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Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia.

Hayashi KG, Ushizawa K, Hosoe M, Takahashi T - Reprod. Biol. Endocrinol. (2011)

Bottom Line: The expression profiles of six identified SERPIN genes were further confirmed by QPCR analysis.QPCR analysis confirmed that mRNA expression of four SERPINs (SERPINA5, SERPINB6, SERPINE2 and SERPINF2) was greater in healthy than in atretic follicles, while two SERPINs (SERPINE1 and SERPING1) had greater expression in atretic than in healthy follicles.Our results demonstrate a characteristic expression of SERPIN superfamily member genes in bovine healthy and atretic follicles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Reproductive Biology Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.

ABSTRACT

Background: SERPINs (serine protease inhibitors) regulate proteases involving fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation and apoptosis. This study aimed to investigate differentially expressed genes of members of the SERPIN superfamily between healthy and atretic follicles using a combination of microarray and quantitative real-time PCR (QPCR) analysis. In addition, we further determined mRNA and protein localization of identified SERPINs in estradiol (E2)-active and E2-inactive follicles by in situ hybridization and immunohistochemistry.

Methods: We performed microarray analysis of healthy (10.7 +/- 0.7 mm) and atretic (7.8 +/- 0.2 mm) follicles using a custom-made bovine oligonucleotide microarray to screen differentially expressed genes encoding SERPIN superfamily members between groups. The expression profiles of six identified SERPIN genes were further confirmed by QPCR analysis. In addition, mRNA and protein localization of four SERPINs was investigated in E2-active and E2-inactive follicles using in situ hybridization and immunohistochemistry.

Results: We have identified 11 SERPIN genes expressed in healthy and atretic follicles by microarray analysis. QPCR analysis confirmed that mRNA expression of four SERPINs (SERPINA5, SERPINB6, SERPINE2 and SERPINF2) was greater in healthy than in atretic follicles, while two SERPINs (SERPINE1 and SERPING1) had greater expression in atretic than in healthy follicles. In situ hybridization showed that SERPINA5, SERPINB6 and SERPINF2 mRNA were localized in GCs of E2-active follicles and weakly expressed in GCs of E2-inactive follicles. SERPING1 mRNA was localized in both GCs and the theca layer (TL) of E2-inactive follicles and a weak hybridization signal was also detected in both GCs and TL of E2-active follicles. Immunohistochemistry showed that SERPINA5, SERPINB6 and SERPINF2 were detected in GCs of E2-active and E2-inactive follicles. SERPING1 protein was localized in both GCs and the TL of E2-active and E2-inactive follicles.

Conclusions: Our results demonstrate a characteristic expression of SERPIN superfamily member genes in bovine healthy and atretic follicles. The cell-type-and stage-specific expression of SERPINs may be associated with bovine follicular growth and atresia.

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QPCR analysis of SERPINA5, SERPINB6, SERPINE1, SERPINE2, SERPINF2 and SERPING1 in healthy and atretic follicles. Total RNA from the follicular wall (i.e., granulosa plus theca interna) was extracted from three healthy follicles and three atretic follicles. The expression of mRNA was normalized to the expression of GAPDH measured in the same RNA preparation. The black and white bars indicate healthy and atretic follicles, respectively. Data are shown as the mean ± SEM. Different superscript letters denote significant differences (P < 0.05).
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Figure 1: QPCR analysis of SERPINA5, SERPINB6, SERPINE1, SERPINE2, SERPINF2 and SERPING1 in healthy and atretic follicles. Total RNA from the follicular wall (i.e., granulosa plus theca interna) was extracted from three healthy follicles and three atretic follicles. The expression of mRNA was normalized to the expression of GAPDH measured in the same RNA preparation. The black and white bars indicate healthy and atretic follicles, respectively. Data are shown as the mean ± SEM. Different superscript letters denote significant differences (P < 0.05).

Mentions: Among 14 genes coding for the SERPIN superfamily represented on a bovine oligonucleotide array, 11 SERPIN genes were identified after data normalization: SERPINA1, SERPINA5, SERPINB1, SERPINB6, SERPINB8, SERPINE1, SERPINE2, SERPINF1, SERPINF2, SERPING1 and SERPINH1. Of these 11 SERPIN genes, SERPINA5, SERPINB6, SERPINE2 and SERPINF2 were consistently up-regulated at least 2.0-fold, while SERPINE1 and SERPING1 were consistently down-regulated at least 0.5-fold in all healthy follicles compared with all atretic follicles. We compared the expression levels of these six SERPINs between healthy and atretic follicles by QPCR. Figure 1 shows the results of QPCR analysis. Consistent with the microarray analysis, SERPINA5, SERPINB6, SERPINE2 and SERPINF2 mRNA expression was greater in healthy than in atretic follicles (P < 0.05). On the other hand, SERPINE1 and SERPING1 mRNA expression was greater in atretic than in healthy follicles (P < 0.05).


Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia.

