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. 2015 Mar;82(3):251-64.
doi: 10.1002/mrd.22468. Epub 2015 Feb 20.

Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development

Mohamed Ashry  1 , KyungBon Lee, Mohan Mondal, Tirtha K Datta, Joseph K Folger, Sandeep K Rajput, Kun Zhang, Nabil A Hemeida, George W Smith
Affiliations

Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development

Mohamed Ashry et al. Mol Reprod Dev. 2015 Mar.

Abstract

Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFβ-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence.

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Figures

Figure 1
Figure 1
Expression of FST (A), JY1 (B) BMP15 (C), and GDF9 mRNAs (D) in BCB-screened GV-stage bovine oocytes. Quantitative reverse transcriptase PCR analysis of mRNA abundance was performed on samples of control, BCB+ and BCB− GV-stage oocytes (n = 4 pools of 10 oocytes for each oocyte group). Data were normalized relative to abundance of endogenous control (RPS18) and are shown as mean ± standard error. Values with different superscripts across treatments indicate significant differences (P < 0.05).
Figure 2
Figure 2
Cumulus cell cathepsins expression in BCB-screened GV-stage COCs. Quantitative reverse transcriptase PCR analysis of mRNA abundance for CTSB (A) , CTSK (B) , CTSS (C) and CTSZ (D) in bovine cumulus cells harvested from GV stage control, BCB+ and BCB− bovine COCs (n = 4 pools of 10 COCs for each oocyte group). Data were normalized relative to abundance of endogenous control (RPS18) and are shown as mean ± standard error. Values with different superscripts across treatments indicate significant differences (P < 0.05)
Figure 3
Figure 3
Cumulus cell expression of FST (A) and oocyte expression of CTSB (B), CTSS (C), and CTSZ (D) in oocytes and cumulus cells from BCB-screened, GV-stage bovine COCs. Quantitative reverse-transcriptase PCR analysis of mRNA abundance for FST in cumulus cells, and CTSB, CTSS, and CTSZ in oocytes from control, BCB+ and BCB− groups (n = 4 pools of 10 oocytes/cumulus cells from 10 COCs each per group). Data were normalized relative to abundance of endogenous control (RPS18) and are shown as mean ± standard error. Values with different superscripts across treatments indicate significant differences (P < 0.05)
Figure 4
Figure 4
Expression of mRNAs for SMAD1 (A), SMAD2 (B), SMAD3 (C) and SMAD5 (D) in BCB-screened, GV-stage oocytes. Quantitative reverse-transcriptase PCR analysis was on oocytes from control, BCB+ and BCB− groups (n = 7 pools of 10 oocytes each per group). Data were normalized relative to abundance of endogenous control (RPS18) and are shown as mean ± standard error. Values with different superscripts across treatments denote significant differences (P < 0.05).
Figure 5
Figure 5
SMAD 2/3 phosphorylation in BCB-screened, GV-stage oocytes. Samples of control, BCB+ and BCB− GV stage oocytes (n=6 replicates of 20 oocytes/group) were subjected to Western blot analysis of total (t)-SMAD2/3 (A), phosphorylated (p)-SMAD2/3 (B). Expression levels were normalized relative to abundance of endogenous control (actin). Phosphorylation level (C) was expressed as p -SMAD2/3 / t-SMAD2/3 Data are shown as mean ± standard error. Values with different superscripts across treatments indicate significant differences (P < 0.05). Representative Western blot (D).
Figure 6
Figure 6
SMAD1/5 phosphorylation in BCB-screened, GV-stage oocytes. Samples of control, BCB+ and BCB− GV stage oocytes (n=6 replicates of 20 oocytes/group) were subjected to Western blot analysis of total (t)-SMAD1/5 (A), phosphorylated (p)-SMAD1/5 (B). Expression levels were normalized relative to abundance of endogenous control (actin). Phosphorylation level (C) was expressed as p-SMAD1/5/t-SMAD1/5 Data are shown as mean ± standard error. Values with different superscripts across treatments indicate significant differences (P < 0.05). Representative Western blot (D).
Figure 7
Figure 7
Effects of exogenous follistatin supplementation on indices of development for embryos derived from BCB-screened, GV-stage oocytes. Presumptive zygotes derived from in vitro fertilization of oocytes from control, BCB+ and BCB− groups were cultured with or without 10 ng/ml follistatin (n=25-30 presumptive zygotes/group; n = 4 replicates) for 72 hr, then cultured in fresh medium (minus FST) until d 7. Effect of exogenous follistatin on (A) proportion of embryos that reached the 2-cell stage within 30 h post fertilization (early cleaving), (B) total cleavage rate (determined 48 hpi), (C) proportion of embryos developing to the 8-16 cell stage (72 hpi) and (D) proportion of embryos developing to the blastocyst stage (d 7). Data are shown as mean ± standard error. Values with different superscripts across treatments indicate significant differences (P < 0.05).
Figure 8
Figure 8
Effects of cathepsin inhibitor (E-64) treatment during meiotic maturation on developmental capacity of in vitro fertilized embryos derived from BCB-screened, GV-stage oocytes. Cumulus oocyte complexes (COCs) from control, BCB+ and BCB− oocytes were cultured with or without 10 µM cathepsin inhibitor E-64 during in vitro maturation (45-50 COCs per group, 4 replicates). After subsequent in vitro fertilization, presumptive zygotes were cultured until d 7. Effect of E-64 supplementation during meiotic maturation on (A) early cleavage rate (determined at 30 hpi), (B) total cleavage rate (determined 48 hpi), (C) proportion of embryos developing to the 8-16 cell stage (72 hpi) and (D) blastocyst formation rate (d 7). Data are shown as mean ± standard error. Values with different superscripts across treatments indicate significant differences (P < 0.05).
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References

    1. Abir R, Fisch B, Johnson MH. BMP15, fertility and the ovary. Reprod BioMed Online. 2014;29(5):525–526. - PubMed
    1. Alm H, Torner H, Lohrke B, Viergutz T, Ghoneim IM, Kanitz W. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology. 2005;63(8):2194–2205. - PubMed
    1. Balboula AZ, Yamanaka K, Sakatani M, Kawahara M, Hegab AO, Zaabel SM, Takahashi M. Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus-oocyte complexes exposed to heat shock during in vitro maturation. Reproduction. 2013;146(4):407–417. - PubMed
    1. Balemans W, Van Hul W. Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators. Dev Biol. 2002;250(2):231–250. - PubMed
    1. Barzegari A, Atashpaz S, Ghabili K, Nemati Z, Rustaei M, Azarbaijani R. Polymorphisms in GDF9 and BMP15 associated with fertility and ovulation rate in Moghani and Ghezel sheep in Iran. Reprod Domest Anim. 2010;45(4):666–669. - PubMed

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