PRISCILA HELENA DOS SANTOS EFEITOS DA PROTEÍNA OVIDUTAL HSPA5 NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS Botucatu – SP 2022 PRISCILA HELENA DOS SANTOS EFEITOS DA PROTEÍNA OVIDUTAL HSPA5 NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS Tese apresentada ao programa de Pós-graduação do Instituto de Biociências de Botucatu, Universidade Estadual Paulista – UNESP, para a obtenção do título de Doutor em Farmacologia e Biotecnologia. Orientador: Prof. Doutor Anthony César de Souza Castilho Botucatu – SP 2022 Palavras-chave: BiP; GRP78; Heat shock protein; MIV; PIVE. Santos, Priscila Helena dos. Efeitos da proteína ovidutal HSPA5 na produção in vitro de embriões bovinos / Priscila Helena dos Santos. - Botucatu, 2022 Tese (doutorado) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Instituto de Biociências de Botucatu Orientador: Anthony César de Souza Castilho Capes: 21005001 1. Bovinos - Reprodução. 2. Proteínas de choque térmico. 3. Homeostase celular. 4. Fertilização in vitro. 5. Transcrição genética. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSEMEIRE APARECIDA VICENTE-CRB 8/5651 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. Nome do autor: Priscila Helena dos Santos Título: EFEITOS DA PROTEÍNA OVIDUTAL HSPA5 NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS COMISSÃO EXAMINADORA Prof. Dr. Anthony César de Souza Castilho Presidente e Orientador Unoeste – Presidente Prudente – São Paulo Prof. Dr. João Carlos Pinheiro Ferreira Membro FMVZ, Unesp – Botucatu – SP Dra. Raquel Zaneti Puelker Membro Progest Biotecnologia em Reprodução Animal – Botucatu – SP Prof. Dr. Mateus José Sudano Membro UFSCar – São Carlos – SP Isabele Picada Emanuelli Membro UniCesumar – Maringá - SP Data da defesa: 11 de abril de 2022. “O que prevemos raramente ocorre; o que menos esperamos geralmente acontece. ” (Benjamin Disraeli) AGRADECIMENTOS Aos meus pais, meus maiores apoiadores desta jornada, que mesmo não entendendo o percurso, confiaram, acreditaram e me incentivaram do início ao fim. Ao meu marido, Guilherme, que além de estar pacientemente ao meu lado, me deu suporte, incentivo e deixou a vida mais leve. Às minhas avós pelas constantes orações e amor incondicional. Aos meus avôs, que levo no coração, pelo carinho que será sempre lembrado. Aos meus irmãos e cunhados por serem inspiração. Ao meu orientador, por acreditar em mim desde o início, pela oportunidade de fazer parte de um time de excelência e pela amizade além da pós-graduação. À minha amiga, madrinha, companheira de todas as horas, que mesmo na distância se faz presente, Fernanda (Tico). Obrigada por ouvir, respeitar e estar comigo. Às amigas que além do suporte científico, estiveram comigo nas alegrias e tristezas fora da pós-graduação, Sarah e Patrícia. Botucatu não seria a mesma sem vocês. Durante os cinco anos de estudo, tive a oportunidade (e sorte) de conviver com muitas pessoas que deixaram marcas e aprendizados. Agradeço a cada colega e amigo do LaMem, FitoFarmaTec e Biotera que compartilhou conhecimento, risadas e cafés filosóficos comigo. Talita, Ketlyn, Eduardo, Carol, Elisa, Erika, Vitória, Ana Elise e Alan. À Raquel, pelo companheirismo, por ser prestativa e salvar nossas vidas nas intercorrências. Ao professor Di Stasi pela confiança e acolhimento em seu laboratório. À Janete, que sempre cuidou tão bem dos alunos da Farmacologia e aos demais funcionários. À seção-técnica de pós- graduação do Instituto de Biociências de Botucatu, em especial ao Davi. Aos professores do Departamento de Farmacologia do Instituto de Biociências de Botucatu pelas disciplinas. Aos professores João Ferreira, Mateus Sudano, Isabele Picada e a Raquel Puelker, por atenderem prontamente meu convite para compor a banca de doutorado. À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo financiamento à pesquisa (# 2018/06674-7; 2013/11480-3; 2015/04505-5) e à Coordenação de aperfeiçoamento de Pessoal de Nível Superior – CAPES (Financiamento Cód 001) pelo tempo de bolsa concedida. RESUMO A proteína HSPA5 (do inglês, heat shock 70 kDa protein 5), também conhecida como GRP78 (do inglês, 78 kDa glucose-regulated protein) e BiP (do inglês, immunoglobulin-binding protein), é uma chaperona do retículo endoplasmático que desenvolve um papel fundamental na manutenção da homeostase celular. Relativamente recente, essa proteína foi descrita como um dos componentes do fluido ovidutal com capacidade de se ligar aos espermatozoides e aumentar a concentração de cálcio intracelular. Considerando que o ambiente ovidutal fornece substratos e fatores importantes para a fusão dos gametas e desenvolvimento inicial embrionário in vivo e que a proteína HSPA5está em alta concentração no fluido ovidutal, nós acreditamos que essa proteína pode exercer efeitos benéficos na produção de embriões in vitro. Hipotetizando que a HSPA5 pode interagir com o complexo cumulus-oócito (COC) durante a maturação in vivo (MIV) e também com o embrião em desenvolvimento durante o cultivo in vitro (CIV), a temática primordial desta tese foi testar a adição da HSPA5 recombinante humana na produção in vitro de embriões (PIVE). Em um primeiro momento, nós analisamos o efeito da HSPA5 nas últimas 4 horas da MIV sobre os níveis de espécies reativas de oxigênio (ROS) e potencial de membrana mitocondrial (MMP) dos oócitos e subsequentes embriões, os níveis de glutationa (GSH) nos COCs e meio de cultivo, a solubilidade da zona pelúcida e penetração espermática, sobre a taxa de embriões produzidos e o perfil de transcritos destes embriões. Na segunda abordagem nós testamos a adição da rHSPA5 nos oito dias de CIV e comparamos com o tratamento na MIV e a associação dos dois tratamentos. Foi avaliado a taxa de embriões produzidos e a transcrição gênica destes embriões. Em suma, os resultados indicaram que a rHSPA5 é capaz de interagir com o COC durante a MIV e com os embriões em desenvolvimento no CIV. A proteína foi capaz de aumentar taxa de embriões produzidos quando adicionada nas últimas 4 horas de MIV, manter os níveis de ROS e GSH nos COCs, mas não aumentou o tempo de solubilidade da zonna-pellucida (ZP) ou alterou a porcentagem de monospermia, mas a rHSPA5 na MIV foi capaz de modular positivamente a expressão gênica dos embriões produzidos. Já a inclusão da proteína no CIV, apesar de haver uma pequena modulação na expressão gênica, não foi capaz de aumentar a taxa de embriões produzidos. Com os presentes achados, concluímos que, a HSPA5 pode interagir com o COC e ser capaz de exercer funções ainda não determinadas, assim como o uso da rHSPA5 nas últimas 4 horas da MIV pode otimizar a PIVE e potencializar a qualidade embrionária. Palavras-chave: GRP78, BiP, heat shock protein, MIV, CIV, PIVE ABSTRACT The HSPA5 protein (heat shock 70 kDa protein 5), also known as GRP78 (78 kDa glucose- regulated protein) and BiP (immunoglobulin-binding protein), is a chaperone from the endoplasmic reticulum with an important role in cellular homeostasis. Relatively recent, this protein was described as an oviductal fluid component as well as, its ability to hardly bind the spermatozoa, and then, increase the intracellular calcium abundance. The oviduct provides an appropriate environment for gametes fusion and early embryo development. Once the HSPA5 is one of the main heat shock proteins from oviductal fluid, we have a hypothesis that this protein is able to be beneficial on in vitro embryo production. Hypothesizing that HSPA5 can interact with cumulus-oocyte complex (COC) during the in vitro maturation (MIV) and, also with developing early embryo on in vitro culture (CIV), the primordial thematic of this thesis tested the HSPA addition on in vitro embryo production. First of all, we analyzed the HSPA5 effect on the last 4 hours of MIV under reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels and on the subsequent embryos. The glutathione (GSH) levels on COC and culture medium, the zona-pellucida (ZP) solubility, sperm penetration, and last, the rate of embryo production and transcript profile of them. The second point was tested the HSPA5 effect on the eight days of CIV to compare with MIV treatments and an association of both treatments (on the last 4 hours of MIV and 8 days of CIV). We assessed the embryo rate and transcriptome profile of them. In conclusion, the results indicated that HSPA5 was able to interact with the COC and the developing early embryo. This protein was capable to increase the embryo rate on the MIV treatment and maintaining the ROS and GSH levels on COC, but it did not increase the ZP solubility or monospermy rate. Mainly, the HSPA5 was able to positively modulate the gene expression of embryos. On the other hand, the HSPA5 on the CIV did not alter the embryo rate and found a lower modulation on the transcript profile. We conclude that HSPA5 interacts with COC and develops an unknown role and also, the inclusion of rHSPA5 on the last 4 hours of MIV can improve the in vitro embryo production and enhance the embryo quality. Keywords: GRP78, BiP, heat shock protein, MIV, CIV, PIVE SUMÁRIO PREFÁCIO ................................................................................................................... 