Identification of Molecular Profile of Ear Fibroblasts Derived from Spindle-Transferred Holstein Cattle with Ooplasts from Taiwan Yellow Cattle under Heat Stress

Abstract

Global warming has a significant impact on the dairy farming industry, as heat stress causes reproductive endocrine imbalances and leads to substantial economic losses, particularly in tropical-subtropical regions. The Holstein breed, which is widely used for dairy production, is highly susceptible to heat stress, resulting in a dramatic reduction in milk production during hot seasons. However, previous studies have shown that cells of cows produced from reconstructed embryos containing cytoplasm (o) from Taiwan yellow cattle (Y) have improved thermotolerance despite their nuclei (n) being derived from heat-sensitive Holstein cattle (H). Using spindle transfer (ST) technology, we successfully produced ST-Yo-Hn cattle and proved that the thermotolerance of their ear fibroblasts is similar to that of Y and significantly better than that of H (p < 0.05). Despite these findings, the genes and molecules responsible for the different sensitivities of cells derived from ST-Yo-Hn and H cattle have not been extensively investigated. In the present study, ear fibroblasts from ST-Yo-Hn and H cattle were isolated, and differentially expressed protein and gene profiles were compared with or without heat stress (hs) (42 °C for 12 h). The results revealed that the relative protein expression levels of pro-apoptotic factors, including Caspase-3, -8, and -9, in the ear fibroblasts from the ST-Yo-Hn-hs group were significantly lower (p < 0.05) than those from the H-hs group. Conversely, the relative expression levels of anti-apoptotic factors, including GNA14 protein and the CRELD2 and PRKCQ genes, were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Analysis of oxidative phosphorylation-related factors revealed that the relative expression levels of the GPX1 gene and Complex-I, Complex-IV, CAT, and PGLS proteins were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Taken together, these findings suggest that ear fibroblasts from ST-Yo-Hn cattle have superior thermotolerance compared to those from H cattle due to their lower expression of pro-apoptotic factors and higher expression of oxidative phosphorylation and antioxidant factors. Moreover, this improved thermotolerance is attributed, at least partially, to the cytoplasm derived from more heat-tolerant Y cattle. Hence, using ST technology to produce more heat-tolerant H cattle containing Y cytoplasm could be a feasible approach to alleviate the negative impacts of heat stress on dairy cattle in tropical-subtropical regions.

Keywords: Holstein; Taiwan yellow cattle; antioxidant; apoptotic factors; cytoplasmic origin; heat stress; oxidative phosphorylation; thermotolerance.

Figure 1

Differentially expressed protein profiles in ear fibroblasts derived from cattle with different cytoplasmic origins in response to heat shock (42 °C for 12 h). H: ear fibroblasts derived from Holstein cattle; Y: ear fibroblasts derived from Taiwan yellow cattle; ST: ear fibroblasts derived from ST cattle produced by embryos reconstructed with Taiwan yellow cattle ooplasm and Holstein nuclei (ST-Hd-Yo). Each treatment was repeated three times.

Figure 2

The relative expression of Caspase-3 (A), -8 (B), and -9 (C) proteins in ear fibroblasts derived from cattle with different cytoplasmic origins in response to heat shock (42 °C for 12 h). All values were calculated using the relative expression compared to non-heat-shocked ear fibroblasts from the same individual. H: ear fibroblasts derived from Holstein cattle; Y: ear fibroblasts derived from Taiwan yellow cattle; ST: ear fibroblasts derived from ST cattle produced by embryos reconstructed with Taiwan yellow cattle ooplasm and Holstein nuclei (ST-Hd-Yo). ^{a, b, c, d} Values without the same superscripts are significantly different (p < 0.05). Each treatment was repeated three times.

Figure 3

Relative mRNA abundance of CRELD2 (A) and PRKCQ (B) genes in ear fibroblasts derived from cattle with different cytoplasmic origins in response to heat shock (42 °C for 12 h). All values were calculated using the relative expression compared to non-heat-shocked ear fibroblasts from the same individual. H: ear fibroblasts derived from Holstein cattle; Y: ear fibroblasts derived from Taiwan yellow cattle; ST: ear fibroblasts derived from ST cattle produced by embryos reconstructed with Taiwan yellow cattle ooplasm and Holstein nuclei (ST-Hd-Yo). ^{a, b, c, d, e, f} Values without the same superscripts are significantly different (p < 0.05). Each treatment was repeated three times.

Figure 4

The relative expression of GNA14 protein in ear fibroblasts derived from cattle with different cytoplasmic sources after heat shock (42 °C for 12 h). Western blot analysis of GNA14 from matching samples (original Western blot figures are presented in Figure S4). β-actin served as an internal control, and all values were calculated using the relative expression compared to non-heat-shocked ear fibroblasts from the same individual. H: ear fibroblasts derived from Holstein cattle; Y: ear fibroblasts derived from Taiwan yellow cattle; ST: ear fibroblasts derived from ST cattle produced by embryos reconstructed with Taiwan yellow cattle ooplasm and Holstein nuclei (ST-Hd-Yo). ^{a, b, c} Values without the same superscripts are significantly different (p < 0.05). Each treatment was repeated three times.

Figure 5

The relative expression of Complex-I (A), -II (B), -III (C), and -IV (D) proteins in ear fibroblasts derived from cattle with different cytoplasmic origins in response to heat shock (42 °C for 12 h). (E) Western blot analysis of Complex-I (A), -II (B), -III (C), and -IV (D) from matching samples (original Western blot figures are presented in Figure S5). β-actin served as an internal control, and all values were calculated using the relative expression compared to non-heat-shocked ear fibroblasts from the same individual. H: ear fibroblasts derived from Holstein cattle; Y: ear fibroblasts derived from Taiwan yellow cattle; ST: ear fibroblasts derived from ST cattle produced by embryos reconstructed with Taiwan yellow cattle ooplasm and Holstein nuclei (ST-Hd-Yo). ^{a, b, c, d} Values without the same superscripts are significantly different (p < 0.05). Each treatment was repeated three times.

Figure 6

Relative mRNA abundance of GPX1 gene in ear fibroblasts derived from cattle with different cytoplasmic origins in response to heat shock (42 °C for 12 h). All values were calculated using the relative expression compared to non-heat-shocked ear cells from the same individual. H: ear fibroblasts derived from Holstein cattle; Y: ear fibroblasts derived from Taiwan yellow cattle; ST: ear fibroblasts derived from ST cattle produced by embryos reconstructed with Taiwan yellow cattle ooplasm and Holstein nuclei (ST-Hd-Yo). ^{a, b, c, d, e} Values without the same superscripts are significantly different (p < 0.05). Each treatment was repeated three times.

Figure 7

The relative expression of SOD1 (A), CAT (B), and PGLS (C) proteins in ear fibroblasts derived from cattle with different cytoplasmic origins in response to heat shock (42 °C for 12 h). (D) Western blot analysis of SOD1 (A), CAT (B), and PGLS (C) from matching samples (original Western blot figures are presented in Figure S7). β-actin served as an internal control, and all values were calculated using the relative expression compared to non-heat-shocked ear fibroblasts from the same individual. H: ear fibroblasts derived from Holstein cattle; Y: ear fibroblasts derived from Taiwan yellow cattle; ST: ear fibroblasts derived from ST cattle produced by embryos reconstructed with Taiwan yellow cattle ooplasm and Holstein nuclei (ST-Hd-Yo). ^{a, b, c, d} Values without the same superscripts are significantly different (p < 0.05). Each treatment was repeated three times.