Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development.
The availability of viable oocytes is the limiting factor in the development of new reproductive techniques. Many attempts have been made to grow immature oocytes in vitro during recent decades. Recently, a modified alginate-based three-dimensional culture system was designed to support the growth and maturation of multilayered secondary follicles. This system was able to produce oocytes that successfully completed meiosis, fertilization, and development to the blastocyst stage. Subsequent attempts to culture two-layered secondary follicles were unsuccessful under the original conditions. Herein, we investigated the effect of alginate consistency on two-layered follicle growth and oocyte developmental competence by encapsulating follicles into alginate scaffolds of various concentrations. Although there were no significant differences in survival rates, 0.25% and 0.5% alginate supported more rapid growth of follicles and antrum formation compared with 1.5% and 1.0% alginate after 8 days of culture. Alginate scaffold concentration also affected the proliferation and differentiation of somatic cells (theca and granulosa cells), measured in terms of morphological changes, steroid profiles (androstenedione, estradiol, and progesterone), and specific molecular markers (Fshr, Lhcgr, and Gja1). Theca cell proliferation and steroid production were hindered in follicles cultured in 1.5% alginate. In vitro fertilization and embryo culture revealed that oocytes obtained from 0.25% alginate retained the highest developmental competence. Overall, the present study showed that the alginate scaffold consistency affects folliculogenesis and oocyte development in vitro and that the alginate culture system can and should be tailored to maximally support follicle growth depending on the size and stage of the follicles selected for culture.
Xu
Min
M
Center for Reproductive Science, Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, USA.
West
Erin
E
Shea
Lonnie D
LD
Woodruff
Teresa K
TK
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D047108:Embryonic Development; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D020869:Gene Expression Profiling; D008540:Meiosis / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D009866:Oogenesis / Q000502:physiology; D009924:Organ Culture Techniques; D006080:Ovarian Follicle / Q000166:cytology* / Q000254:growth & development* / Q000502:physiology; D011374:Progesterone / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000735:Androstenedione; D011374:Progesterone; D004958:Estradiol