11 CAPÍTULO I ................................................................................................................ 12 The functionality of oviductal heat shock proteins on gametes and embryos: an integrative review ......................................................................................................... 13 ABSTRACT ................................................................................................................ 13 INTRODUCTION ...................................................................................................... 14 METHODOLOGY ..................................................................................................... 15 RESULTS ................................................................................................................... 17 DISCUSSION ............................................................................................................. 25 CONCLUSION ........................................................................................................... 29 REFERENCES ........................................................................................................... 30 CAPÍTULO II ............................................................................................................... 35 EVIDENCE THAT HEAT SHOCK PROTEIN A5 (HSPA5) PLAYS A ROLE DURING BOVINE IN VITRO EMBRYO PRODUCTION ..................................................... 36 ABSTRACT ................................................................................................................ 36 INTRODUCTION ...................................................................................................... 37 RESULTS ................................................................................................................... 39 DISCUSSION ............................................................................................................. 43 MATERIALS AND METHODS ................................................................................ 48 REFERENCES ........................................................................................................... 55 CAPÍTULO III ............................................................................................................. 59 AS NOVAS ESTRATÉGIAS DO USO DA rHSPA5 HUMANA NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS ........................................................................... 60 RESUMO .................................................................................................................... 60 INTRODUÇÃO .......................................................................................................... 61 MATERIAL E MÉTODO .......................................................................................... 63 RESULTADOS E DISCUSSÃO ................................................................................ 67 CONCLUSÃO ............................................................................................................ 79 REFERÊNCIAS .......................................................................................................... 80 CONSIDERAÇÕES FINAIS ....................................................................................... 83 ATIVIDADES ACADÊMICAS DESENVOLVIDAS DURANTE O DOUTORADO85 11 PREFÁCIO A presente tese a ser defendida pelo programa de Pós-graduação em Farmacologia e Biotecnologia está dividida em quatro sessões. A primeira, denominada Capítulo I, compreende uma Revisão Integrativa que traz um compilado de publicações que introduz a relação das Heat Shock proteins com os gametas e embriões, e mostra como essas proteínas, que estão presentes no oviduto, estão envolvidas com o processo reprodutivo. Essa revisão além de ser a base para uma futura revisão sistemática, foi utilizada como uma introdução à temática desta tese. Das duas partes subsequentes, o Capítulo II - “Evidences that heat shock protein A5 (HSPA5) plays role during bovine in vitro embryo production” e Capítulo III – “As novas estratégias do uso da rHSPA5 humana na produção in vitro de embriões”, correspondem um como uma proposta de manuscrito a ser submetido como artigo no periódico “Reproduction in Domestic Animals”, e o outro a descrição dos resultados recém obtidos pelo grupo, que será utilizado como base para o próximo artigo. Por fim, a sessão “Considerações finais” compreende as conclusões gerais (descrevendo de forma sucinta os resultados obtidos). Conforme normas do programa de Pós-graduação, o presente exemplar consiste na versão definitiva da tese a ser encaminhada à biblioteca e fará parte do conjunto de publicações do Instituto de Biociências de Botucatu da UNESP. No entanto, o manuscrito aqui apresentado está no prelo, mas não comprometido com a publicação em quaisquer periódicos, estando, assim, passível de correções e modificações por parte da banca de defesa. 12 CAPÍTULO I 13 THE FUNCTIONALITY OF OVIDUCTAL HEAT SHOCK PROTEINS ON GAMETES AND EMBRYOS: AN INTEGRATIVE REVIEW Priscila H Santos a a São Paulo State University (UNESP), Department of Pharmacology, Institute of Biosciences, 5 Botucatu, SP, Brazil. Running title: How the HSPs from oviductal fluids operate on gametes and embryos. ABSTRACT 10 The fusion of gametes to generate an embryo takes place on the oviduct. For a long period, this tubal-like organ was forgotten once the in vitro embryo production reaches a great success rate. After it become a study target, their oviductal fluid (OF) was assessed and a range of proteins with an important role on gametes and embryos became known. Some of them were the Heat-15 Shock proteins (HSPs), a chaperone kind protein responsible to maintain viability and cellular homeostasis. Different HSPs can be found at OF and in the oviductal epithelial cells (OEC) at different portions of the oviduct but few of them were assessed about their possible role on gametes or embryos. This integrative review was conducted examining 323 publications from three databases, to merge relevant publications about HSPs localization on OF and OEC, the 20 interaction, and effects on gametes and early embryos. This study reveals that even HSPs are present on OF, only three proteins have more than one publication about a direct effect on spermatozoa capacity or fertilization process (HSPA5, HSPA8, and HSP60), but a range of them were predicted as actively participating in the reproduction process and a possible additive 14 on a culture medium to improve the in vitro embryo production and quality. After all, more 25 studies are necessary to completely understand the OF HSPs` role on gametes and embryos. INTRODUCTION In the in vivo embryo development, a range of coordinated processes occurs. Sperm 30 capacitation, fertilization, and embryo development take place in the oviduct during the peri- ovulatory period of the estrous cycle and to each step, the oviduct provides an optimal environment. The oviduct is a dynamic structure of the reproductive system that connects the ovary to the uterus and is divided into three anatomical regions where each physiological process occurs: the infundibulum, ampulla, and isthmus (Avilés, 2010; Pillai et al., 2017). 35 When the spermatozoa enter into the oviduct in mammalians, they form a sperm reservoir on the isthmus portion. It is possible because the sperm cells bind to the oviductal-cell membranes and can remain adhered for hours to be released only near the time of ovulation. In the isthmus portion, the spermatozoa undergo hyperactivation and early acrosome reaction. As soon as the complex oocyte-cumulus (COC) is picked-up by adhesion at the cilia of the 40 infundibulum its cumulus matrix becomes compacted to enter into the oviduct. When the oocyte reaches the ampulla, it immediately attaches to the oviductal epithelium and its zona-pellucida becomes more accessible to the OF and the spermatozoa are released from the reservoir and migrate driving to the oocyte (Kolle et al., 2009; Kolle et al., 2010). From the gamete’s passage to the fusion until the early embryo development, the 45 structures maintain contact with the OF, which is essential to these physiological processes (Avilés, 2010). The OF is a complex mixture of components produced by secretions from OEC and plasm transudate with a spatiotemporally dynamic process (Mondejar et al., 2013; Pillai et al., 2017). Multiple proteins are secreted by the oviductal cells, including HSPs (Killian, 2011; Yanagimachi, 2015; Soleilhavoup et al., 2016). 