Hayashi KG, Ushizawa K, Hosoe M, Takahashi T - Reprod. Biol. Endocrinol. (2011)

QPCR analysis of SERPINA5, SERPINB6, SERPINE1, SERPINE2, SERPINF2 and SERPING1 in healthy and atretic follicles. Total RNA from the follicular wall (i.e., granulosa plus theca interna) was extracted from three healthy follicles and three atretic follicles. The expression of mRNA was normalized to the expression of GAPDH measured in the same RNA preparation. The black and white bars indicate healthy and atretic follicles, respectively. Data are shown as the mean ± SEM. Different superscript letters denote significant differences (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117774&req=5

Figure 1: QPCR analysis of SERPINA5, SERPINB6, SERPINE1, SERPINE2, SERPINF2 and SERPING1 in healthy and atretic follicles. Total RNA from the follicular wall (i.e., granulosa plus theca interna) was extracted from three healthy follicles and three atretic follicles. The expression of mRNA was normalized to the expression of GAPDH measured in the same RNA preparation. The black and white bars indicate healthy and atretic follicles, respectively. Data are shown as the mean ± SEM. Different superscript letters denote significant differences (P < 0.05).
Mentions: Among 14 genes coding for the SERPIN superfamily represented on a bovine oligonucleotide array, 11 SERPIN genes were identified after data normalization: SERPINA1, SERPINA5, SERPINB1, SERPINB6, SERPINB8, SERPINE1, SERPINE2, SERPINF1, SERPINF2, SERPING1 and SERPINH1. Of these 11 SERPIN genes, SERPINA5, SERPINB6, SERPINE2 and SERPINF2 were consistently up-regulated at least 2.0-fold, while SERPINE1 and SERPING1 were consistently down-regulated at least 0.5-fold in all healthy follicles compared with all atretic follicles. We compared the expression levels of these six SERPINs between healthy and atretic follicles by QPCR. Figure 1 shows the results of QPCR analysis. Consistent with the microarray analysis, SERPINA5, SERPINB6, SERPINE2 and SERPINF2 mRNA expression was greater in healthy than in atretic follicles (P < 0.05). On the other hand, SERPINE1 and SERPING1 mRNA expression was greater in atretic than in healthy follicles (P < 0.05).

Bottom Line: The expression profiles of six identified SERPIN genes were further confirmed by QPCR analysis.QPCR analysis confirmed that mRNA expression of four SERPINs (SERPINA5, SERPINB6, SERPINE2 and SERPINF2) was greater in healthy than in atretic follicles, while two SERPINs (SERPINE1 and SERPING1) had greater expression in atretic than in healthy follicles.Our results demonstrate a characteristic expression of SERPIN superfamily member genes in bovine healthy and atretic follicles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Reproductive Biology Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.

ABSTRACT

Background: SERPINs (serine protease inhibitors) regulate proteases involving fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation and apoptosis. This study aimed to investigate differentially expressed genes of members of the SERPIN superfamily between healthy and atretic follicles using a combination of microarray and quantitative real-time PCR (QPCR) analysis. In addition, we further determined mRNA and protein localization of identified SERPINs in estradiol (E2)-active and E2-inactive follicles by in situ hybridization and immunohistochemistry.

Methods: We performed microarray analysis of healthy (10.7 +/- 0.7 mm) and atretic (7.8 +/- 0.2 mm) follicles using a custom-made bovine oligonucleotide microarray to screen differentially expressed genes encoding SERPIN superfamily members between groups. The expression profiles of six identified SERPIN genes were further confirmed by QPCR analysis. In addition, mRNA and protein localization of four SERPINs was investigated in E2-active and E2-inactive follicles using in situ hybridization and immunohistochemistry.

Results: We have identified 11 SERPIN genes expressed in healthy and atretic follicles by microarray analysis. QPCR analysis confirmed that mRNA expression of four SERPINs (SERPINA5, SERPINB6, SERPINE2 and SERPINF2) was greater in healthy than in atretic follicles, while two SERPINs (SERPINE1 and SERPING1) had greater expression in atretic than in healthy follicles. In situ hybridization showed that SERPINA5, SERPINB6 and SERPINF2 mRNA were localized in GCs of E2-active follicles and weakly expressed in GCs of E2-inactive follicles. SERPING1 mRNA was localized in both GCs and the theca layer (TL) of E2-inactive follicles and a weak hybridization signal was also detected in both GCs and TL of E2-active follicles. Immunohistochemistry showed that SERPINA5, SERPINB6 and SERPINF2 were detected in GCs of E2-active and E2-inactive follicles. SERPING1 protein was localized in both GCs and the TL of E2-active and E2-inactive follicles.

Conclusions: Our results demonstrate a characteristic expression of SERPIN superfamily member genes in bovine healthy and atretic follicles. The cell-type-and stage-specific expression of SERPINs may be associated with bovine follicular growth and atresia.

Show MeSH
Related in: MedlinePlus