Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water.
The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.
Faculty of Veterinary Medicine, PPGCV, State University of Ceara, Fortaleza, CE, Brazil. j.r.v.silva@vet.uu.nl
José R V
JR
Silva
Robert
R
van den Hurk
Sonia H F
SH
Costa
Evelyn R
ER
Andrade
Ana P A
AP
Nunes
Francisco V A
FV
Ferreira
Raimundo N B
RN
Lôbo
José R
JR
Figueiredo
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D003059:Cocos; D003470:Culture Media; D046508:Culture Techniques; D005260:Female; D005973:Glutamine / Q000494:pharmacology; D006041:Goats / Q000502:physiology*; D006107:Granulosa Cells / Q000166:cytology; D019271:Hypoxanthine / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D006080:Ovarian Follicle / Q000187:drug effects / Q000502:physiology*; D010053:Ovary / Q000502:physiology*; D019289:Pyruvic Acid / Q000494:pharmacology; D012643:Selenium / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D012996:Solutions; D014168:Transferrin / Q000494:pharmacology; D014867:Water
D003470:Culture Media; D007328:Insulin; D012996:Solutions; D014168:Transferrin; D014867:Water; D005973:Glutamine; D012710:Serum Albumin, Bovine; D019271:Hypoxanthine; D019289:Pyruvic Acid; D012643:Selenium