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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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Protein localization of SERPINA5, SERPINB6, SERPINF2 and SERPING1 in E2-active and E2-inactive follicles. Localization of SERPINA5 (A and B), SERPINB6 (C and D), SERPINF2 (E and F) and SERPING1 (G and H) protein was detected by immunohistochemistry. Sections (7 μm) of bovine E2-active (A, C, E and G) and E2-inactive follicles (B, D, F and H) were incubated with anti-SERPINA5, anti-SERPINB6, anti-SERPINF2 and anti-SERPING1 polyclonal antibodies. SERPINA5, SERPINB6 and SERPINF2 were detected in the GCs of E2-active and E2-inactive follicles. SERPING1 was detected in both GCs and the TL of E2-active and E2-inactive follicles. Negative control (I and J) was incubated without anti-SERPIN antibodies. Scale bars = 20 μm.
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Figure 3: Protein localization of SERPINA5, SERPINB6, SERPINF2 and SERPING1 in E2-active and E2-inactive follicles. Localization of SERPINA5 (A and B), SERPINB6 (C and D), SERPINF2 (E and F) and SERPING1 (G and H) protein was detected by immunohistochemistry. Sections (7 μm) of bovine E2-active (A, C, E and G) and E2-inactive follicles (B, D, F and H) were incubated with anti-SERPINA5, anti-SERPINB6, anti-SERPINF2 and anti-SERPING1 polyclonal antibodies. SERPINA5, SERPINB6 and SERPINF2 were detected in the GCs of E2-active and E2-inactive follicles. SERPING1 was detected in both GCs and the TL of E2-active and E2-inactive follicles. Negative control (I and J) was incubated without anti-SERPIN antibodies. Scale bars = 20 μm.

Mentions: SERPINA5, SERPINB6 and SERPINF2 were detected in GCs of E2-active and E2-inactive follicles (Figure 3A, B, C, D, E and 3F). SERPING1 protein was localized in both GCs and the TL of E2-active and E2-inactive follicles (Figure 3G and 3H).


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)

Protein localization of SERPINA5, SERPINB6, SERPINF2 and SERPING1 in E2-active and E2-inactive follicles. Localization of SERPINA5 (A and B), SERPINB6 (C and D), SERPINF2 (E and F) and SERPING1 (G and H) protein was detected by immunohistochemistry. Sections (7 μm) of bovine E2-active (A, C, E and G) and E2-inactive follicles (B, D, F and H) were incubated with anti-SERPINA5, anti-SERPINB6, anti-SERPINF2 and anti-SERPING1 polyclonal antibodies. SERPINA5, SERPINB6 and SERPINF2 were detected in the GCs of E2-active and E2-inactive follicles. SERPING1 was detected in both GCs and the TL of E2-active and E2-inactive follicles. Negative control (I and J) was incubated without anti-SERPIN antibodies. Scale bars = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Protein localization of SERPINA5, SERPINB6, SERPINF2 and SERPING1 in E2-active and E2-inactive follicles. Localization of SERPINA5 (A and B), SERPINB6 (C and D), SERPINF2 (E and F) and SERPING1 (G and H) protein was detected by immunohistochemistry. Sections (7 μm) of bovine E2-active (A, C, E and G) and E2-inactive follicles (B, D, F and H) were incubated with anti-SERPINA5, anti-SERPINB6, anti-SERPINF2 and anti-SERPING1 polyclonal antibodies. SERPINA5, SERPINB6 and SERPINF2 were detected in the GCs of E2-active and E2-inactive follicles. SERPING1 was detected in both GCs and the TL of E2-active and E2-inactive follicles. Negative control (I and J) was incubated without anti-SERPIN antibodies. Scale bars = 20 μm.
Mentions: SERPINA5, SERPINB6 and SERPINF2 were detected in GCs of E2-active and E2-inactive follicles (Figure 3A, B, C, D, E and 3F). SERPING1 protein was localized in both GCs and the TL of E2-active and E2-inactive follicles (Figure 3G and 3H).

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