EFFECT OF BOVINE PITUITARY EXTRACT ON HATCHING AND POST-HATCHING DEVELOPMENT OF IVP BOVINE EMBRYOS

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References and Acknowledgements

The authors thank Dr. Robert Wall for assistance with statistical analysis.

Cezar, G.G. et al. (2003) Biol. Reprod. 68: 1009-1014. Cibelli, J.B. et al. (1998) Science 280: 1256-1258.Edwards, J. L. and Hansen, P. J. (1996) Biol. Reprod. 55: 340-346.Hawk, H.W. et al. (1992) Theriogenology, 38: 989-998.Humpherys, D. et al. (2002) Proc Natl Acad Sci, USA. 99:12889-12894.Kang, Y.K. et al. (2002) EMBO J. 21:1092-1100.Wilmut, I. et al. (1997) Nature 385, 810-813.

Abstract

The effect of bovine pituitary extract (BPE) on development of in vitro-produced (IVP) bovine blastocysts after Day 7 was studied. IVP embryos were produced from in vitro matured COC’sprocessed from local slaughterhouse ovaries or obtained from Bomed, Inc., Madison, WI. The first 7 days of embryo culture following in vitro fertilization were in G1/G2 medium in 5% O2 + 5% CO2 + 90% N2. Embryos that had reached the compacted morula or early blastocyst stage on Day 7 were identified and randomly assigned to four media treatments. DMEM/199 medium (D2) or G2, each supplemented by the addition of BSA, insulin, transferrin, and selenium (ITS), were used with or without with BPE at 30 µg/ml. Extended embryo culture was continued until day 11. Further development was assessed by counting hatched blastocysts and measuring the size (cross-sectional index) and the number of cells per blastocyst. Size diameters of blastocysts ranged from 200 µm to 400 µm across treatments. Cell counts were performed by staining the fixed blastocysts with Hoechst 33342 and counting the nuclei of squashed blastocysts with a fluorescent microscope. The percentage of hatched blastocysts was significantly greater (p < 0.05) in G2 with BPE (63.7 ± 6.8%) than in G2 alone (29.6 ± 7.3%). D2 with BPE (59.3 ± 6.8%) was not significantly different than D2 alone (56.8 ± 7.9%). Area index gave similar results; G2 + BPE (267 ± 168), G2 alone (76 ± 28), D2 + BPE (192 ± 139) and D2 alone (155 ± 106). Cell number had the same trend; G2 + BPE (303 ± 164), G2 alone (123 ± 47), D2 + BPE (254 ± 166) and D2 alone (233 ± 120). A second experiment randomly assigned 7 day embryos to extended culture in G2 or D2 medium with or without irradiated STO feeder cells and under either 5% O2 or 20% O2 (ambient air). All treatments contained BPE and a layer of agarose. Hatched blastocysts were counted on Day 11 and percentages were similar for both media without STO in low O2 (G2 = 75 ± 12 %; D2 = 67 ± 8%) and in air (G2 = 82 ± 19%; D2 = 83 ± 11%). Hatching was similar with STO in both media in air (G2 = 73 ± 10%; D2 = 68 ± 15%), but was significantly reduced (p < 0.05) when STO was combined with low oxygen (G2 = 28 ± 14%; D2 = 4 ± 6%). Further culture to 18 days was completed but many of the embryos collapsed by day 12 and thereafter grew as lobed bodies and occasionally attached to each other. A few embryos continued uncomplicated growth and were composed of over 5000 cells. These results indicate that promotion of in vitro bovine blastocystdevelopment in a minimal medium such as G2 requires BPE while a more complex medium, D2, supports further development without BPE. STO feeder cells in combination with low oxygen culture was inhibitory to blastocyst hatching and survival.

N.C. Talbot and A.M. Powell

USDA, ARS, Biotechnology and Germplasm Laboratory, Beltsville Agricultural Research Center, Beltsville, MD 20705

Introduction

Somatic cell nuclear transfer (NT) can create animals with complete ontogeny from cultured somatic cells by using the cultured cell’s nucleus to replace the oocyte’s nucleus (Wilmut et al., 1997; Cibelli et al., 1998). However, NT embryo development to term and post-natal survival is usually reported as being dramatically lower than that of embryos derived from in vitro fertilization (IVF). It is possible that extensive epigenetic changes resulting from incomplete reprogramming of the transferred nucleus are responsible for this poor survival rate. This incomplete reprogramming appears to be manifested by the wide spread changes in DNA methylation, and by the aberrant expression of hundreds of genes in NT embryos (Kang et al., 2002; Humpherys et al., 2002; Cezar et al., 2003).

One strategy for dealing with the apparently numerous epigenetic problems inherent to NT embryos would be to pre-select NT embryos that are more normal, i.e., more like IVF embryos, prior to transfer into the recipient cow. An extended embryo culture system might allow the assay of embryo secreted proteins that could act as indicators of successful reprogramming prior to NT embryo transfer.

OBJECTIVE: Test the effect of bovine pituitary extract (BPE) on the post-7-Day culture of bovine IVF embryos.