50 15 The HSPs are widely expressed and present in the genome of all cellular organisms with the main role in protein degradation, breakdown, and transmembrane transport. There are about ten types of these molecular chaperones and they are divided into seven families according to molecular mass (sHSP, HSP40, HSP60, HSP70, HSP90, HSP110, and ubiquitin) (Shan et al., 2020). For a long period, the HSPs were reported to be an exclusive intracellular component, 55 but later the HSPs were described at the plasmatic membrane and secreted, including in OEC and OF (Buhi et al., 2000; Boilard et al., 2004; Marín-Briggiler, 2010). Studies report the influence and the essential role of HSPs on OF. Some of these proteins can interact with gametes and improve fertilization (Mondejar et al., 2013), the spermatozoa capacitation and viability (Lloyd et al., 2009; Lamy et al., 2018), and possibly, play a role in 60 early embryo development (Alminana et al., 2017). Although embryo production is possible without the oviduct, this environment provides factors that a simple in vitro culture cannot supply and that improve the embryo quality (Mahdavinezhad et al., 2019). Then, better knowledge about the interaction of HSPs and gametes and embryos allows benefits on in vitro embryo production efficiency and enhances the embryo quality. This integrative review aimed 65 to merge relevant publications about HSPs localization on OF and OEC, the interaction, and effects on gametes and early embryos. METHODOLOGY 70 This article is an integrative review that was conducted following the steps below: 1. identification of the theme and formulation of the guiding question; 2. establishment of criteria to include or exclude the studies; 3. definition of keywords to research in databases; 4. evaluation and categorization of data; 5. interpretation of the results obtained; 6. presentation of results. 75 16 The search was carried out in December 2021 using the PubMed, Scopus, and Web of Science databases and involved an extensive review to address the main question: What is the function of heat shock protein from oviductal fluid on gametes and embryos? For it, the search was electronically performed for the following terms in the titles, abstracts, and keywords: ‘heat shock protein’ and ‘oviduct’. In vitro or in vivo research with mammals that demonstrate the 80 interaction of HSP with gametes or embryos were included. Studies that used proteomic or immunolocalization analyses that demonstrate the presence of HSP on the oviduct were also included. There was no restriction about the period of publication. First of all, duplicated articles were removed, and then publications were selected by title and abstract. Exclusion criteria included publication not in English language, use of no mammalian as a model, 85 investigations of environmental effect and pathologies on HSP levels, or induction of higher levels of HSP as thermal stress. A total of 32 studies met the criteria, but once five publications were a kind of revision that cited studies already included in this work, they were removed. Then, twenty-seven studies were eligible for this integrative review at the end of the selection process (fig 1). 90 17 Fig 1. Flowchart of search and selection process. Thirty-seven studies met the criteria and investigate HSPs on the oviduct or its function and were used to review. RESULTS 95 Of twenty-seven studies that met the criteria (Table 1), twelve were studies that addressed two or more HSPs. Five mainly focused or at least quote HSPA8, three the HSPA5, two the HSP70, and five studies approached another HSP. About it, in eight of the studies, one Integrative review – Flow Diagram Th em e se le ct io n sc re en in g El ig ib ili ty In cl u si o n Records identified through database searching (n= 323) Records after duplication removed (n= 217) Records screened (n= 217) Records excluded (n= 154) Studies included in qualitative synthesis (n= 27) Full-text assessed for elegibility (n= 63) Full-texte articles excluded, with reasons (n=36) 18 or more proteins was found by proteomic, transcriptomic, or localization analysis and, besides 100 implying a functionality of the proteins on gametes and/or embryo, it did not show data to clarify. Table 1. Articles that met the criteria and were selected for the integrative review. Article Article Title Authors Protein 1 Constitutive expression of heat-shock proteins hsp25 and hsp70 in the rat oviduct during neonatal development, the oestrous cycle and early pregnancy Mariani et al., 2000 HSP25 and HSP70 2 Localization of the chaperone proteins GRP78 and Hsp60 on the luminal surface of bovine epithelial cells and their association with spermatozoa Boilard et al., 2004 HSPA5 and HSP60 3 Expression of Hsp60 and GRP78 in the human endometrium and oviduct, and their effect in sperm functions Lachance et al., 2007 HSP60 and HSPA5 4 Identification of potential oviductal factors responsible for zona pellucida hardening and monospermy during fertilization in mammals Mondéjar et al., 2013 HSP90A, HSPA5, HSP90B and HSP70 5 Viable and morphological normal boar spermatozoa alter the expression of heat-shock protein genes in oviductal epithelial cells during co-culture in vitro Yeste et al., 2014 HSPA8, HSP90AA1 and HSPA5 6 Proteomes of the female genital tract during the oestrous cycle Soleilhavoup et al., 2016 HSPA8, HSP90B1 and HSP70 7 Regulation of the bovine oviductal fluid proteome Lamy et al., 2016 HSPA5, HSP90AA1, HSPA8, HSP90AB1, HSP90B1, HSPA6, HSPA4 and HSP70 8 Oviductal extracellular vesicle protein content and their role during oviduct-embryo cross-talk Almiñana et al., 2017 HSPA1A, HSP60, HSPA4, HSPA5, HSPA8, HSP90AA1, HSP90AB1 and HSP90B1 9 Semen modulated secretory activity of oviductal epithelial cells is linked to cellular proteostasis network remodeling: Proteomic insights into the early phase of interaction in the oviduct in vivo Steinberger et al., 2017 HSPA5 and HSP90B1 10 Identification by proteomics of oviductal sperm-interacting proteins Lamy et al., 2018 HSPA5, HSP27 and HSP90B1 19 11 Addition of exogenous proteins detected in oviductal secretions to in vitro culture medium does not improve the efficiency of in vitro fertilization in pigs García- Martinez et al., 2020 HSP90α and HSP70 12 Oviductal fluid during IVF moderately modulates polyspermy in vitro-produced goat embryos during the non- breeding season Bragança et al., 2021 HSPA8 and HSPA1A 13 Effects of oviductal proteins, including heat shock 70kDa protein 8, on survival of ram spermatozoa over 48 h in vitro Lloyd et al., 2009 HSPA8 14 Effects of HSPA8, an evolutionarily conserved oviductal protein, on boar and bull spermatozoa Elliott et al., 2009 HSPA8 15 The oviductal protein, heat-shock 70-kDa protein 8, improves the longterm survival of ram spermatozoa during storage at 17°C in a commercial extender Lloyd et al., 2011 HSPA8 16 Heat-shock protein A8 restores sperm membrane integrity by increasing plasma membrane fluidity Moein-Vaziri et al., 2014 HSPA8 17 Heat Shock protein A8 stabilizes the bull sperm plasma membrane during cryopreservation: Effect of breed, protein concentration, and mode of use Holt, Valle and Fazeli 2015 HSPA8 18 Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm-zona pellucida binding Marín- Briggiler et al., 2010 HSPA5 19 GRP78 expression and the immunohistochemical localization in the female reproductive tract of the mice Lin et al., 2012 HSPA5 20 Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows Fontes et al., 2019 HSPA5 21 Expression of the testis-specific HSP70-related gene (HST70 gene) in somatic non-testicular rat tissue revealed by RT-PCR and transgenic mice analysis Scieglinska et al., 1997 HSP70 22 Gametes alter the oviductal secretory proteome Georgiou et al., 2005 HSP70 23 Expression and cellular localization of the mRNA for the 25- kDa heat-shock protein in the mouse Wakayama and Iseki 1998 HSP25 24 An immunohistochemical study of the rete ovarii and epoophoron Woolnough et al., 2000 HSP27 25 The expression pattern of the 70-kDa heat shock protein Hspa2 in the mouse tissues Vydra et al., 2009 HSPA2 20 26 The effect of HSP60 on fertilization and pre-implantation embryo development in mice: an in vitro study Abdi et al., 2019 HSP60 27 Which low-abundance proteins are present in the human milieu of gamete/embryo maternal interaction? Canha- Gouvea et al., 2019 HSP90A 105 Among the selected article, ten studies only demonstrated the localization or the expression of one or more HSP in the oviduct, three reports an altered expression of HSP on epithelial cells or oviductal secretory proteome in a presence of spermatozoa, and one only demonstrated the interaction of oviductal proteins with spermatozoa after co-incubation, and not the effect of proteins. So, from twenty-seven selected studies, thirteen reports some effect 110 and interaction of HSP on the gametes. From this, seven studies showed the effect of HSP on spermatozoa and four on in vitro fertilization. Only one study showed a possible effect of HSP on the embryo through extracellular vesicles, and one publication suggests the effect of HSP on the oocyte pre IVF (Table 2; Fig 2). 