hormonal and neuropeptidergic compounds as well as cytokines and nutritional compo- Ž
. nents that initiate and maintain follicular growth Hurk et al., 1997 . There is report of
Ž .
using insulin–transferrin–selenium ITS to maintain granulosa cells within the follicu- lar microenvironment. Insulin is essential for follicle culture; lack of insulin results in
follicle degeneration. However, insulin alone is insufficient to maintain healthy follicles. Moreover, selenium acts as an acceptor of free radicals generated during cell metabolism
Ž .
and it improves culture conditions Roy and Treacy, 1993 . The presence of additives such as FSH, ITS, glutamine and sodium pyruvate greatly improved development of the
follicles. This was manifested by a twofold increase in the percentage of growth and Ž
survival time of follicles cultured in the supplemented medium Katska and Rynska, .
1998 . The number of morphologically normal oocytes increases when bovine preantral Ž
. follicles are cultured in hypoxanthine-supplemented medium Figueiredo et al., 1994 .
Ž .
Eppig and Downs 1987 demonstrated that hypoxanthine promoted the association of oocytes and accompanying granulosa cells during the culture of mouse preantral
follicles. The maintenance of oocyte–granulosa cell communication via gap junctions Ž
. has been shown to promote oocyte growth in vitro Buccione et al., 1990 . It is probable
that hypoxanthine improves the utilization of additional energy substrate by maintaining Ž
interactions between the oocyte and the surrounding granulosa cells Figueiredo et al., .
1994 . Larger variations in the increase of follicular diameter were observed among Ž
. follicle cultured in the absence of hypoxanthine Jewgenow, 1998 .
Ž There are several reports using wide range of culture media Daniel et al., 1989;
. Ž
Eppig, 1977; Torrance et al., 1989 , pyruvate Daniel et al., 1989; Eppig, 1977; Eppig .
Ž .
and Schroeder, 1989; Torrance et al., 1989 and glutamine Nayudu and Osborn, 1992 as energy substrate. The addition of pyruvate or glutamine to the culture medium
increased the percentage of intact follicle. It was shown that pyruvate was the predomi- Ž
nant substrate used by immature and mature bovine oocytes and zygotes Khurana and .
Ž .
Niemann, 1992 . Eppig 1976 reported that isolated growing mouse oocytes utilized exogenously administered pyruvate more efficiently than glucose, lactate or succinate. It
has been shown that glutamine is an efficient energy substrate for oocytes and embryos Ž
. Figueiredo et al., 1994 .
Cellular maturation and follicular development from the primary to tertiary stages and their regulation by endocrine and paracrine factors are areas of limited knowledge. The
ability to culture isolated preantral follicles constitute a new tool for investigation of paracrine and autocrine factors involved in early folliculogenesis. In this experiment, we
have studied the effects of some endocrine and growth factors on follicular growth by culturing preantral follicles over a period of 10 days.
2. Materials and methods
2.1. Isolation of preantral follicles Ovaries were obtained from a local slaughterhouse and placed in isotonic sodium
Ž .
Ž .
chloride solution 0.9 NaCl supplemented with penicillin 200 IUrml and strepto- Ž
. mycin 200 mgrml at 30–358C. Sections of the ovarian cortex were excised by scalpel
Ž and placed into a tissue chopper The McIlwain Tissue Chopper, The Mickle Laboratory
. Engineering, Gomshal, Surrey, England adjusted to produce 500-mm sections. These
small to minute segments were placed in Dulbecco’s phosphate-buffered solution Ž
. Nissui Pharmaceutical, Tokyo, Japan supplemented with 36 mgrml sodium pyruvate,
1 mgrml glucose and 3 mgrml BSA. After several washings and repeated pipetting, the Ž
samples were filtered first through a 500-mm filter Testing Sieve, Tokyo Screen, .
Tokyo, Japan to remove all large fragments and debris, then through a 38-mm filter Ž
. Testing Sieve to exclude blood cells and other minute particles. The tissue remaining
Ž on the 38-mm filter was washed and collected in an embryo searching dish 100 = 20
. mm, Falcon, Becton Dickinson Labware, Becton Dickinson, NJ, USA .
2.2. Culture of isolated preantral follicles Freshly isolated morphologically normal follicles appearing healthy, spherical under
the inverted microscope with one or more compact layers of granulosa cells around the Ž
. central part containing oocytes within an intact basal membrane, with no apparent sign
of necrosis and no antrum were used for present experiment. Preantral follicles with diameters of 40, 60, 80 and 100 mm were collected, selected on the above criteria and
Ž .
cultured in vitro using one of the four treatments culture media . The treatments were: Ž
. Ž
FSH 100 ngrml; pFSH, Denka Pharmaceuticals, Kawasaki, Japan ; EGF 100 ngrml; .
Ž .
q
Ž Toyobo, Osaka, Japan q FSH 100 ngrml ; ITS
1; Insulin–Transferrin–Selenium- .
Ž .
Ž X, Gibco, Grand Island, NY 14072, USA q FSH 100 ngrml ; and hypoxanthine 4
. Ž
. mM; Kohjin, Tokyo, Japan q FSH 100 ngrml . The basal medium for all treatments
Ž was tissue culture medium TCM 199 with Earle’s salts,
L
-glutamine, 2200 mgrl .
sodium bicarbonate, 25 mM HEPES buffer; Gibco supplemented with 10 FCS, 0.1 mgrml sodium pyruvate, 100-IUrml of penicillin and 100 mgrml streptomycin. The
basal medium without any supplements for treatments was considered as control. Preantral follicles were cultured in groups of one to three in 500 ml of culture medium
Ž .
in 24-well dishes Falcon at 38.58C in 5 CO for 10 days. Half the medium was
2
removed and replaced by fresh medium every third day. Follicular diameters were measured under a microscope with an ocular micrometer on
the day of collection and onset of culture, on days 5 and 11 to monitor volume, morphology and any deformity or degeneration. At the end, half of the cultured preantral
follicles were stained with trypan blue to categorize their viability on the basis of the degree of dye exclusion. Unstained follicles were classified as viable and fully stained
follicles as dead. Follicles with medium staining were regarded as damaged. And the
Ž .
rest of the cultured preantral follicles were double stained by bisbenzimide H 33342 Ž
. plus propidium iodine 10 mgrml each in PBS and compared with fresh controls to
examine the morphological aspects as to determine the presence of an intact follicle membrane, a flattened or cuboidal granulosa cell layer, and the presence of a nucleus.
Double staining helped to analyze the proportions of live and dead granulosa cells and
Ž .
also the nuclear components Fig. 1 . 2.3. Statistical analysis
Ž .
Statistical analyses were carried out by one-way analysis of variance ANOVA and Ž
Fisher’s protected least significant difference test using the STATVIEW Abacus
Fig. 1. A healthy, morphologically normal bovine preantral follicle with circular granulosa cell layers visible Ž
. after 10 days of in vitro culture after staining with Hoechst 33342 =400 .
. Concepts, Berkeley, CA, USA for the diameter gain by follicles on different days of
culture. The analysis for maintenance of viability and morphology was done by Chi-square test.
3. Results
