Bovine embryo production and evaluation of cell number

4.1.1 Bovine embryos generated by in vitro fertilization and parthenogenesis

In vitro fertilized and parthenogenetic preimplantation bovine embryos were generated routinely 1 to 2 times per week from 2001 to 2003. The blastocyst rate was 24%±2.4 for the in vitro fertilized embryos and 27%±2.2 for the parthenogenetic embryos. The embryos were initially used to optimize the bisulfite sequencing method and the semi-quantitative RT-PCR assay. 69 zygotes and 81 4-cell embryos were generated by in vitro fertilization and analyzed for their methylation patterns.

4.1.2 Differential staining of in vitro produced bovine blastocysts

Cell numbers of in vitro fertilized and parthenogenetic blastocysts and expanded blastocysts are shown in table 9 and fig. 31. The total cell number of single embryos was calculated from the results obtained from the TE and ICM cells.

Figure 31: Differential staining of expanded blastocysts generated by in vitro fertilization or parthenogenesis

Inner cell mass cells are stained blue due to the fluorochrome Hoechst 33342 and the outer trophectoderm cells are stained in red by propidium iodide. In vitro fertilized expanded blastocyst (left) and parthenogenetic expanded blastocyst (right).

Table 9: Cell number of day 7 blastocysts and expanded blastocysts, trophectoderm and inner cell mass generated by in vitro fertilization and parthenogenesis

Values with different superscripts (a, b) indicate significant differences (p≤0.05). One way ANOVA, Tukey test. (TE: trophectoderm; ICM: Inner cell mass; SEM: standard error of means)

Embryos

Total number of blastocysts

analyzed

Mean ± SEM of total cell number/embryo

Mean ± SEM of trophectoderm cell number

%

Trophectoderm cells/embryo

Mean ± SEM of ICM cell number/embryo

% Inner cell mass/embryo

Ratio TE : ICM

Blastocyst

(IVF) 22 126±7.2^ab 78±4.9^b 62% 48±2.9 38% 1:0.6

Blastocyst (PA)

17 107±7.1^b 70±5.4^b 65% 37±2.8 35% 1:0.5

Expanded blastocyst

(IVF)

13 144±5.4^a 103±4.4^a 72% 41±3.5 28% 1:0.4

Expanded blastocyst

(PA)

18 125±7.1^ab 82±4.7^b 66% 43±3.6 34% 1:0.5

79 Results

4.2 mRNA gene expression analyses of the bovine IGF2, IGF2R and MASH2 genes

Semi-quantitative RT-PCR was performed to determine differences in the relative amount of transcriptional products due to mono- or bi-allelic gene expression.

The bovine IGF2, IGF2R and MASH2 genes were analyzed in expanded blastocysts derived by in vitro fertilization (IVF) or parthenogenesis (PA). As shown in figure 32, relative amounts of transcriptional products of the MASH2 gene were different (p≤0.05) between IVF (48.5±12) and PA (7.7±9.2) derived expanded blastocysts. No differences were detected in the mRNA expression pattern of the IGF2 and IGF2R genes. We did not distinguished between blastocysts and expanded blastocysts in this experiment.

Figure 32: Relative amounts of transcriptional products of the IGF2, IGF2R and MASH2 genes as determined by semi-quantitative RT-PCR

Significant differences are indicated by superscripts (a:b, p≤0.05). One way ANOVA, Dunn's method. (IVF: in vitro fertilization; PA: parthenogenesis)

Results of the mRNA gene expression analyses of blastocysts and expanded blastocysts are presented in fig. 33. Trophectoderm and inner cell mass cells and in vivo collected expanded blastocysts were included in the analyses. Relative amounts of transcriptional products were calculated on a per cell basis. The cell number for the in vivo expanded blastocysts was taken from the publication of LAZZARI et al.

(2002) who counted 158 cells per in vivo expanded blastocyst.

Increased relative amounts of IGF2 mRNA were detected in IVF expanded and hatched blastocysts compared to their parthenogenetic counterparts. Differences (p≤0.05) were found in the expression patterns of the IGF2 gene between expanded

0.2

IVF blastocysts and both types of PA blastocysts (blastocysts, expanded blastocysts). In contrast to IVF blastocysts, in which decreased relative amounts of transcriptional products of the IGF2 gene were determined from expanded blastocysts to hatched blastocysts, the relative amounts of mRNA increased in expanding to hatched parthenogenetic blastocysts.

ICM cells from IVF expanded blastocysts expressed the IGF2R gene at a significantly higher level (p≤0.05) than did IVF blastocysts, expanded blastocysts and IVF trophectoderm cells. The expression levels of the IGF2R gene in ICM cells from IVF and PA blastocysts were not different. Hatched PA blastocysts contained significantly more IGF2R mRNA (p≤0.05) than did IVF or PA blastocysts and expanded blastocysts. No differences were detected between in vitro produced and in vivo collected blastocysts.

Hatched PA blastocysts showed a significantly higher MASH2 mRNA level (p≤0.05) than blastocysts and expanded blastocysts. PA trophectoderm cells contained increased amounts of MASH2 transcriptional products than PA-ICM cells.

This was in contrast to the trophectoderm and ICM cells of IVF blastocysts, which expressed MASH2 at a similar level. Comparison of IVF blastocysts revealed that the MASH2 gene was higher expressed in expanded and hatched blastocysts (p≤0.05) than in blastocysts. No differences were detected between the in vitro produced blastocysts and in vivo expanded blastocysts.

Figure 33: Differences in mRNA expression of the bovine IGF2, IGF2R and MASH2 genes determined by semi-quantitative RT-PCR

Blastocysts, expanded blastocysts and hatched blastocysts derived from in vitro fertilization and parthenogenesis were included in the analyses. Isolated ICM and TE were additionally investigated. In vivo collected expanded blastocysts served as controls.

Bars with different superscripts indicate significant differences in the relative amount of transcriptional products. The results obtained from the semi-quantitative RT-PCR analyses were calculated on a per cell basis and statistically analyzed using one way ANOVA, Dunn's method, Tukey test: IGF2 – a:b p≤0.05; IGF2R – a:b p≤0.05; MASH2 - a:b:c:d p≤0.05

(IVF: in vitro fertilization; PA: parthenogenesis; ICM: inner cell mass; TE: trophectoderm; BL:

blastocyst; eBL: expanded blastocyst; hBL: hatched blastocyst; V: in vivo collected blastocyst)

In summary, blastocysts and expanded blastocysts derived from in vitro fertilization or parthenogenesis consisted of similar total cell numbers and similar numbers of trophectoderm and inner cell mass cells. The only exception was that expanded in vitro fertilized blastocysts showed a significantly increased total cell number and contained a significantly higher number of trophectoderm cells (p≤0.05).

Semi-quantitative RT-PCR revealed no differences in the relative abundance of transcriptional products of the IGF2 and IGF2R genes between in vitro fertilized and parthenogenetic blastocysts. In contrast, IVF blastocysts expressed the MASH2

PA

gene to a significantly higher relative amount than parthenogenetic embryos (p≤0.05).

A significantly increased relative amount of transcriptional products from IGF2 gene was detected in in vitro fertilized expanded blastocysts (p≤0.05) compared to blastocysts and hatched blastocysts. The IGF2R gene was predominantly (p≤0.05) expressed in ICM cells isolated from IVF blastocysts and in hatched parthenogenetic blastocysts. A significantly increased relative amount of MASH2 mRNAs was found in hatched blastocysts derived from in vitro fertilization and parthenogenesis (p≤0.05).

4.3 Sequencing of three fragments of the bovine IGF2 gene