Ultrastructure of in-vivo fertilization in superovulated cattle

Heifers were induced to superovulate by treatment with PMSG or FSH. Subsequently, oestrus was induced with prostaglandins and artificial insemination was performed. Ova were collected from the oviducts and their ultrastructural features were related to an estimated time of ovulation based on the time of the LH peak. With the insemination schedule used, the estimated time of ovulation defined the time at which fertilization was expected to occur. The ova were characterized as unfertilized, fertilized or possibly fertilized, and a sequence of nuclear and cytoplasmic changes associated with fertilization was revealed. Within 4 h after the estimated time of ovulation formation of the female and male pronucleus was initiated, and at 5-7 h swelling of the pronuclei occurred. At 19 h the pronuclei were closely apposed and synkaryosis was seen, and at 23 h the first two-cell stage was obtained. Within 2-3 h after the estimated time of ovulation cortical granule release, development of conspicuous Golgi complexes, and transformation of the smooth endoplasmic reticulum occurred. At approximately 7 h parallel arrays of annulate lamellae appeared. In one third of the unfertilized ova deviant oocyte maturation was noticed.     

Preovulatory plasma estradiol-17beta concentrations and ovulation rates in PMSG/anti-PMSG treated heifers

Possibilities for early characterization of the superovulatory response were studied in 41 PMSG/PG-treated dairy heifers, of which 21 received an additional treatment of PMSG-antiserum. Plasma was obtained at 33, 36, 41, 47 and 51 h after PG for hormone analyses. After slaughter at 6 or 7 d after insemination, the number of follicles and corpora lutea (CL) were recorded, and ova were recovered for morphological evaluation. Significant correlations were demonstrated between plasma concentrations of estradiol-17beta (E2) at 33, 36 and 41 h after PG and the ovulation rate (number of CL). Each of these correlations was equal to the one found by using the peak concentration of E2 achieved during the preovulatory E2 surge. In heifers with preovulatory E2 surges, as determined with the blood sampling scheme used, both the ovarian response (number of CL and follicles) and the quality of ova recovered (number of transferable embryos) was clearly better compared to heifers without this surge. None of the parameters studied was affected significantly by treatment with PMSG-antiserum. It is concluded that plasma E2 determinations at fixed times in relation to prostaglandin treatment can be used to characterize the superovulatory response in donor cattle in terms of the ovulation rate and the quality of ova recovered. No evidence was found in favor of using PMSG-antiserum for improving either the superovulatory response or such characterization.     

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

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 nucieolus 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 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, electrondense material was found. It is concluded that (1) the S-phase in porcine zygotes is initiated around 56 hr post-hCG injection and is of a duration of 4.5–7.5 hr and (2) the progress of the S-phase is paralleled by the appearance of and complex interaction between different granules in the nucleoplasm. © 1995 Wiley-Liss, Inc.     

Character of preovulatory follicles and oocytes after different superovulatory treatments in heifers

The relationship between the opaqueness of the surface of bovine preovulatory follicles, degree of expansion of oocyte-cumulus investment, presence of perivitelline space, and abstriction of the first polar body was examined in heifers treated with PMSG (Group I), FSH-P (Group II), and FSH-P/GnRH (Group III). Follicles greater than 8 mm in diameter were inspected by laparoscopy 65 hours after treatment with cloprostenol in all groups and were classified as clear or opaque based on their surface appearance. Subsequently, oocytes were recovered from the follicles and characterized. The proportion of clear exceeded that of opaque follicles in all groups. The oocyte recovery rate was highest for clear follicles in Group I and II, while in Group III the rates were identical for clear and opaque follicles. The proportion of oocytes with expanded cumulus investment was highest in Group III. In Group II and I decreased proportions of expansion was seen especially among oocytes from opaque follicles. The proportion of oocytes with perivitelline space was highest in Group II and III. Again, this effect was most pronounced among oocytes from "opaque" follicles. The proportion of oocytes with a polar body was highest in Group III followed by Group II while only few oocytes in Group I displayed a polar body. It is concluded that treatment with FSH-P and especially in combination with GnRH reduced the incidence of follicular atresia as measured by opaqueness and improved oocyte quality as indicated by cumulus expansion, formation of the perivitelline space, and abstriction of the first polar body.     

