in inhibiting follicular atresia, in which apoptosis is suggested to be the underlying mechanism. It is known that FSH inhibits apoptosis in mouse preantral and antral follicles cultured in vitro [7]. In the rat, FSH appears to be the most important survival factor for early antral follicles [9] and will suppress apoptosis in serum-free culture of preantral [44] and antral [45] follicles. In isolated human preantral follicles, FSH was also seen to reduce atresia [6]. In agreement with the results from Wu et al. [25] and from other species [42], the present experiments demonstrated that addition of FSH to the culture medium significantly enhanced follicular growth and improved antrum formation. Furthermore, it suppressed follicular cell apoptosis, suggesting that FSH may prevent atresia of cultured follicles. Hence, an appropriate concentration of FSH is important for preantral follicular development. These data support the previous findings that FSH was required for the growth of preantral follicles in the human [6] and in the mouse [46].
The development of a culture system that supports growth of small follicles in the pig is an ambitious one. The span of oocyte development is lengthy in the pig when compared with the period in the mouse [38]. In vivo studies have shown that the early antral follicle requires approximately 40 days to grow to the preovulatory stage [38]. This means that the culture of large porcine preantral follicles may take many weeks, while the culture of small preantral follicles will require months to reach full maturity. From our experimental results, the follicular growth rate was higher during the first 2 days of culture and a gradual reduction of these rates was noticed during the last 2 days of culture in both TCM199 and NCSU23 medium, implying that these culture conditions were not ideal to sustain a long-term follicular culture. It is also evident that fetal calf serum and the high FSH concentration used, while significantly improving the intact COC recovery rate, had no effect on the proportion of oocytes resuming meiosis in vitro. Obviously, a 4-day culture was not long enough to culture oocytes to full maturity, which is in disagreement with the results reported by Wu et al. [25]. Moreover, in experiment 4, we isolated and cultured 981 preantral follicles using the method as described by Wu et al. [25]. Three hundred forty-three COCs were isolated at the end of culture and 181 COCs were cultured in IVM medium. However, we were not able to obtain any metaphase II stage oocytes. Furthermore, evidence has been reported that pig oocytes in follicles >1 mm in diameter could acquire the competence to resume meiotic maturation in vitro, but only those from follicles of about 2 mm in diameter complete the first meiotic division in vitro [47]. A close association was established between the oocyte diameter and the ability to resume meiosis in vitro [47]. Oocytes with a mean diameter of about 100 µm cannot complete germinal vesicle breakdown; those with a mean diameter of 115 µm, which is nearly equal that of full-sized pig oocytes [48], seem to be able to complete meiotic maturation [15, 47]. In the current study, oocyte diameter from cultured follicles was 100.7 ± 11.4 µm, and their inability to undergo germinal vesicle breakdown supports those observations [15, 47, 48]. It is a lengthy process for early antral follicles to grow to the preovulatory stage [38] and for the oocytes to gain developmental competency. Therefore, we conclude that 4-day culture of porcine preantral follicles was not long enough and longer term culture system (>4 days) may be required to allow oocytes to grow to maturity and to obtain developmentally competent fertilizable oocytes. It is essential that new methods be developed that ensure complete growth of these follicles in vitro. Alternatively, some other factors that may play an important role in oocyte growth and development, such as growth factors, steroid hormones, and LH, were absent in the media used, and their effects need to be investigated in future studies.
Culture systems for domestic animals are at an early stage of development and are at present being used to define the characteristics of preantral follicular growth and development rather than as a method of producing meiotically competent oocytes [16]. More research is necessary for reaching the final goal of establishing a culture system(s) that will support granulosa cell differentiation and maintain granulosa-oocyte cell contact to enable oocyte development to occur as has been demonstrated in the murine culture system [22, 29].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Billy N. Day, 159 Animal Science Research Center, University of Missouri-Columbia, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827; dayb{at}missouri.edu
Received: 3 January 2002.
First decision: 12 February 2002.
Accepted: 13 May 2002.
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REFERENCES |
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