115 120 Figure 2: The oviductal HSPs that interact, were predicted, or play some effect on spermatozoa, oocyte, fertilization process, or on the embryo, described by studies included in this integrative 125 review. Spermatozoa: HSPA5 HSPA8 HSP70 HSP60 Fertilization: HSPA5 HSPA8 HSP60 HSP90 HSPA1A HSP60 Embryo: HSPA5 HSPA8 HSPA4 HSP70 HSP60 HSP90AA1 HSP90AB1 HSP90B1 Oocyte: HSPA5 HSPA1A HSP90B1 21 Table 2. Objectives of included studies, animal models, and a summary of main results. Article Objective Animal model Main results 1 Identify hsp25 and hsp70 in the development of the oviduct; to compare and study hsp25 and hsp70 during the estrous cycle and early pregnancy, and examine hsp25 and hsp70 association with estrogen receptor α regulation. Rats HSP70 is modulated by the estrous cycle and is more responsive to hormonal changes. 2 Identify oviduct originating factors that associate with spermatozoa and putatively affect their cellular activity. Bovine This demonstrates that both HSP60 and GRP78 are expressed on the surface of oviduct epithelial cells and they strongly associate with spermatozoa. 3 Determine whether HSP60 and HSPA5 are expressed by the epithelial cells from the endometrium and oviducts with a cellular localization that would allow interaction with the sperm surface. And determine their effect of them on human sperm functions and investigate whether they modulate the acquisition of fertilizing ability of human spermatozoa. Human Both proteins are present in the oviduct epithelial cells, bind to spermatozoa, and increased the sperm intracellular calcium concentration. 4 Investigate whether variations in oocyte hardening in different oviductal fluids are correlated with variations in levels of monospermy after IVF and identify the factors responsible for this effect. Porcine The mechanism of pre fertilization ZP hardening in OF is directly related to monospermy levels and suggest a role of proteins from the HSP family. 5 Determine if boar spermatozoa influence the expression of proteins in oviductal epithelial cells (OECs) during in vitro co-culture. Porcine Bind spermatozoa induced the upregulation of the HSPs after co- culture. 6 Provide an integrated analysis of the luminal proteomes of the female genital tract by a proteomic study of inner cervical mucus, uterine fluid, and oviduct fluid and quantify the abundance of these luminal proteins throughout the estrous cycle. Sheep - 7 Investigate the regulation of the proteome in the bovine oviductal fluid according to the stage of the estrous cycle, to the side relative to ovulation, and local concentrations of steroid hormones Bovine - 22 8 Deciphering the oviduct EVs protein content from both sources and analyzing their functional effect on embryo development. Bovine Showed that in vitro- produced embryos were able to internalize in vivo EVs during culture with a functional effect in the embryo development. 9 Assess the effect of semen on the cellular and molecular mechanisms in rabbit OECs Rabbit - 10 Develop a strategy to identify and quantify oviductal proteins that interact with spermatozoa and study the regulation of these interactions in three stages of the estrous cycle: the post-, pre-ovulatory, and luteal phases. Bovine - 11 Study whether proteins previously identified in porcine oviductal secretions have a role in zona pellucida resistance to enzymatic digestion, in vitro fertilization, and sperm viability. Porcine Sperm penetration and polyspermy rates decreased in presence of HSP proteins. 12 Determined the presence of proteins known to be involved in early reproduction in the oviduct fluid of anestrous goats; and the functional effect of IVF on polyspermy modulation and embryonic development. Goat The supplementation with OF during IVF may modulate the polyspermy incidence and enhance IVF efficiency. 13 Decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. Sheep Recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation. 14 Assess the effects of HSPA8 on boar and bull spermatozoa. Boar and bovine The HSPA8 has an important role in the maintenance of boar and bull sperm survival in vitro implying that the mechanism of action could be highly conserved across species. 15 Determine whether supplementing extenders with HSPA8 would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 178C. Ram The extender supplemented with HSPA8 maintained sperm viability significantly better than alone 16 Characterize the immediate impact of exogenous HSPA8 on spermatozoa. Boar The HSPA8 rapidly promotes the viability of uncapacitated spermatozoa, the ability of spermatozoa to bind to oviductal epithelial cells, enhances IVF 23 performance, and decreases sperm mitochondrial activity. 17 Explore the possibility that bovine recombinant HSPA8 might therefore protect bull spermatozoa during cryopreservation through its beneficial effects on the sperm plasma membrane. Bovine The study underlined the potential benefits of controlling plasma membrane permeability, especially in the more fragile semen samples from the beef cattle. 18 Determine the secretion of GRP78 by human epithelial cells, its association to spermatozoa, and its involvement in gamete interaction Human Soluble GRP78 is present in OF in the periovulatory period and recombinant GRP78 was able to bind to the sperm acrosomal cap. 19 Determine the distribution and cyclic variations of GRP78 in the mouse oviduct and uterus throughout the estrous cycle. Mouse - 20 Evaluate the effect of two superstimulatory protocols on the oviductal levels of E2 and P4 and their outcome on oviductal cells. Bovine The FSH/eCG treatment showed a higher concentration of E2 and a higher abundance of mRNA HSPA5 in the infundibulum and ampulla. 21 Detecting the hst70 gene transcripts in various male and female non-spermatogenic rat tissues. Rat - 22 Test the hypothesis that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. Porcine The oviductal response to spermatozoa was different from its response to oocytes and both of them altered the secretion of specific proteins. 23 Examine the expression and cellular localization of Hsp25 mRNA in mice under physiological and unstressed conditions. Mouse - 24 Compare the immunohistochemical profile of the human rete ovarii, and epoophoron, with the Fallopian tube and ovarian surface epithelium. Human - 25 Assess the extra-testicular expression pattern of mouse Hspa2. Mouse - 26 Assess the importance of HSP60 in sperm capacitation and the effect of HSP60 on the Mouse HSP60 in low doses had a positive effect on 24 rate of in vitro fertilization and the cleavage rate in the mouse. cleavage embryos. But it had adverse effects at higher doses. 27 Performer a comparative study of the low abundance proteins in plasma, uterine, and oviductal fluid collected, simultaneously, from healthy and fertile women that underwent a salpingectomy. Human - 130 The most employed animal model was bovine with 25.92% of studies followed by mouse, human and porcine (14.81%; Table 3), and the most studied protein on the effect on the gamete was HSPA8 (25.92%) followed by HSPA5 (18.51%) and HSP60 (14.81%) on publications. All other protein that exhibits some effect on gamete or embryo was cited once. When we add publications about interactions and expression/localization of HSP, 135 seventeen proteins were cited in the selected studies (Fig 3), with two belonging to small HSPs (sHSP; HSP25 and HSP27), seven to HSP70 (HSP70, HSPA1A, HSPA2, HSPA4, HSPA5, HSPA6, HSPA8), one to HSP60 and six to HSP90 family when four is from HSP90-alpha (HSP90A, HSPA1A, HSP90AA1, and HSPAB1) and two from HSP90-beta (HSP90B and HSP90B1). 140 Table 3: Species approached on selected publications 25 Specie Bovine* Mouse Human Porcine Rat Sheep Boar* Rabbit Goat Ram Study (n) 7 4 4 4 2 2 2 1 1 1 Percent (%) 25.92 14.81 14.81 14.81 7.40 7.40 7.40 3.70 3.70 3.70 * One publication approaches both species. 145 Figure 3: The localization of oviductal HSPs from mammals according to publications included in this integrative review. Figure adapted from Patricia Fontes DISCUSSION 150 Isthmus: HSPA5 HSP70 HSP25 HSP60 Ampulla: HSPA5 HSP70 HSP25 HSP60 Infundibulum HSPA5 HSP70 HSP25 HSP60 Oviduct al Fluid: HSPA8 HSP90B1 HSP70 HSPA5 HSP90AB1 HSP90B1 HSPA6 HSPA4 HSP90 26 The HSPs protein family is a well-established intra-cellular protein that develops a fundamental role in the maintenance of cellular homeostasis (Shan et al., 2020). Relatively recently the HSPs have been demonstrated to be released under nonpathological conditions and exert protective roles (Holt e Fazeli, 2016). On the oviduct, a range of HSP was reported expressed on OEC and present on oviductal fluid, and mainly, that the abundance of these HSP 155 as other proteins from the oviduct, can be different into the estrous cycle. This approach leads to the study of the possible interaction of HSP with embryos and gametes, and, its possible role on fertilization and early embryo development. Our integrative review gathers for the first time publications that demonstrated the interaction and effects of HSP on gametes and embryos. The HSPs are widely expressed and present in the genome of all cellular organisms with 160 the main role in protein degradation, breakdown, and transmembrane transport. There are about ten types of these molecular chaperones and they are divided into seven families according to molecular mass (sHSP, HSP40, HSP60, HSP70, HSP90, HSP110, and ubiquitin) (Shan et al., 2020). In this review, four families of HSP were cited: sHSP, HSP70, HSP60, and HSP90. From this, HSP70 and HSP60 families were the most cited proteins. 