Nucleoli from growing oocytes support the development of enucleolated full‐grown oocytes in the pig

Abstract

Recent research has shown that the maternal nucleolus is essential for embryonic development. The morphology of the nucleolus in growing oocytes differs from that in full-grown oocytes. We determined the ability of nucleoli from growing oocytes to substitute for nucleoli of full-grown oocytes in terms of supporting embryonic development in this study. Growing (around 100 microm in diameter) and full-grown porcine oocytes (120 microm) were collected from small (0.6-1.0 mm) and large antral follicles (4-5 mm), respectively. The nucleolus was aspirated from full-grown oocytes by micromanipulation, and the resulting enucleolated oocytes were matured to metaphase II; the nucleoli originating from full-grown and growing oocytes were then injected into the oocytes. The Chromatin of growing oocytes was aspirated with the nucleolus during the enucleolation process. Growing oocytes were thus treated with actinomycin D to release the chromatin from their nucleoli, and the nucleoli were collected and transferred to the enucleolated and matured full-grown oocytes. After activation by electro-stimulation, nucleoli were formed in pronuclei of sham-operated oocytes. Enucleolated oocytes that had been injected with nucleoli from either full-grown or growing, however, did not form any nucleoli in the pronuclei. No enucleolated oocytes developed to blastocysts, whereas enucleolated oocytes injected with nucleoli from full-grown oocytes (15%) or growing oocytes (18%) developed to blastocysts. These results indicate that the nucleoli from growing oocytes can substitute for nucleoli from full-grown oocytes during early embryonic development.

Homeobox gene Sax2 deficiency causes an imbalance in energy homeostasis

“…The nucleoli of growing pig oocytes, which were treated with actinomycin D, supported embryonic development to a similar extent as nucleoli collected from full‐grown oocytes (Kyogoku et al, 2010). This report also showed that it was possible to collect growing oocyte nucleoli, when the oocytes were treated with actinomycin D. Using this enucleolation method, we examined the effect of growing oocyte nucleoli on oocyte maturation.…”

“…Oocytes were enucleolated as described previously (Kyogoku et al, 2010). Briefly, full‐grown and growing oocytes were transferred into HEPES‐199 containing 12.5 µg/ml cytochalasin B and 0.01 µg/ml demecolcine for 10 min.…”

“…Thus, enucleolation of full‐grown oocytes did not cause irreparable damage to the chromosomes for development. The nucleoli of growing oocytes can support early embryonic development (Kyogoku et al, 2010), indicating that these nucleoli have already accumulated the materials that are required for early embryonic cleavages.…”

Nucleoli from growing oocytes inhibit the maturation of enucleolated, full‐grown oocytes in the pig

“…This acquisition of full meiotic competence coincides with the morphological change in the nucleolus. In the present experiment, growing pig oocytes collected from 0.6 to 1.0 mm follicles were around 100 µm in diameter (Kyogoku et al, 2010). Oocytes of this size do not have the ability to complete the maturation (Kanayama et al, 2002).…”

“…In the GV, the oocyte chromatin again enclosed the reassembled nucleolus. Nucleoli are formed in pronuclei of activated, enucleolated‐matured oocytes after their re‐injection at the MII stage (Ogushi et al, 2008; Kyogoku et al, 2010). Since the oocyte nucleoli disappear during oocyte maturation and reappear in the pronucleus of zygotes, it is logical that injected nucleoli disappear in the oocyte cytoplasm at the MII stage.…”