Mammalian ovaries are endowed with a large number of primordial follicles, each containing a nongrowing oocyte. Only a small population of primordial oocytes (oocytes in primordial follicles) is activated to enter the growth phase throughout a female's reproductive life. Little is known about the mechanism regulating the activation of primordial oocytes. Here, we found that the primordial oocytes from infant pigs (10-to 20-day-old) grew to full size at 2 months after xenografting to immunodeficient mice, whereas those from prepubertal pigs (6-month-old) survived without initiation of their growth even after 4 months; thereafter, they started to grow and reached full size after 6 months. These results suggest that the mechanism regulating the activation of primordial oocytes in prepubertal pigs is different from that in infant pigs. In this regard, the involvement of FOXO3, a forkhead transcription factor, was studied. In prepubertal pigs, FOXO3 was detected in almost all (94G2%) primordial oocyte nuclei, and in infant pigs, 42G7% primordial oocytes were FOXO3 positive. At 4 months after xenografting, the percentage of FOXO3-positive primordial oocytes from prepubertal pigs had decreased to the infant level. Further, siRNA was designed to knock down porcine FOXO3. FOXO3-knockdown primordial follicles from prepubertal pigs developed to the antral stage accompanied by oocyte growth at 2 months after xenografting. These results suggest that primordial oocytes are dormant in prepubertal pigs by a FOXO3-related mechanism to establish a nongrowing oocyte pool in the ovary, and that a transient knockdown of the FOXO3 activates the primordial oocytes to enter the growth phase.

Purpose The mechanism underlying primordial follicle activation is poorly understood. In this study, in-vitro culture and subsequent xenotransplantation were conducted to determine whether testosterone promotes the activation of porcine primordial follicles. Methods Prepubertal porcine ovarian cortical strips containing primordial follicles were cultured in the presence of testosterone for 7 days, and subsequently transplanted to immunodeficient mice for 2 months. After culture and transplantation, development of follicles was examined histologically. The presence of androgen receptors in oocytes was assessed by use of western blot and immunohistochemical analyses. Results Testosterone at 10 -6 M induced the primordial follicle transition to the intermediate (19 ± 4%) and primary (3 ± 1%) stages after 7-day culture, while 56 ± 5% of primordial follicles remained in the initial pool. Higher concentrations, above 10 -5 M, or lower concentrations, below 10 -6 M, did not induce follicle transition to the primary stage. After 7-day culture with 10 -6 M testosterone, ovarian cortical strips were transplanted to immunodeficient mice. Some of the follicles developed to the secondary (15 ± 3%) and antral (10 ± 3%) stages, whereas 44 ± 7% of primordial follicles remained in the initial pool. In the culture experiment, estradiol-17b (10 -7 -10 -5 M) had no significant effect on follicle activation. The androgen receptor antagonist, cyproterone acetate, inhibited the stimulatory effect of testosterone on primordial follicle activation, suggesting an androgen receptormediated action of testosterone. Western blot and immunohistochemical analyses revealed that androgen receptors were present in the oocytes of primordial follicles. Conclusions These results suggest that testosterone at 10 -6 M promotes the activation of porcine primordial follicles in vitro through the androgen receptors in the oocytes.

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