165 Among the selected publications, ten executed studies about localization or expression study on OEC or OF and founded HSPs. Five of them demonstrated that the abundance of HSPs is higher on estrus (Mariani et al., 2000; Lin et al., 2012; Lamy et al., 2016; Soleilhavoup et al., 2016; Fontes et al., 2019), and more abundant on the ipsilateral side (Lamy et al., 2016). In all five studies, the HSP70 family is present in all of them, closely with proteins from the HSP90 170 family and sHSP. The rest of localization and expression publications were not studies that aimed to compare the estrous cycle HSPs abundance, and presented results about specifics proteins as HSP70 (Scieglinska et al., 1997), HSP25 (Wakayama e Iseki, 1998), HSP27 (Woolnough et al., 2000), HSPA2 (Vydra et al., 2009) and HSP90 (Canha-Gouveia et al., 2019). 175 27 Besides studies about localization, some authors performed proteomic or transcriptomic analysis reporting alterations on HSP levels on OEC or OF after gamete OEC interaction. It was unanimous that the presence of gametes influences proteins abundance. Authors demonstrate that in vitro co-incubation of OEC (Yeste et al., 2014) and oviduct (Georgiou et al., 2005) with spermatozoa can alter the expression and abundance of proteins, among them 180 the HSPs, HSP70, HSPA8, and HSPA5 belonging HSP70 family and HSP90AA1. About the oocyte, it was also showed that it is also able to alter the secretory proteome of the oviduct, but not about HSPs expression (Georgiou et al., 2005). In in vivo study, Steinberger and colleagues identified two HSP on cellular surface 2 hours after intrauterine inseminations, the HSPA5 and HSP90B1 (Steinberger et al., 2017). Besides the studies that demonstrate the influence of 185 gametes on oviductal proteins, Lamy et al (2018) also reveal that when spermatozoa were co- incubated with OF, an interaction occurs between HSPs proteins and spermatozoa as HSPA5 and HSP90B1, from all estrous phases OF (Lamy et al., 2018). The HSPA5 was also found interacting with spermatozoa by Boilard (2004) along with HSP60 (Boilard et al., 2004). These results point to the importance of HSPs on reproductive processes and allow studies to clarify 190 the impact of HSPs on gametes and embryos. When the oocyte is incubated for 30 minutes in undiluted OF, the levels of monospermy increase, as well as the time of membrane digestion. These effects were related to the protein content of OF, which includes HSP70 (HSPA5 and HSPA1A) and HSP90 family - HSP90B1 (Mondejar et al., 2013). Besides the presence of HSPs on OF, little is known about the 195 interaction of oocyte and HSPs. This is the unique publication included in this work that demonstrates a probable direct effect of oviductal chaperones on the oocyte, having a lack about the theme. Another point that needs more studies, is the interaction of HSPs and the embryo. The oviduct supplies biochemical substrates (carbohydrates, ions, proteins, enzymes, etc) and 200 28 physical requirements (pH, viscosity, osmolarity) to the early embryo development (Besenfelder et al., 2020). Relative recently, extracellular vesicles (EVs) have been described to participate in maternal-embryo communication transferring their molecular cargo (proteins, mRNA, miRNA) from cell-to-cell (Valadi et al., 2007). From this, Alminana and colleagues (2017) demonstrate that EVs from OF content HSPs (HSPA8, HSPA5, HSP70, HSP60, 205 HSP90AA1, HSP90AB1, and HSP90B) (Alminana et al., 2017), and mainly, that it was internalized by the embryo during in vitro culture and enhance their ability to reach the blastocyst stage. Although the EVs have a range of proteins, the data support the hypothesis of interaction and effect of HSP from OF under the embryo. Some HSPs have a well-established role in the reproductive process, specifically on 210 spermatozoa. The HSPA8 was the most cited protein on publications included at this review, on data presented above describing proteins present on oviduct or interacting on gametes and others publication, that will be discussed next, that present their direct effect on spermatozoa. Lloyd et al (2009) showed through preincubation of spermatozoa and the soluble OEC apical plasma membrane from the ampullar region, that HSPA8 is an active component and supports 215 the survival of ram spermatozoa, enhancing its viability (Lloyd et al., 2009). The same results on sperm viability were assessed by different authors, in a varied way as on 17°C (Lloyd et al., 2012), on 39°C (Moein-Vaziri et al., 2014), or during cryopreservation (Holt e Fazeli, 2016). The HSPA8 protein also showed effects on polyspermy modulation, increasing the membrane fluidity and enhancing the IVF efficiency and embryo rate (Elliott et al., 2009; Lloyd et al., 220 2012; Moein-Vaziri et al., 2014; Holt e Fazeli, 2016; Braganca et al., 2021). These studies demonstrate the presence of HSPA8 on OF is not only an effect for stress as were believed, but there are reasons to oviduct cells released these HSPs. The HSPA5 proteins were the second most cited protein on publications eligible to our work, are present at all oviduct segments, and seem to be related to all stages of reproductive 225 29 processes. As cited above this protein interact with spermatozoa and was found increased at the estrous phase (Boilard et al., 2004; Lin et al., 2012; Fontes et al., 2019). Lachance et al., (2007) did not find an increase in sperm viability after sperm were incubated with the protein but demonstrated that exogenous HSPA5 alters the spermatic physiology in the process of acquisition of fertilizing ability, as increased intracellular calcium concentration (Lachance et 230 al., 2007). In the same way, Marin-Briggiler (2010) found similar results and demonstrate that the protein seems to facilitate sperm binding to zona pellucida, influencing fertilization (Marín- Briggiler, 2010). The other two publications reached the effects of specific HSPs proteins on fertilization. Garcia-Martinez (2020) assessed the effect of HSP90 and HSPA1A on ZP digestion (Garcia-235 Martinez et al., 2020). Both proteins increased the time of ZP digestion but only HSPA1A increased the monospermy rate. On the other hand, none of them enhance the IVF efficiency. The exogenous HSP60 protein was not able to enhance the fertilization ability of spermatozoa but seems to elevate the two-cell embryo rate in mice (Abdi et al., 2019). In a general way, the results found here demonstrate that HSPs play an active and 240 fundamental role in the reproductive process, and these proteins seem to influence some specific aspects of spermatozoa capacitation and in the fertilization that could directly impact embryo development. In addition, the use of exogenous HSPs on in vitro embryo production can enhance the IVF efficiency and embryo rate but there is a lot to clarify about their mechanism of action of them. 245 CONCLUSION Fertilization involves a range of processes on gametes and the oviduct should be able to receive them and provide the required substrate as carbohydrates, ions, enzymes, and proteins. 250 The HSPs are expressed on OEC and present on OF and their concentration fluctuates into the 30 estrous cycle. 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A. The common and species-specific roles of oviductal proteins in mammalian fertilization and embryo development. Oxford University Press, 2015. YESTE, M. et al. Viable and morphologically normal boar spermatozoa alter the expression of heat- shock protein genes in oviductal epithelial cells during co-culture in vitro. Mol Reprod Dev, v. 81, n. 9, 425 p. 805-19, Sep 2014. ISSN 1098-2795 (Electronic) 1040-452X (Linking). Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/24945888 >. http://www.ncbi.nlm.nih.gov/pubmed/10101046 http://www.ncbi.nlm.nih.gov/pubmed/10840824 http://www.ncbi.nlm.nih.gov/pubmed/24945888 35 CAPÍTULO II 36 EVIDENCE THAT HEAT SHOCK PROTEIN A5 (HSPA5) PLAYS A ROLE DURING BOVINE IN VITRO EMBRYO PRODUCTION Priscila Helena dos Santos1, Fernanda Fagali Franchi1, Sarah Gomes Nunes1, Patrícia Kubo Fontes1, Ana Elisa Valencise Quaglio1, Anthony César de Souza Castilho2. 1. Botucatu São Paulo State University (Unesp), Institute of Bioscience, Botucatu, São Paulo, Brazil. 