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Purpose <p>The current study aimed to investigate whether cathepsin B (cath B) inhibition in cumulus cells of morphologically graded buffalo cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) could modulate apoptotic markers and enhance developmental potential.</p> Methods <p>Cumulus cells from grade A and B buffalo COCs were collected at 0, 12, and 24&#xa0;h of IVM, with or without supplementation of the optimized cysteine protease inhibitor E-64. Cathepsin B and selected apoptotic regulators were quantified by qRT-PCR. Cathepsin B protein levels and apoptotic signals were assessed by immunostaining and TUNEL assay, respectively. Following 24&#xa0;h of IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were performed, and developmental competence was evaluated based on oocyte maturation, cleavage rate, blastocyst formation rate, and blastocyst quality.</p> Results <p>Expression of cath B and pro-apoptotic genes (BID, BCL2, BAX, caspase-3) was found higher along with lower expression of anti-apoptotic genes (BCL2 and XIAP) in the cumulus cells of B grade COCs. The addition of 10&#xa0;µM E-64 significantly (<i>P</i> &lt; 0.05) reduced cath B and pro-apoptotic gene expression, lowered the BAX/BCL2 ratio, and decreased apoptosis signals in cumulus cells of both COC grades. E-64 supplementation improved blastocyst yield from grade A COCs and enhanced blastocyst quality in both grades, without affecting maturation and cleavage rates.</p> Conclusion <p>E-64 supplementation during IVM modulates apoptotic markers in cumulus cells of different grades of buffalo COCs by inhibiting cath B, thereby enhancing the developmental competence and offering valuable insights for improving in vitro embryo production programs in this species.</p>

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Cathepsin B expression during in vitro maturation of buffalo COCs: apoptosis and developmental competence

  • 期刊论文

  • 发表日期：

    2026年2月9日

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  • Asit Jain,
  • Tripti Jain,
  • Sachinandan De,
  • Rakesh Kumar,
  • Tirtha Kumar Datta

摘要

Purpose

The current study aimed to investigate whether cathepsin B (cath B) inhibition in cumulus cells of morphologically graded buffalo cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) could modulate apoptotic markers and enhance developmental potential.

Methods

Cumulus cells from grade A and B buffalo COCs were collected at 0, 12, and 24 h of IVM, with or without supplementation of the optimized cysteine protease inhibitor E-64. Cathepsin B and selected apoptotic regulators were quantified by qRT-PCR. Cathepsin B protein levels and apoptotic signals were assessed by immunostaining and TUNEL assay, respectively. Following 24 h of IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were performed, and developmental competence was evaluated based on oocyte maturation, cleavage rate, blastocyst formation rate, and blastocyst quality.

Results

Expression of cath B and pro-apoptotic genes (BID, BCL2, BAX, caspase-3) was found higher along with lower expression of anti-apoptotic genes (BCL2 and XIAP) in the cumulus cells of B grade COCs. The addition of 10 µM E-64 significantly (P < 0.05) reduced cath B and pro-apoptotic gene expression, lowered the BAX/BCL2 ratio, and decreased apoptosis signals in cumulus cells of both COC grades. E-64 supplementation improved blastocyst yield from grade A COCs and enhanced blastocyst quality in both grades, without affecting maturation and cleavage rates.

Conclusion

E-64 supplementation during IVM modulates apoptotic markers in cumulus cells of different grades of buffalo COCs by inhibiting cath B, thereby enhancing the developmental competence and offering valuable insights for improving in vitro embryo production programs in this species.