Conclusions1. Day-7 to day-11 embryo culture in G2 medium was improved by BPE (30 µg/ml).2. Day-7 to day-11 embryo culture in the more complex D2 medium was not significantly

improved by BPE.3. STO co-culture in combination with low O2 was inhibitory to blastocysts hatching and

survival.4. Culture past 11 days (Exp. 2) resulted in most blastocysts collapsing at day-12 with

extensive cell death. A few did not and these were composed of over 5000 cells.

Materials and MethodsCulture materials.

IVF-TL solution (BSS-010-D) and G1/G2 media were purchased from Specialty Media, Lavalette, NJ. Dulbecco’s phosphate buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM) and Medium 199 were obtained from In Vitrogen (Gibco), Gaithersburg, MD. Fetal bovine serum (FBS) was obtained from Hyclone, Logan, UT. Four-well plates were purchased from Nunc, Naperville, IL.

Production of in vitro matured/in vitro fertilized (IVM/IVF) bovine embryos.Oocytes were purchased from BoMed, Madison, WI, and matured during transit. Alternatively, ovaries were

obtained from a commercial slaughterhouse (MoPAC, Allentown, PA), were washed in 1% Nolvasan in 0.9% saline, and were transported to the laboratory at rt in 3 to 5 h of slaughter for oocyte collection. Cumulus oocyte complexes (COCs) were isolated using a method previously described by Edwards and Hansen (1996). The cumulus cells were partially removed by drawing the COCs through a slightly smaller diameter Pasteur pipette (Hawk et al., 1992). COCs were cultured in 4-well plate wells in 0.5 ml maturation medium (Ham’s F10 with 10% FBS, 0.3 µg/ml USDA-bLH-B6, and penicillin/streptomycin) for 22 h at 38.5 C in 5% CO2 in a humidified incubator. For fertilization, commercially prepared bull semen from one straw was washed in 10 ml DPBS by centrifugation (800 x g for 5 min). Spermatozoa were resuspended in 400 µ l of IVF-TL containing heparin (50 µ g/ml) and capacitated by incubation for 15 min at 38.5 C in humidified 5% CO2 atmosphere. The sperm were diluted by the addition of 11 ml of IVF-TL to give approximately 1 x 106 spermatozoa/ml and 0.5 ml of this suspension was placed on about 50 COCs after aspiration of most of the maturation medium from the culture wells (Hawk et al., 1992). Twenty h after the addition of sperm the putative zygotes were vortexed for 3 min in DPBS supplemented with 1% bovine serum albumin (BSA, Fraction V, Sigma, St. Louis, MO) to remove the cumulus cells.

Presumptive embryos were cultured in G1 medium. On day 3 embryos were move to G2 medium. All embryos in G1/G2 were cultured at 5% O2 + 5% CO2 + 90% N. Embryos were evaluated for morula or early blastocyst formation on Day 7 and transferred to fresh G2 medium or a 1:1 mixture of DMEM low glucose and Medium 199 (D2 medium) both supplement with BSA (0.05%) and with 1x ITS [insulin (10 µg/ml), transferrin (5.5 µ g/ml), and selenium (0.005 µg/ml); Sigma]. For Experiment 1, media were or were not supplemented with 30 µg/ml BPE. For Experiment 2, both media contained BPE and the effects of ± STO feeder cells and high or low O2 atmosphere were tested.

ResultsExp. 1. Development of Day-7 bovine embryos after extended culture to Day-11 Media N % Hatched Area Index Cell Number

Blastocysts of Blastocysts of BlastocystsG2 no BPE1 20 b29.6 ± 7.3 b76 ± 28 b123 ± 47 G2 + BPE 54 a63.7 ± 6.8 a267 ± 168 a303 ± 164 D2 no BPE 37 a56.8 ± 7.9 a155 ± 106 a233 ± 120 D2 + BPE 50 a59.3 ± 6.8 a192 ± 139 a254 ± 166 1Treatment means with different letters (a, b) are different at the 0.05 significance level

Exp. 2. Hatched blastocysts (HB) from Day-7 bovine embryos cultured to Day-11 in media with BPE

Media/atmosphere1 (+) STO feeder cells (% HB±std) (-) STO feeder cells (% HB±std)

G2/5% O2b28 ± 14% (n = 50) a75 ± 12% (n = 53)

G2/20% O2 a73 ± 10% (n = 49) a82 ± 19% (n = 50)

D2/5% O2b4 ± % (n = 48) a67 ± 8% (n = 51)

D2/20% O2a68 ± 15% (n = 52) a83 ± 11% (n = 50)

___________________________________________________________________________________ 1Treatment means with different letter (a, b) are different at the 0.05 significance level.

Experimental Method

Experiment 1

1. Zygotes cultured until day-7 in G1/G2 in 5% O2. 2. Morula or early blastocysts randomly assigned to D2 or G2 medium ±30 µg/ml BPE.3. Embryo culture continued until day-11 in 5% O2.4. Embryo development assessed by:

a. counting hatched blastocysts.b. measuring diameter (µm).c. counting total cells per blastocyst.

Experiment 2

1. Zygotes cultured until day-7 in G1/G2 in 5% O2.2. Morula or early blastocysts randomly assigned to D2 or G2 medium with 30 µg/ml BPE

±STO feeder cells and under either 5% O2 or 20% O2.3. Embryos culture continued until day-184. Embryos assessed for percentage hatched at day-11.