5 2. University of Western São Paulo (Unoeste), Presidente Prudente, São Paulo, Brazil. Corresponding author: Anthony César de Souza Castilho, Universidade do Oeste Paulista (Unoeste), Pró- Reitoria de Pesquisa e Pós-Graduação – Campus II Rodovia Raposo Tavares, km 572 – Bairro Limoeiro, 19067-10 175, Presidente Prudente, São Paulo, Brasil. Email: castilho.anthony@gmail.com Grants: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and by São Paulo Research Foundation (FAPESP; grant numbers: 2018/06674-7; 2013/11480-3; 2015/04505-5. 15 ABSTRACT Proteins derived from oviductal fluid (OF) have shown an essential role in reproductive physiology. Some studies demonstrate that heat shock proteins as HSPA5 are secreted on OF and participate in zona pellucida (ZP)-sperm interaction. Hypothesizing that HSPA5 can 20 interact with the oocyte, we assessed its effect during the last four hours of bovine in vitro complex cumulus-oocyte maturation and subsequence embryo yield/quality. The COCs were aspirated from cow ovaries obtained in a slaughterhouse (n= 20 COC/group) and matured for 20 hours in base medium (BM). After that, the medium was part exchanged to BM (control) or BM + recombinant HSPA5 (100 ng/ml) and continued in vitro maturation to complete 24 hours. 25 In vitro matured COCs were mechanically separated from cumulus cells or followed to the in vitro fertilization (IVF) and in vitro culture (IVC) until 8 days to obtain expanded blastocyst and its rate. To gain insight into the presumptive role of HSPA5, we investigated reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) from oocytes and mailto:castilho.anthony@gmail.com 37 expanded blastocysts, the GSH content from COCs and IVM medium, the oocyte penetration, 30 the zona pellucida solubility, the blastocyst rate, and its gene expression analysis. The rHSPA5 modulated mitochondrial activities from oocytes (p= 0.0006) and increased blastocyst yield (p= 0.04). Besides that, in blastocysts derived from oocytes matured with rHSPA5, we figure out an upregulation of 19 genes involved in embryo quality, DNA methylation, cell growth, cellular development, and oxidative stress. Taken all data together, we infer that rHSPA5 during 35 the final time of oocyte in vitro maturation, even though it did not change monospermic fertilization and oocyte oxidative stress, plays a role in oocyte mitochondrial function, improves blastocyst development, and modulates low-scale embryonic transcriptional profile. Keywords: GRP78, heat shock protein, in vitro maturation, bovine, embryo production 40 INTRODUCTION During the ovulation, the complex cumulus-oocyte (COC) is released from the ovary and attached by cilia of the infundibulum, an initial portion of the oviduct (P. Coy, Garcia-45 Vazquez, Visconti, & Aviles, 2012; Kolle et al., 2009; Li & Winuthayanon, 2017). The oviduct is a tubal-like structure that connects the ovary to the uterus. It is divided into three main regions, the infundibulum, the ampulla, and the isthmus (ordered from the ovary to uterus), where each region differs in morphology and fluid composition according to function during the gamete and embryo transport (Li & Winuthayanon, 2017). The oviducts play a crucial role 50 in ensuring the gametes' transport, fertilization, development, and transport of early embryos. Once in the oviduct, the ovulated COC directly contacts oviductal epithelial cell (OEC) and oviductal fluid (OF). The OF is a complex mixture of components produced by secretions from OEC and plasm transudate (M. G.-A. Avilés, A; Coy, Pilar, 2010; Mondejar, Martinez-Martinez, Aviles, 55 38 & Coy, 2013). In contact of gametes, some contents of OF can be modified to support all gametes necessities and, besides that, the regions tend to have a different pattern of secretion (Alminana et al., 2017; M. G.-A. Avilés, A; Coy, Pilar, 2010; Buhi, Ashworth, Bazer, & Alvarez, 1992). The ampulla is the region that fertilization takes place. The passage of bovine COC through the oviduct was estimated at 72 hours, but the arrival to ampulla seems to last 60 only a few minutes (Croxatto, 2002). In the ampulla, the COC is firmly attached to OEC for a variable time (Kolle et al., 2009). The ZP becomes more accessible to the OF during this process, permitting its modification by different molecules, including proteins (P. Coy et al., 2012). Among a range of proteins in OF, there is heat shock protein A5 (HSPA5). The HSPA5, 65 also known as glucose-regulated protein (GRP78), is a member of the HSP70 family of proteins and share partial amino acid sequence identity with the 70-kDa heat shock protein (HSP70) that is also present in the oviduct (C. Zhang, 2017). The HSPA5 is a well-characterized chaperone from endoplasmic reticulum (ER) that play an essential role in maintaining cellular homeostasis (Gonzalez-Gronow, Selim, Papalas, & Pizzo, 2009). The protein plays a crucial role in the 70 unfolded protein response (UPR), which is activated when proteins are misfolded and accumulate in the ER (Hebert-Schuster, Rotta, Kirkpatrick, Guibourdenche, & Cohen, 2018). However, as a member of the HSP family, the HSPA5 performs a diverse array of functions in multiple cellular compartments (Hebert-Schuster et al., 2018), including on cell surface (Gonzalez-Gronow et al., 2009). 75 Studies have shown that HSPA5 is present on the cell surface where it acts as a receptor- like function and regulates cell proliferation and survival (Gonzalez-Gronow et al., 2009; Sato, Yao, Arap, & Pasqualini, 2010). Besides that, (Boilard et al., 2004; Lachance, Bailey, & Leclerc, 2007; Lin et al., 2012; Marín-Briggiler, 2010) have shown that HSPA5 is located at OEC, is secreted on OF (Boilard et al., 2004; Lamy et al., 2016; Marín-Briggiler, 2010) 80 39 promoting sperm capacitation and sperm-zona pellucida (ZP) binding (Bromfield & Nixon, 2013; Mondejar et al., 2013). Moreover, the presence of HSPA5 protein in the oolemma from mature mouse oocytes was described by (Calvert, Digilio, Herr, & Coonrod, 2003), but how the protein can modulate the final maturation of COC is until unknown. Considering that the studies about secreted HSPA5 on OF demonstrate their role on 85 sperm membrane and that COCs co-cultured with ampullary cells is beneficial to the oocyte (Azari, Kafi, Asaadi, Pakniat, & Abouhamzeh, 2021), as well as the subsequence embryo quality (Kidson et al., 2003), the influence of oviductal proteins on oocyte should be investigated. Based on that, to gain insight into the effect of HSPA5 on the final maturation of oocytes, we add the protein on the last 4 hours of in vitro maturation (IVM), and we 90 hypothesized that protein affects bovine oocyte maturation and influences embryo production and blastocyst quality. Specifically, we aimed to investigate the impact of this heat shock protein on oocyte maturation, oxidative stress, polyspermy control, mitochondrial functionality, and embryonic gene expression. 95 RESULTS Effect of HSPA-5 protein on COCs IVM on ROS and mitochondrial activities oocyte and embryo and GSH content determination from COCs and IVM medium. The HSPA5 is a protein-related to maintaining cellular homeostasis under stress 100 conditions. To gain insight into the effect of rHSPA5 protein on COC stress during IVM, we assessed ROS and MMP levels on oocyte and GSH content from culture medium and COC. The ROS and MMP were also verified from expanded blastocysts to assess the effect of rHSPA5 on the stress of subsequence embryos. The presence of protein for the last 4 hours of maturation decreased the MMP levels in oocytes (p = 0.0006, Figure 2A). Nevertheless, HSPA-105 40 5 did not affect ROS levels on treated oocytes (p= 0.539, Figure 2A), even changing the GSH contents on COCs and IVM medium (Table 1). The rHSPA5 also did not modulate the ROS and MMP from subsequence embryos (p= 0.88 and p= 0.98, respectively) as demonstrated on Figure 2B. 110 A B Figure 2. Effects of addiction of HSPA-5 protein on the last 4 hours of bovine COCs IVM on A) MMP from oocytes (n= 92-109 oocytes/group; n= 5 replicates; p= 0.0006) detected using MitoTracker® red CMXRox dye; and ROS levels from oocytes (n= 94-111 oocytes/group; 5 replicates; p= 0.539) detected using CellRox™ Green 115 dye. B) MMP from embryos (n= 36 embryos/group; n= 5 replicates; p= 0.9880) detected using MitoTracker® red CMXRox dye; and ROS levels from embryos (n= 36 embryos/group; 5 replicates; p= 0.8839) detected using CellRox™ Green dye. Data are means ± SEM of pixel intensity, and differences were considered significant when p ≤ 0.05. Different letters indicate statistical difference. 120 41 TABLE 1. Total GSH content (mean ± SEM) in COCs and IVM medium from COC maturation treated with HSPA-5 on the last 4 hours or not. 125 Cumulus cells pool: cumulus cells from 20 COCs; Oocytes pool: 20 oocytes. Effect of HSPA-5 during the last 4 hours of COCs IVM on zona pellucida solubility and oocyte 130 penetration The oviduct contains a range of proteins, but some have a lacking or an unknown role. Studies have been demonstrated that the HSPA5 can bind to the sperm membrane and possible is related to sperm-ZP interaction. So, to figure out the effect of protein on ZP and fertilization, we tested ZP solubility and oocyte penetration. The presence of HSPA-5 protein on COCs IVM 135 did not modulate the time of digestion of zona pellucida on treated oocytes (259.58 sec ± 12.29) compared to control (276.09 sec ± 11.16; p= 0.4093; Figure 3). Conversely, the protein did not alter the standard of sperm block on zona pellucida, once the means of polyspermy zygote (p= 0.9669) was not different between the group and the monospermy zygote (p= 0.9692). 