Resumen
- Folliculogenesis in the mammalian ovary is one of the most complex development processes in biology. Some in vitro data suggest that growth hormone (GH) plays a role in follicular growth during early gonadotropin-independent folliculogenesis and could have a direct inhibitory action on follicular apoptosis. Although some GH ovarian actions have been well-analyzed in some mammalian species, little is known about the effect of GH on the survival and morphology of bovine preantral follicles. Thus, the objective of the present study was to evaluate different concentrations of GH in the morphology of bovine preantral follicles cultured in situ. Ovaries (n = 12) from crossbred cows at different ages and phases of the estrous cycle were collected from a local slaughterhouse. Tissue samples from each ovarian pair were cut into slices (3 x 3 x 1 mm) and were directly fixed for histological analysis (non-cultured) or placed in culture for one or seven days. Bovine ovarian fragments were cultured on basic culture medium (α-MEM) supplemented with 20 mM HEPES; 3 mM glutamine; 10 ng/mL insulin; 2.5 g/mL transferrin; 4 ng/mL selenium; 0.1% bovine serum albumin; 0.1 mg/mL penicillin and 0.1 mg/mL streptomycin, at 37 °C and 5% CO2 in a humidified incubator. The culture medium was supplemented with GH at different concentrations (ng/mL): 0 (GH0), 10 (GH10), 25 (GH25) and 50 (GH50). The culture media was renewed every day. After culture, all the pieces were fixed in Carnoy’s solution for 4 h and then dehydrated with increasing concentrations of ethanol, diaphonized in xylene and embedded in paraffin to produce serial sections with thicknesses of 5 μm. The mounted slides were stained with PAS-hematoxylin and examined by a light microscope (Olympus BX50, Japão) with a 400X magnification for an evaluation of the follicular morphology, including the organization of the granulosa cells and the antral cavity as well as the maintenance of the basal membrane and oocyte integrity. Follicle stage and survival were examined on an optical microscopy and the percentages of preantral follicles were submitted to the analysis of variance (ANOVA); subsequently, comparisons between means were made by using Dunnett’s test (non-cultured control vs cultured treatments) and Tukey’s test (among cultured treatments). A total of 1620 preantral follicles were analyzed. After one day of culture, the percentage of normal follicles in the GH0 (48.89%) and GH10 (50.56%) treatments was reduced compared to fresh control (75.56%); unlikely the GH25 (57.78%) and GH50 (65.00%) treatments were equivalent to fresh control (P> 0.05). Moreover, after seven days of culture, GH50 treatment (75.00%) showed equivalent efficiency in morphologically normal follicles with the fresh control (75.56%, P > 0.05). Besides, GH50 treatment decreased the percentage of primordial follicles after seven days of culture and concomitantly, it was observed a significant (P < 0.05) increase in the developing follicles, differing from both fresh control and the other GH treatments. Thus, it follows that 50 ng/ml of GH is the most effective concentration for the development and morphology of cultured bovine preantral follicles included in ovarian tissue. Keywords: Folliculogenesis, in vitro culture, Ovary.