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Development of bovine blastocysts following in vitro oocyte maturation on a dynamic microfluidic platform
DESIGN: The timing of development of mono-andric and mono-gynic conceptuses was analyzed. Epigenetic marks were assessed and compared with ICSI generated conceptuses. MATERIALS AND METHODS: To obtain mono-andric pseudo-embryos, enucleated mouse MII oocytes were injected with single sperm heads, while mono-gynic counterparts were generated by SrCl2 treatment. Cleavage of these constructs individually cultured was monitored at hourly interval up to 120. Some pseudo-embryos were processed for cytogenetic analysis at the second and third mitosis, while genome-wide DNA methylation patterns were detected by monoclonal antibodies raised against the methyl cytosine group. ICSI conceptuses served as control. RESULTS: Among the experimental groups, the proportion of constructs that reached the octet stage were comparable to the bi-parental counterparts. While the mono-gynic that reached the morula stage were comparable to the control, only 63.2% of the paternally derived constructs reach this stage. At the blastocyst stage, both mono-andric (15.8%) and mono-gynic (17.6%) development was remarkably compromised (P < 0.001), and required a longer time (P < 0.01). The delay in cleavage was prominent among the sperm-derived constructs. Karyotypic analysis revealed that pseudo-blastomeres of mono-andric at second (88.2%, 15/17) and third mitosis (91.3%, 21/23) confirmed their haploid status. The bi-parental embryos demonstrated active demethylation of the sperm nucleus earlier than the maternal genome, while both underwent progressive loss of methylation to regain it in synchrony at the blastocyst stage. Interestingly, the genome of mono-andric embryos failed to show any methylation changes until the octet stage, while monogynic maintained a methylation profile similar to the female genome of biparental embryos. CONCLUSIONS: We confirmed the compromised development of haploid embryos irrespective of gender. Differences in epigenetic marks as assessed by DNA methylation patterns are responsible for the restricted development of mono-andric embryos, possibly due to the absence or inactivation of the X chromosome. Supported by: Institutional.
P-653 DEVELOPMENT OF BOVINE BLASTOCYSTS FOLLOWING IN VITRO OOCYTE MATURATION ON A DYNAMIC MICROFLUIDIC PLATFORM. C. L. Bormann, L. M. Cabrera, Y. S. Heo, N. Kato, S. Takayama, G. D. Smith. Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI; Departments of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI; Departments of Obstetrics and Gynecology, Molecular and Integrative Physiology, Urology, Reproductive Sciences Program, University of Michigan, Ann Arbor, MI. OBJECTIVE: The objective of this experiment was to determine the effect of a dynamic microfluidic culture environment during in vitro maturation (IVM) has on bovine oocyte maturation and subsequent blastocyst development. DESIGN: Experimental. MATERIALS AND METHODS: Bovine cumulus oocyte complexes (COCs) from 2-10 mm follicles were matured in tissue culture medium 199, supplemented with 10% fetal calf serum, bovine FSH, LH and EGF. Oocytes were randomly distributed in groups of 10 to standard culture drops (control) and microfluidic chips with media flow (dynamic). In each treatment, oocytes were matured in 50 ml drops of maturation medium covered with mineral oil at 39 C in 5% CO2 and 100% humidity. No oocyte/embryo selection was performed beyond this point. At 22 h post IVM oocytes were inseminated at a concentration of 1x106 sperm/ ml in IVF-TALP media. Presumptive zygotes were washed and cultured in groups of 10 in KSOM þ amino acids supplemented with 3mg/ml crystallized BSA in 50 ml drops under mineral oil at a temperature of 39 C, 100% humidity and in an atmosphere of 10% CO2, 5% O2 and balanced N2. On day 7 of culture, embryos were morphologically assessed for cleavage and blastocyst development. Developmental data were analyzed using Chi-square statistics. All values given are significant at P<0.05, unless otherwise stated. RESULTS: Oocytes matured on a dynamic microfluidic chip resulted in statistically higher rates of blastocyst development than those cultured under control conditions (34 and 22%: P<0.05). There were no differences in rate of oocyte nuclear maturation between dynamic, static and control IVM treatments. CONCLUSIONS: Results from this experiment suggest that a dynamic IVM environment better supports cytoplasmic maturation than conventional static microdrop culture. This experiment further demonstrates the impor-
FERTILITY & STERILITYÒ
tance of a dynamic chemical, physical, and mechanical environment on preimplantation embryo development. Supported by: The National Institutes of Health (NIH; HD 049607-01 – S.T & G.D.S), United States Department of Agriculture (USDA; 200535203-16148 – G.D.S & S.T), the Michigan Economic Development Corporation (MEDC; GR 696 – S.T & G.D.S. Support for C.L.B. was provided by NIH Training Grant in Reproductive Sciences T32-HD07048 (G.D.S).
P-654 ENDOPLASMIC RETICULUM AND GOLGI DYNAMICS DURING MATURATION OF HUMAN OOCYTES: IMPLICATIONS FOR CELLULAR STRESS DURING CULTURE. S. Abdelmassih, A. P. Dos Reis, A. Lass-Napionkawska, C. Alvarez, C. Wojcik, V. Rawe. Clınica e Centro de Pesquisa em Reproducao, Sao Paulo, Brazil; Dept. of Anatomy and Cell Biology, Indiana Univ., Evansville, IN; CEGyR, Buenos Aires, Argentina; Dept. of Anatomy and Cell Biology, Indiana Univ., Evansville. 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. Supported by: IU School of Medicine – Evansville and CEGyR.