140 TOTAL GSH CONTENT Group IVM medium (pMol/ml) Cumulus cells (pMol/pool) Oocytes (pMol/pool) Control 0.069 ± 0.02 0.87 ± 0.20 0.11 ± 0.01 rHSPA5 0.039 ± 0.0092 0.87 ± 0.03 0.08 ± 0.02 p value 0.37 0.33 0.85 42 Figure 3. Effects of addiction of HSPA-5 protein on the last 4 hours of bovine COCs IVM on zona pellucida digestion time (n=16-20 oocytes/group; n= 4 replicates); monospermy and polyspermy rate (n=15-21 zygotes/group; n=4 replicates). Digestion of zona pellucida by pronase was continued observed and counted the time (in seconds). Data are means ± SEM, and differences were considered significant when p ≤ 0.05. 145 Effect of HSPA-5 on IVM on embryo production and transcript profile of embryo produced An adequate COC maturation is necessary to obtain a competent oocyte to be fertilized and a subsequence development of an embryo of good quality. The rHSPA5 in the last 4 hours 150 of IVM was able to increase the blastocysts yield (p= 0.04; Figure 4). Regarding the transcript profile of embryos derived from the treated oocyte, 19 genes were up-regulated on the embryo, as demonstrated in Table 2. 155 160 165 Figure 4. Effects of addiction of HSPA-5 protein on the last 4 hours of bovine COCs IVM on subsequence embryo development. Percentage of blastocysts produced at 8 days of culture (7 replicates). Data are means ± SEM. Differences were considered significant when p ≤ 0.05. 170 43 TABLE 2. Relative mRNA abundance of differently expressed genes on blastocyst (p≤ 0.05). Target genes were normalized with the reference genes (HMBS, PPIA, and RPL30) geometric means using the 2(-ΔCt) method. Data are means ± SEM of four biological replicates. 175 Gene symbol Control Treated p- Value Function Gene ID ATP5L 2.30±0.08 2.56±0.10 0.04 Transmembrane transport Bt03210836_g1 IGFBP2 1.97±0.12 2.79±0.28 0.02 Cell growth regulation Bt01040719_m1 IMPDH1 0.04±0.004 0.06±0.005 0.04 Cell growth regulation Bt00995384_m1 IMPDH2 0.23±0.01 0.31±0.03 0.03 Cell growth regulation Bt03226238_g1 MAPK1 0.18±0.01 0.25±0.01 0.01 Cell differentiation Bt03216718_g1 HSP90AA1 4.46±0.23 5.15±0.09 0.002 Cellular survive Bt0.218068_g1 NANOG 0.04±0.01 0.10±0.01 0.006 Pluripotency Bt03220541_m1 ATF4 0.29±0.06 0.47±0.02 0.02 ER stress Bt03221057_m1 GFPT2 0.04±0.001 0.05±0.005 0.04 Cellular Stress Bt03223996_m1 PRDX3 0.10±0.01 0.17±0.021 0.01 Oxidative Stress Bt03214402_m1 XBP1 0.32±0.05 0.45±0.01 0.04 Oxidative Stress Bt03227621_g1 SLC2A3 0.82±0.11 1.16±0.14 0.05 Energetic metabolism Bt03259514_g1 SLC2A1 1.66±0.28 2.32±0.15 0.05 Energetic Metabolism Bt03215314_m1 SREBF2 0.55±0.06 0.81±0.09 0.03 Lipid metabolism Bt04283467_m1 DNMT3A 0.63±0.09 0.87±0.06 0.04 Active DNA methylation Bt01027164_m1 REST 0.37±0.04 0.61±0.08 0.02 Gene expression control Bt03278318_s1 GLRX2 0.31±0.02 0.39±0.01 0.03 Oxidative Stress Bt03250351_m1 HPRT1 0.26±0.02 0.37±0.04 0.04 Others Bt03225311_g1 S100A10 0.72±0.10 1.12±0.11 0.02 Others Bt03215645_m1 DISCUSSION 180 The HSPA-5 protein is secreted on OF and has been demonstrated an interaction and develops an essential role in capacitation and activation of sperm. In our study, for the first time, we showed evidence that this oviductal secreted protein seems to interact with bovine COC. The rHSPA5 protein in the last 4 hours of COCs IVM medium, in general, increased blastocyst yield and could improve blastocyst quality modulating target genes involved with 185 keys cellular pathways. 44 The HSPA5 protein is an ER chaperone well-known for the unfolded proteins response (UPR). First, we need to clarify that cells can transcript specific genes during stress conditions to maintain homeostasis. In normal conditions, the HSPA5 from ER is binding to an intraluminal portion of IRE1 (endoplasmic reticulum to nucleus signaling-1), PERK (protein 190 kinase RNA-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6) that are membrane receptors from ER. Under stress conditions, the HSPA5 dissociates from receptors to bind to the unfolded proteins. The IRE1, PERK, and ATF6 trigger UPR vias to maintain the cell or reach apoptosis (J. Y. Zhang, Diao, Kim, & Jin, 2012; X. Zhang, Zhang, Qi, Huang, & Zhang, 2016). Besides the HSPA5 is best known as an ER luminal protein, the 195 HSPA5 is also found on cytoplasm, mitochondria, the nucleus, plasma membrane, and secreted (Casas, 2017; Delpino & Castelli, 2002; Gonzalez-Gronow et al., 2009), including on OF (Marín-Briggiler, 2010). Then, why did we test HSPA5 protein during oocyte IVM? First of all, during in vitro embryo production, COCs are exposed to a range of stressors, resulting in a high level of ROS 200 (Goto, Noda, Mori, & Nakano, 1993; Nasr-Esfahani, Aitken, & Johnson, 1990). Although the HSPA5 is responsible for maintaining the cellular homeostasis on stress (Basar et al., 2014; Latham, 2016), in the present study, we demonstrated that HSPA-5 neither influences the total GSH content of ROS concentration but modulate MMP levels. Based on these findings, we could propose, as previously described (Dumollard, Carroll, Duchen, Campbell, & Swann, 205 2009; Quinlan, Perevoshchikova, Hey-Mogensen, Orr, & Brand, 2013), that basal ROS production, on control or HSPA-5 groups can act as a natural regulatory mechanism for the cell, involved in general housekeeping and regulation of redox state, which no cause excess of ROS production, no increased oxidative stress ROS production must be kept to a minimum. On the other hand, oocytes treated with HSPA-5 showed lower levels of MMP but did not influence 210 oocyte competence to be fertilized and promote embryo development. Indeed, we know that 45 mitochondria have diverse activities in the regulation of cellular homeostasis, including ATP production, calcium control homeostasis, redox regulation, etc. (Demant, Trapphoff, Frohlich, Arnold, & Eichenlaub-Ritter, 2012) and that ROS induces mitochondrial damages, resulting in low values of MMP (Hao et al., 2011; Loor et al., 2010). So, how to explain lower values of 215 MMP and no negative impact of oocyte competence to reach more blastocysts? First, we need to reinforce that there is may also discrepancies in results due to variations in MMP measurement techniques. Further, it is interesting that we showed no ROS production changes despite an apparent decrease in mitochondrial activity, as usually, a reduced MMP coincides with lowered ROS generation as described (Korshunov, Skulachev, & Starkov, 1997). 220 Some studies have evaluated the role of HPA5 protein on OF. An important finding regarding fertilization controlling was that HSPA5 protein did not influence ZP hardening and not changes rates of polyspermy. It is known that the hardening of ZP is directly related to monospermy fertilization (P. Coy et al., 2008). Studies described a difference between oocytes from the ovary compared to that transit through the oviduct, and that OF influence in ZP 225 bindings and develop the role on ZP in the control of polyspermy (P. Coy et al., 2012). Together, these results caused us an astonishment once HSPA5 protein was described to be on the surface of mature mouse oolema (Calvert et al., 2003) and seems to be involved in preparing oocytes for fusion with spermatozoa (Bromfield & Nixon, 2013). In humans, (Marín-Briggiler, 2010) and colleagues demonstrated that the protein is secreted by OEC. It is associated with sperm-230 ZP binding modulation and proposed that HSPA5 bind to the gametes modulates their interaction in a calcium-dependent manner. Moreover, (Mondejar et al., 2013) suggested that this binding could modify the ZP chemical properties such as resistance to enzymatic hardening. Even though our findings did not corroborate with previous studies, we need to highlight that these works always used the HSPA-5 during fertilization or previously on spermatozoa, which 235 could modify interactions and modulation of ZP hardening during oocyte IVM. 46 As know, oviduct takes place the gametes meeting to fertilization. According to (Croxatto, 2002), the COC transport through the oviduct can spend approximately 72 hours in cows. During this time, COCs maintain contact with the OF and its proteins. The OF has been shown a fundamental role in the reproduction process because the presence of OEC during IVM 240 improves the quality of cytoplasmic maturation of the oocyte and subsequence quality of embryos produced, probably because of their secretions (Azari et al., 2021; Kidson et al., 2003). Indeed, here we found that HSPA5 protein could increase the blastocyst yield and further alter the transcriptional pattern of