Endoplasmic reticulum and Golgi dynamics during maturation of human oocytes: implications for cellular stress during culture

Abstract

OBJECTIVE: Proteasomes degrade the bulk of proteins as part of a quality control process. Of particular interest is the cytosolic degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER-associated degradation, ERAD). ERAD is induced as part of the unfolded protein response. ERAD involves elements of the retrotranslocation machinery: ER transmembrane membrane proteins, luminal (BiP) and cytosolic (Hsp70) chaperones. We explored the distribution of BiP and Hsp70 during human oocyte maturation and inhibition of ER and Golgi functions with Tunicamicyn (TNC) and Brefeldin A (BFA) respectively.DESIGN: Immunofluorescence microscopy of human oocytes maturated in vitro.MATERIALS AND METHODS: Confocal study was performed in a total of 118 human oocytes. In vitro oocyte maturation (IVM) was performed at 37°C for 24hs. Oocytes were assessed after culture and IVM was terminated by fixation. Fixation and permeabilization were done with 2% formaldehyde and 0.1% Triton X-100 respectively. ER and Golgi were identified using Calreticulin and GM-130 antibodies respectively. BiP and Hsp70 were detected by monoclonal antibodies. DNA was labeled using TOTO-3 and samples were imaged with a confocal microscope. BiP and Hsp70 dynamics was studied after TNC and BFA treatment.RESULTS: Germinal Vesicle (GV) oocytes have Golgi structures (mini Golgies) that break down and disperse following GV breakdown (GVBD) leading to punctate clusters. BiP and Hsp70 were distributed in the cytoplasm associated with the cortex (punctuated pattern). A complete overlapping in the distribution of Calreticulin and BiP was observed. TNC and BFA don´t affect oocyte maturation rate but increases the expression of BiP and Hsp70. BiP distribution was seen as clusters around the cytoplasm and Hsp70 as punctuate pattern. Oocytes degeneration and vacuoles formation were observed after treatment with both drugs.CONCLUSIONS: Dynamics of ER and Golgi during human IVM, suggest that they have an important role remodeling the cytoplasm. Qualitatively, levels of BiP and Hsp70 raise following ER stress induced by TNC and BFA. Despite inducing ER stress and formation of vacuoles, TNC and BFA treatment did not significantly interfere with GVBD and oocytes maturation rate. Probably, oocytes' competence is severely affected in ER stressed oocytes. These investigations will enhance our understanding of human oocytes biology with emphasis on clinically valuable applications. OBJECTIVE: Proteasomes degrade the bulk of proteins as part of a quality control process. Of particular interest is the cytosolic degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER-associated degradation, ERAD). ERAD is induced as part of the unfolded protein response. ERAD involves elements of the retrotranslocation machinery: ER transmembrane membrane proteins, luminal (BiP) and cytosolic (Hsp70) chaperones. We explored the distribution of BiP and Hsp70 during human oocyte maturation and inhibition of ER and Golgi functions with Tunicamicyn (TNC) and Brefeldin A (BFA) respectively. DESIGN: Immunofluorescence microscopy of human oocytes maturated in vitro. MATERIALS AND METHODS: Confocal study was performed in a total of 118 human oocytes. In vitro oocyte maturation (IVM) was performed at 37°C for 24hs. Oocytes were assessed after culture and IVM was terminated by fixation. Fixation and permeabilization were done with 2% formaldehyde and 0.1% Triton X-100 respectively. ER and Golgi were identified using Calreticulin and GM-130 antibodies respectively. BiP and Hsp70 were detected by monoclonal antibodies. DNA was labeled using TOTO-3 and samples were imaged with a confocal microscope. BiP and Hsp70 dynamics was studied after TNC and BFA treatment. RESULTS: Germinal Vesicle (GV) oocytes have Golgi structures (mini Golgies) that break down and disperse following GV breakdown (GVBD) leading to punctate clusters. BiP and Hsp70 were distributed in the cytoplasm associated with the cortex (punctuated pattern). A complete overlapping in the distribution of Calreticulin and BiP was observed. TNC and BFA don´t affect oocyte maturation rate but increases the expression of BiP and Hsp70. BiP distribution was seen as clusters around the cytoplasm and Hsp70 as punctuate pattern. Oocytes degeneration and vacuoles formation were observed after treatment with both drugs. CONCLUSIONS: Dynamics of ER and Golgi during human IVM, suggest that they have an important role remodeling the cytoplasm. Qualitatively, levels of BiP and Hsp70 raise following ER stress induced by TNC and BFA. Despite inducing ER stress and formation of vacuoles, TNC and BFA treatment did not significantly interfere with GVBD and oocytes maturation rate. Probably, oocytes' competence is severely affected in ER stressed oocytes. These investigations will enhance our understanding of human oocytes biology with emphasis on clinically valuable applications.