P-655 HUMAN OOCYTES AND THE DYNAMICS OF MICROTUBULE MOTOR PROTEINS. S. Abdelmassih, R. Abdelmassih, A. P. Dos Reis, C. Alvarez, S. Racedo, V. Rawe. Clınica e Centro de Pesquisa em Reproducao, Sao Paulo, Brazil; CEGyR, Buenos Aires, Argentina. OBJECTIVE: In vitro oocyte maturation in humans is associated with a loss of developmental competence and reduced pregnancy rates. Meiotic spindle structure and the segregation of oocytes chromosomes have gained attention in the last years. In the present project, we aim to explore the presence and distribution of Eg5 and cytoplasmic dynein and dynactin in human
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P-653 DEVELOPMENT OF BOVINE BLASTOCYSTS FOLLOWING IN VITRO OOCYTE MATURATION ON A DYNAMIC MICROFLUIDIC PLATFORM. C. L. Bormann, L. M. Cabrera, Y. S. Heo, N. Kato, S. Takayama, G. D. Smith. Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI; Departments of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI; Departments of Obstetrics and Gynecology, Molecular and Integrative Physiology, Urology, Reproductive Sciences Program, University of Michigan, Ann Arbor, MI. OBJECTIVE: The objective of this experiment was to determine the effect of a dynamic microfluidic culture environment during in vitro maturation (IVM) has on bovine oocyte maturation and subsequent blastocyst development. DESIGN: Experimental. MATERIALS AND METHODS: Bovine cumulus oocyte complexes (COCs) from 2-10 mm follicles were matured in tissue culture medium 199, supplemented with 10% fetal calf serum, bovine FSH, LH and EGF. Oocytes were randomly distributed in groups of 10 to standard culture drops (control) and microfluidic chips with media flow (dynamic). In each treatment, oocytes were matured in 50 ml drops of maturation medium covered with mineral oil at 39 C in 5% CO2 and 100% humidity. No oocyte/embryo selection was performed beyond this point. At 22 h post IVM oocytes were inseminated at a concentration of 1x106 sperm/ ml in IVF-TALP media. Presumptive zygotes were washed and cultured in groups of 10 in KSOM þ amino acids supplemented with 3mg/ml crystallized BSA in 50 ml drops under mineral oil at a temperature of 39 C, 100% humidity and in an atmosphere of 10% CO2, 5% O2 and balanced N2. On day 7 of culture, embryos were morphologically assessed for cleavage and blastocyst development. Developmental data were analyzed using Chi-square statistics. All values given are significant at P<0.05, unless otherwise stated. RESULTS: Oocytes matured on a dynamic microfluidic chip resulted in statistically higher rates of blastocyst development than those cultured under control conditions (34 and 22%: P<0.05). There were no differences in rate of oocyte nuclear maturation between dynamic, static and control IVM treatments. CONCLUSIONS: Results from this experiment suggest that a dynamic IVM environment better supports cytoplasmic maturation than conventional static microdrop culture. This experiment further demonstrates the impor-
FERTILITY & STERILITYÒ
tance of a dynamic chemical, physical, and mechanical environment on preimplantation embryo development. Supported by: The National Institutes of Health (NIH; HD 049607-01 – S.T & G.D.S), United States Department of Agriculture (USDA; 200535203-16148 – G.D.S & S.T), the Michigan Economic Development Corporation (MEDC; GR 696 – S.T & G.D.S. Support for C.L.B. was provided by NIH Training Grant in Reproductive Sciences T32-HD07048 (G.D.S).
P-654 ENDOPLASMIC RETICULUM AND GOLGI DYNAMICS DURING MATURATION OF HUMAN OOCYTES: IMPLICATIONS FOR CELLULAR STRESS DURING CULTURE. S. Abdelmassih, A. P. Dos Reis, A. Lass-Napionkawska, C. Alvarez, C. Wojcik, V. Rawe. Clınica e Centro de Pesquisa em Reproducao, Sao Paulo, Brazil; Dept. of Anatomy and Cell Biology, Indiana Univ., Evansville, IN; CEGyR, Buenos Aires, Argentina; Dept. of Anatomy and Cell Biology, Indiana Univ., Evansville. 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. Supported by: IU School of Medicine – Evansville and CEGyR.
P-655 HUMAN OOCYTES AND THE DYNAMICS OF MICROTUBULE MOTOR PROTEINS. S. Abdelmassih, R. Abdelmassih, A. P. Dos Reis, C. Alvarez, S. Racedo, V. Rawe. Clınica e Centro de Pesquisa em Reproducao, Sao Paulo, Brazil; CEGyR, Buenos Aires, Argentina. OBJECTIVE: In vitro oocyte maturation in humans is associated with a loss of developmental competence and reduced pregnancy rates. Meiotic spindle structure and the segregation of oocytes chromosomes have gained attention in the last years. In the present project, we aim to explore the presence and distribution of Eg5 and cytoplasmic dynein and dynactin in human
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