Ingrid B. Bøgh

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5 nM TSA for 22-24 h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15 h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.

PURE PREOVULATORY FOLLICULAR FLUID PROMOTES IN VITRO MATURATION OF IN VIVO ASPIRATED EQUINE OOCYTES

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.

High Hydrostatic Pressure Treatment of Porcine Oocytes before Handmade Cloning Improves Developmental Competence and Cryosurvival

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.

In vitro maturation of bovine cumulus-oocyte complexes in undiluted follicular fluid: effect on nuclear maturation, pronucleus formation and embryo development.

Since resumption of meiosis and cytoplasmic maturation of bovine oocytes takes place in close association with follicular fluid, it would be logical to assume that this might be a perfect maturation medium. To test the hypothesis, abattoir-derived cumulus-oocyte complexes (COCs) were in vitro matured in undiluted (i) mixed follicular fluid (FF) from 3 to 15 mm follicles from abattoir ovaries, (ii) preovulatory follicular fluid (POF) from the dominant follicle from a cyclic unstimulated heifer, (iii) preovulatory follicular fluid (OPU) from synchronised and superovulated heifers 60 h after prostaglandin and 20 h after GnRH treatment, and in (iv) TCM-199 with 5% serum. Subsequent to IVM, the COC were subjected to IVF and IVC, and embryo development was followed until the blastocyst stage at Day 8 after insemination. The MII rates in the TCM-199 (69%), POF (69%) and OPU (72%) groups were not different from each other but different from the FF (41%) group (P<0.05). In spite of the high MII rates, none of the follicular fluids supported embryo development: the FF, POF and OPU blastocyst rates were alike (3%, 3%, 2%) and different (P<0.05) from the rates in the TCM-199 (19%). During IVM in follicular fluids but not in TCM-199, the expanded cumulus masses became trapped in a coagulum. Although it could be prevented by the presence of heparin during IVM, it did not improve the blastocyst rates. In conclusion, undiluted preovulatory follicular fluids supported nuclear maturation but not further embryonic development as judged by the high MII and low blastocyst rates.

Comparative Evaluation of Nuclear Morphology of Equine Oocytes Aspirated in vivo and Stained with Hoechst and Orcein

Nuclear maturation of equine oocytes was assessed immediately after in vivo collection. A double-staining technique (Hoechst and orcein) was used on the same oocytes to visualize nuclear morphology, i.e. to evaluate the chromatin configurations of each oocyte after Hoechst in relation to the nuclear morphology after orcein staining. The proportion of oocytes evaluated as germinal vesicle stages was significantly (p < 0.02) lower after Hoechst (14.5%) than after orcein staining (29.0%), while the incidence of the so-called dense chromatin stage was assessed to be higher (p < 0.05) after Hoechst than after orcein staining (14.5 vs. 6.5%). There was no difference between Hoechst and orcein staining in the incidence of diakinesis and germinal vesicle breakdown stages, respectively (44.9 vs. 42.0%), and the same applied for metaphase I (11.6 vs. 8.0%), metaphase II (7.2 vs. 8.0%) and degenerated stages (7.2 vs. 6.5%). It was concluded that the interpretation of the meiotic stages may differ between Hoechst and orcein staining and in a large proportion of equine oocytes the nuclear border may not be visualized on orcein staining.

A Method for Chronological Intravital Imaging of Bovine Oocytes during In Vitro Maturation

Oocyte maturation is known to affect the chances for successful fertilization, embryonic development, establishment of pregnancy and delivery of a live, healthy, and viable offspring. Two-photon laser scanning microscopy (TPLSM) has previously been used to evaluate early embryonic development without a detectable impairment of subsequent development, but has never been applied to assess mammalian oocytes throughout in vitro maturation (IVM). Visualization of structures within live oocytes during IVM, followed by fertilization and embryo culture, may improve the understanding of oocyte maturation. To visualize structures within bovine oocytes using TPLSM, it is necessary to remove the cumulus cells that normally surround the oocyte during maturation. Repeated visualization of structures within the same oocyte is possible, if movement of the oocyte can be avoided. In this article, we describe the development of a method for repeated intravital imaging of denuded bovine oocytes using an upright TPLSM equipped with a specially constructed incubator. Oocytes were stained with Hoechst 33258, and the nuclear structures were evaluated. Oocyte fertilization rate was not affected by TPLSM exposure, but the developmental capacity of the denuded oocytes was significantly reduced. This is, to our knowledge, the first article describing repeated intravital imaging during mammalian oocyte maturation using TPLSM.