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Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis.

Shikina S, Chiu YL, Chung YJ, Chen CJ, Lee YH, Chang CF - Sci Rep (2016)

Bottom Line: Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period.Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity.These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Marine Environment and Ecology, National Taiwan Ocean University, Keelung, 20224, Taiwan.

ABSTRACT
To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction.

No MeSH data available.


Related in: MedlinePlus

Determination of the oocyte stage that starts to express EaRFP.(a) Seasonal variation of EaRFP expression levels during 2010–2011 in female E. ancora polyp tissues, as assessed by quantitative RT-PCR. Data shown are the mean ± SE (n = 3 colonies) relative to the April 2011 group. Groups with different letters are significantly different (P < 0.05). (b–e). SDS-PAGE and Western blotting of EaRFP. (b) SDS-PAGE of protein extract from pET-19b E. coli expressing recombinant EaRFP (Ec) and purified recombinant EaRFP (P). (c) Western blotting of the same protein shown in (b) with an anti-RFP antibody. (d) SDS-PAGE of protein extracts of ovary (Ov) collected in March. Arrow indicates the predicted EaRFP band. (e) Western blotting of ovarian protein extract with an anti-EaRFP antibody. Arrow indicates an EaRFP immunoreactive band of approximately 27 kDa. Molecular weight markers are shown on the left. (f–j) The immunohistochemical analysis of EaRFP with an anti-RFP antibody in stage I–V oocytes. Inset in (f) shows the stage I oocyte. (k) The negative control of immunohistochemical analysis. Immunohistochemistry was performed with the omission of the 1st antibody for this section (stage II oocytes). Oogenic stages I–V were classified according to cell size and morphology 6. Broken lines in (i–k) indicate the oocytes. All bars = 50 μm.
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f4: Determination of the oocyte stage that starts to express EaRFP.(a) Seasonal variation of EaRFP expression levels during 2010–2011 in female E. ancora polyp tissues, as assessed by quantitative RT-PCR. Data shown are the mean ± SE (n = 3 colonies) relative to the April 2011 group. Groups with different letters are significantly different (P < 0.05). (b–e). SDS-PAGE and Western blotting of EaRFP. (b) SDS-PAGE of protein extract from pET-19b E. coli expressing recombinant EaRFP (Ec) and purified recombinant EaRFP (P). (c) Western blotting of the same protein shown in (b) with an anti-RFP antibody. (d) SDS-PAGE of protein extracts of ovary (Ov) collected in March. Arrow indicates the predicted EaRFP band. (e) Western blotting of ovarian protein extract with an anti-EaRFP antibody. Arrow indicates an EaRFP immunoreactive band of approximately 27 kDa. Molecular weight markers are shown on the left. (f–j) The immunohistochemical analysis of EaRFP with an anti-RFP antibody in stage I–V oocytes. Inset in (f) shows the stage I oocyte. (k) The negative control of immunohistochemical analysis. Immunohistochemistry was performed with the omission of the 1st antibody for this section (stage II oocytes). Oogenic stages I–V were classified according to cell size and morphology 6. Broken lines in (i–k) indicate the oocytes. All bars = 50 μm.

Mentions: We subsequently addressed when the E. ancora oocyte starts to express EaRFP during oogenesis. Our previous study revealed that E. ancora has an annual reproductive cycle, and its oogenesis takes approximately 11–12 months in southern Taiwan6. The oogenesis usually starts in June, and the matured oocytes are released the following May6. Quantitative RT-PCR of cDNA from the polyp tissue samples collected in different months showed that EaRFP transcripts were already present in June-August (11–9 months before spawning), increased as April approached, and decreased somewhat during the spawning month (May) (Fig. 4a). Microscopic observation of the the ovaries collected in August, December, and March (9, 5, and 2 months before spawning, respectively) also showed that the oocytes in all the samples express EaRFP (Supplementary Fig. 3a–f).


Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis.