Effects of brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/prostaglandin-treated heifers

The aim of this study was to investigate whether prolongation of the period of preovulatory follicular development after superovulation reduces heterogeneity of oocytes of stimulated follicles with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos. Heifers were treated with eCG on Day 10 and prostaglandin (PG) 48 h later. At the time of eCG administration some of the heifers received a norgestomet implant (N) to suppress the LH surge. After 96 to 104 h, N was removed and an LH surge was induced with GnRH (G) (N/G); the other animals served as controls. Matured oocytes (Experiment A: n=9, 139 [N/G] and 11, 125 [Control] heifers, oocytes), zygotes and oviducts (Experiment B: n=8, 44 [N/G] and 9, 72 [Control] heifers, zygotes) and embryos (Experiment C: n=11, 205 [N/G] and 11, 165 [Control] heifers, embryos) were collected at 22 to 26 h, 38 to 52 h and 7 days after the LH surge, respectively. Hatched blastocyst formation of matured oocytes (Experiment A) was analyzed after 11 days of IVC after IVF. In vivo fertilization rate of zygotes, the presence of periodic acid-Schiff (PAS) positive granules in the oviduct (Experiment B) and stage of development of embryos (Experiment C) were analyzed stereomicroscopically. The mean interval between PG and the LH surge was 53.8+/-3 (SD) (N/G) vs. 42.4+/-4 h (Control). The maximum peripheral estradiol-17beta concentration (529+/-36 [SEM] [N/G] vs. 403+/-17 pmol/L [Control]) and the response to superovulation (25.4+/-2 [N/G] vs. 18.7+/-2 [Control]) were higher in N/G than in Control heifers. Hatched blastocyst formation rate (37.4 [N/G] vs. 33.6% [Control]), in vivo fertilization rate (69.0+/-14 [N/G] vs. 73.0+/-10% [Control]) and the yield of total embryos (3.8+/-1 [N/G] vs. 5.6+/-2 [Control]) did not differ between groups. The percentage of heifers with abundant PAS-positive granules in the distal ampulla (0 [N/G] vs. 31% [Control]) was reduced after N/G treatment. Prolongation of the period of preovulatory follicular development increased the number of mature follicles and ovulations but did not result in higher embryo yield, possibly because of an impaired oviductal environment.     

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.     

Chromosome aberrations in in vitro-produced bovine embryos at days 2-5 post-insemination

Availability of embryos of high quality is required to obtain satisfactory embryonic developmental rates and normal calves following transfer of in vitro-produced (IVP) bovine embryos. One relevant quality parameter is the frequency of chromosome aberrations, which can be evaluated using multicolor fluorescent in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes in cattle. In this study, interphase nuclei (n = 3805) were analyzed from 426 bovine IVP embryos. We found that 73%, 72%, 81%, and 58% of the embryos from Days 2, 3, 4, and 5 post-insemination (pi), respectively, displayed a normal diploid chromosome number in all cells. When looking at the types of chromosome aberrations, the percentages of mixoploidy at Days 2, 3, 4, and 5 pi were 22%, 15%, 16%, and 42%, respectively, whereas the percentages of polyploidy (i.e., all nuclei in an embryo were analyzed and were polyploid) were 5%, 13%, 3%, and 0%, respectively. In conclusion, numerical chromosome aberrations were detected as early as Day 2 pi. The development of polyploid embryos is slow and is apparently arrested during the third cell cycle, whereas the mixoploid embryos seem to continue development.     

Epigenetic regulation of gene expression in porcine epiblast, hypoblast, trophectoderm and epiblast-derived neural progenitor cells

After fertilization, lineage specification is governed by a complicated molecular network in which permissiveness and repression of expression of pluripotency- and differentiation-associated genes are regulated by epigenetic modifications. DNA methylation operates as a very stable repressive mark in this process. In this study, we investigated the relationship between DNA methylation and expression of pluripotency-associated genes (OCT4, NANOG and SOX2), a trophectoderm (TE)-specific gene (ELF5), and genes associated with neural differentiation (SOX2 and VIMENTIN) in porcine Day 10 (E10) epiblast, hypoblast, and TE as well as in epiblast-derived neural progenitor cells (NPCs). We found that OCT4, NANOG, and SOX2 were highly expressed in the epiblast and hypoblast, while VIMENTIN was only highly expressed in the epiblast. Moreover, low expression of OCT4, NANOG, SOX2 and VIMENTIN was noted in the TE. Most CpG sites of OCT4, NANOG, SOX2 and VIMENTIN displayed low methylation levels in the epiblast and hypoblast and, strikingly, also in the TE. Hence, the expression patterns of these genes were not directly related to levels of DNA methylation in the TE in contrast to the situation in the mouse. In contrast, ELF5 was exclusively expressed in the TE and was correspondingly hypomethylated in this tissue. In NPCs, we observed down-regulation of NANOG and OCT4 expression, which correlated with hypermethylation of their promoters, whereas VIMENTIN displayed up-regulation in accordance with hypomethylation of its promoter. In conclusion, DNA methylation is an inconsistently operating epigenetic mechanism in porcine E10 blastocysts, whereas in porcine epiblast-derived NPCs, expression of pluripotency-associated and differentiation genes appear to be regulated by this modification.