DNA Synthesis and pronucleus development in pig zygotes obtained in vivo: An autoradiographic and ultrastructural study

Abstract

Porcine zygotes flushed from oviducts 48, 52, 56, 60, or 64 hr after hCG were incubated 30 min in 3H-thymidine, transferred to nonradioactive medium for 2 hr, and incubated for 30 min with 14C-thymidine. After this procedure, ova were prepared (i.e., at 51, 55, 59, 63, or 67 hr after hCG) for autoradiography and ultrastructural observations, respectively. The first autoradiographic labelling, i.e., DNA synthesis, was observed at 56-56.5 hr after hCG, while the latest labelling was seen at 60-60.5 hr. At 51 hr after hCG, formation of the pronuclear envelope was observed, while no nucleolus precursor bodies or prestages to these structures were found. At 55 hr a few clusters of small electron-dense granules were observed, together with condensed chromatin in the pronuclei. At 59 hr the apposed regions of both pronuclei contained nucleolus precursor bodies and condensed chromatin, in close contact with both clusters of small granules and clusters of an additional category of large granules and the nuclear envelope. Additionally, large accumulations of the small granules were found in the vicinity of similarly sized accumulations of the large granules without chromatin association. At 63 hr the spherical accumulations of large granules on some occasions presented a central vacuole, and condensed chromatin and clusters of small granules were attached to its periphery. Within the vacuole, electron-dense material was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa

“…It has been proposed that human nuclear precursor bodies are formed by the aggregation of small dense bodies, which arise in association with condensed chromatin (Tesarik & Kopecny, 1989). In the present study we found intranuclear granules of varying size and shape as early as the pronuclear stage, similar to those observed in the pronuclei of porcine zygotes in vivo (Laurincik et al, 1995). In the present study we found intranuclear granules of varying size and shape as early as the pronuclear stage, similar to those observed in the pronuclei of porcine zygotes in vivo (Laurincik et al, 1995).…”

“…As observed for fertilisation with isogenic species sperm (Laurincik et al, 1995), characteristic 'undulation' of the nuclear envelope is seen in porcine oocytes following murine sperm injection. This structural change may be required for specialised nucleocytoplasmic transport.…”

“…The onset and duration of zygotic DNA synthesis has been studied in mice (Luthardt & Donahue, 1973) and cattle (Eid et al, 1994) as well as in the pig (Laurincik et al, 1995). In porcine zygotes produced by in vivo fertilisation, S-phase occurs about 55-62.5 h after the injection of hCG, and comparable extents of autoradiographic labelling are observed for both pronuclei in almost all zygotes, suggesting that the maternal and paternal S-phase are synchronised (Laurincik et al, 1995).…”

“…In conventional in vitro fertilisation of the mouse, meiosis is completed within 2 h of insemination, and male and female pronuclei form between 4-7 h and 5-8 h post-insemination, respec-concerning the onset of pronucleus formation and DNA synthesis have not been described (Laurincik et al, 1995). In conventional in vitro fertilisation of the mouse, meiosis is completed within 2 h of insemination, and male and female pronuclei form between 4-7 h and 5-8 h post-insemination, respec-concerning the onset of pronucleus formation and DNA synthesis have not been described (Laurincik et al, 1995).…”

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

“…Also, cryopreservation alters DNA synthesis in different cell types, such as embryonic inner cell mass cells and fetal pancreatic cells [58,59]. In porcine zygotes, it has been suggested that DNA replication starts approximately 6 h after sperm penetration [60] and lasts for 4.5-7.5 h [61]. Although sperm and oocytes are coincubated for 3 h in our IVF system, the sperm penetration rate peaks about 4 h after insemination, probably due to penetration of spermatozoa that are in the zona pellucida or perivitelline space at 3 h after IVF (Kikuchi et al, unpublished observation).…”