Shikina S, Chiu YL, Chung YJ, Chen CJ, Lee YH, Chang CF - Sci Rep (2016)

Determination of the oocyte stage that starts to express EaRFP.(a) Seasonal variation of EaRFP expression levels during 2010–2011 in female E. ancora polyp tissues, as assessed by quantitative RT-PCR. Data shown are the mean ± SE (n = 3 colonies) relative to the April 2011 group. Groups with different letters are significantly different (P < 0.05). (b–e). SDS-PAGE and Western blotting of EaRFP. (b) SDS-PAGE of protein extract from pET-19b E. coli expressing recombinant EaRFP (Ec) and purified recombinant EaRFP (P). (c) Western blotting of the same protein shown in (b) with an anti-RFP antibody. (d) SDS-PAGE of protein extracts of ovary (Ov) collected in March. Arrow indicates the predicted EaRFP band. (e) Western blotting of ovarian protein extract with an anti-EaRFP antibody. Arrow indicates an EaRFP immunoreactive band of approximately 27 kDa. Molecular weight markers are shown on the left. (f–j) The immunohistochemical analysis of EaRFP with an anti-RFP antibody in stage I–V oocytes. Inset in (f) shows the stage I oocyte. (k) The negative control of immunohistochemical analysis. Immunohistochemistry was performed with the omission of the 1st antibody for this section (stage II oocytes). Oogenic stages I–V were classified according to cell size and morphology 6. Broken lines in (i–k) indicate the oocytes. All bars = 50 μm.
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Related In: Results  -  Collection

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f4: Determination of the oocyte stage that starts to express EaRFP.(a) Seasonal variation of EaRFP expression levels during 2010–2011 in female E. ancora polyp tissues, as assessed by quantitative RT-PCR. Data shown are the mean ± SE (n = 3 colonies) relative to the April 2011 group. Groups with different letters are significantly different (P < 0.05). (b–e). SDS-PAGE and Western blotting of EaRFP. (b) SDS-PAGE of protein extract from pET-19b E. coli expressing recombinant EaRFP (Ec) and purified recombinant EaRFP (P). (c) Western blotting of the same protein shown in (b) with an anti-RFP antibody. (d) SDS-PAGE of protein extracts of ovary (Ov) collected in March. Arrow indicates the predicted EaRFP band. (e) Western blotting of ovarian protein extract with an anti-EaRFP antibody. Arrow indicates an EaRFP immunoreactive band of approximately 27 kDa. Molecular weight markers are shown on the left. (f–j) The immunohistochemical analysis of EaRFP with an anti-RFP antibody in stage I–V oocytes. Inset in (f) shows the stage I oocyte. (k) The negative control of immunohistochemical analysis. Immunohistochemistry was performed with the omission of the 1st antibody for this section (stage II oocytes). Oogenic stages I–V were classified according to cell size and morphology 6. Broken lines in (i–k) indicate the oocytes. All bars = 50 μm.
Mentions: We subsequently addressed when the E. ancora oocyte starts to express EaRFP during oogenesis. Our previous study revealed that E. ancora has an annual reproductive cycle, and its oogenesis takes approximately 11–12 months in southern Taiwan6. The oogenesis usually starts in June, and the matured oocytes are released the following May6. Quantitative RT-PCR of cDNA from the polyp tissue samples collected in different months showed that EaRFP transcripts were already present in June-August (11–9 months before spawning), increased as April approached, and decreased somewhat during the spawning month (May) (Fig. 4a). Microscopic observation of the the ovaries collected in August, December, and March (9, 5, and 2 months before spawning, respectively) also showed that the oocytes in all the samples express EaRFP (Supplementary Fig. 3a–f).

Bottom Line: Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period.Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity.These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Marine Environment and Ecology, National Taiwan Ocean University, Keelung, 20224, Taiwan.

ABSTRACT
To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction.

No MeSH data available.


Related in: MedlinePlus