embryos. Also, we would like to highlight that all genes modulated by HSPA5 was up-regulated in the blastocyst, e.g., genes related to embryo quality as ATP5L, 245 IGFBP2, IMPDH1, IMPDH2, MAPK, HSP90AA1 and, NANOG; genes related to DNA methylation (DNMT3B), glucose transporter (SLC2A3 and SLC2A1), gene expression control (REST) lipid metabolism (SREBF2), RE stress (ATF4, XBP1) oxidative stress (PDRX3) and others functional genes (GLRX2 and HPRT1). Although in the present study we demonstrated that HSPA5 did not affect some 250 parameters of oxidative stress on oocytes, we verify that HSPA5 protein up-regulated as Activating transcription factor-4 (ATF4), Glutaredoxin-2 (GLRX2), Peroxiredoxin-3 (PRDX3), Glutamine-fructose-6-phosphate transaminase-2 (GFPT2), and X-box binding protein-1 (XBP1). Based on that, we believe that upregulation of ATF4 and XBP1 by HSPA5 could indicate a possible protective mechanism to prevent ER stress in the embryos derived from 255 oocytes treated with HSPA5 once we reached a higher yield of embryo produced in this group. To summarizing, even though we did not find any phenotypical evidence of HSPA5 on oxidative stress (ROS levels, GSH), indeed, proteins from OF protect gametes from environmental stress to ensure embryo quality and future pregnancy outcome and in our case, these internal anti-stress activities are being performed throughout mechanism controlling 260 transcription of target genes as mentioned above. 47 The other genes up-regulated by treatment have been found in embryos as a marker of quality. The up-regulation of the DNA methyltransferase 3B enzyme could results in well- driven embryo development and implantation rate (Gujar, Weisenberger, & Liang, 2019) once DNA methylation is essential in the embryonic stem cells and post-implantation embryo 265 development periods and early embryos. In the same way, the NANOG enzyme is also essential to embryo development. It is a crucial regulator of pluripotency during early development (Chambers et al., 2003; Karantzali et al., 2011), leaving us to think this upregulation caused by HSPA5 could be critical for normal embryonic development (Carey et al., 2015). Regarding other genes related to multiple pathways to guarantee a better embryo 270 development, we could summarize and suggest that blastocysts derived from oocytes treated with HSPA5 could demonstrate a positive transcriptional panel which would lead to preimplantation embryo development, up-regulating genes as SLC2A1 and SLC2A3 (Solute carrier family 2, facilitated glucose transporter member 1 and 3) to energy support (Kramer et al., 2020), IMPDH1 and IMPDH 2 (monophosphate dehydrogenase 1 and 2) to embryo 275 development and cell growth (Lake, Avetisyan, Zimmermann, & Heuckeroth, 2016), IGFBP2 (Insulin-like growth factor-binding protein-2) to cell proliferation and differentiation (Heyner, Shi, Garside, & Smith, 1993; Yang, Brown, Robcis, Rechler, & de Pablo, 1993), SREBF2 (Sterol regulatory element-binding transcription factor 2) to embryo development (Vergnes et al., 2016). 280 As HSPA5, some other oviductal proteins have been studied to identify their role in fertilization and embryo development (P. Y. Coy, R., 2017). However, there is a lack of these proteins' importance on final oocyte maturation. The co-culture with OEC was demonstrated to be beneficial to oocyte resulting embryos with good quality (Kidson et al., 2003) and indicate that proteins participate in the process of final maturation and preparation to fertilization and 285 embryo development (possible a molecular modulation). In all, our study demonstrated that 48 the rHSPA5 decreased the MMP levels and possibly modulate the final oocyte maturation in a good perspective once it was able to increase the in vitro embryo rate and up-regulated transcripts of genes related to embryo quality. Taken all data together, we conclude that the HSPA5 protein modulates the 290 mitochondrial functionality, possible participation of final oocyte maturation and that rHSPA- 5 on in vitro maturation modulates the final oocyte maturation once it increases the blastocyst rate and the quality of the embryo (Figure 6). Figure 5. Effects of rHSPA-5 on the last 4 hours of bovine COCs IVM on final maturation and subsequence 295 embryo development. The rHSPA5 decreased the MMP levels on oocytes and resulted in a higher blastocyst rate and an up-regulation of genes markers of blastocyst quality. rHSPA5, recombinant HSPA5; COC, cumulus- oocytes complex; IVM, in vitro maturation. 300 MATERIALS AND METHODS Experimental design This study was designed to reach how the oviductal protein HSPA5 can interact with bovine oocyte and its influence on subsequence initial embryo development on in vitro embryo 305 production. The final maturation of oocyte occurs on the oviduct (C. Avilés, and Rizos, 2015; M. G.-A. Avilés, A; Coy, Pilar, 2010; P. Coy et al., 2012), but the precise time that the oocyte 49 can be firmly attached to the OEC is not defined. The passage of bovine COC through the oviduct is about 72 hours (Croxatto, 2002) and, considering that in 20 hours of in vitro maturation, 89% of COC achieve the MII stage (Khatir, Lonergan, & Mermillod, 1998), we 310 proposed to include the HSPA5 protein at this moment. The protein concentration was previously tested for our group (data not published), comparing the 100 ng/ml with 1 ng/ml concentrations and no addition of rHSPA5. The concentration of 100 ng/ml was more effective on embryo production than another one and was not detrimental to the oocyte maturation. 315 Figure 1. Effects of addiction of HSPA-5 protein on the last 4 hours of bovine COCs IVM. The immature COCs (20 COCs/group) were selected and randomly distributed among the groups: control - basic maturation medium (BM); and treated - BM + human rHSPA5 (100 ng/ml). In the first 20 hours of maturation, COCs from both groups 320 were matured in the BM medium. After that, the medium was partially exchanged, and the rHSPA5 protein was added to the specific group. The IVM maturation continued to complete 24 hours. Matured COCs were separated to cumulus cells mechanically or followed to the in vitro fertilization (IVF) and in vitro culture (IVC) until 8 days to obtain expanded blastocyst. The IVM medium was collected to assess the total glutathione (GSH) content. 50 325 COC collection Ovaries from abattoir at Torrinha, São Paulo, Brazil (22º 25' 34" S; 48º 10' 09" W) were collected and transported to the laboratory in saline solution (0.9%) at 36 °C. Follicles in 2-8 mm were aspirated with an 18-gouged needle and the follicular fluid pooled in a conical tube to sedimentation. COCs with homogenous cytoplasm and compacted multilayers of cumulus 330 cells (grade 1 and 2; (Stojkovic et al., 2001) were recovered using a stereomicroscope and randomly divided into experimental groups. In vitro maturation All COCs selected were randomly divided into each experimental group. It was 335 transferred to a 4-well plate with 10 µL/COC of primary maturation medium (BM) containing TCM199 with bicarbonate and Earle's slats, follicular stimulating hormone (0.1 IU/ml solution, Gonal-f, Merck Serono, Bari, Italy), 22 µg/ml of sodium pyruvate, 75 µg/ml of amikacin and 4 mg/ml of fatty acid-free bovine serum albumin –BSA). The groups were cultured for 20 hours at 38.5 °C in humidified air containing 5.5% CO2. After that, both experimental groups had a 340 partially medium exchanged to add BM (control) or BM plus human recombinant HSPA5 (treated; 100 ng/ml; Abcam© plc, Cambridge, MA, USA) of the same volume, and continued the culture until complete 24 hours at identical conditions. The matured COC was propriety collected to subsequent experiments or continue to embryo development. 345 In vitro fertilization The matured COCs were washed in IVF medium (Progest, Botucatu, São Paulo, Brazil) and were placed in plates with 90 µl of IVF medium drops (in a proportion of 25 COCs/drops) and covered with silicone oil (Quimesp Química, Guarulhos, São Paulo, Brazil). 51 Cryopreserved semen samples from different ejaculates of same Nelore bull (CRV 350 Lagoa, Sertãozinho, São Paulo, Brazil) were used in the whole experiment. The sample of semen was thawed at 37 °C for the 30s, enriched for motility using density-gradient centrifugation (BotuFIV Select SPERM – Botupharma, Botucatu, São Paulo, Brazil) at 3,000 rpm for 5 min. After that, the pellet was recovered and extended with the fertilization medium to a final concentration of 1 × 106 spermatozoa ml-1. Co-incubation of oocytes and spermatozoa 355 was performed for 18 h at 38 °C under 5% CO2 in humidified air. In vitro culture After the insemination, the presumptive zygotes were denuded by vortexing, washed, and transferred to four-well plates containing 500 µl of IVC medium (SOF medium, 0.2 mM 360 of sodium pyruvate, 5 mg/ml of BSA, and 2.5% (v/v) of fetal bovine serum). The cultured occurred at 38.5 °C in 5.5% CO2 and low oxygen concentration (5% O2) for 8 days. Embryos in expanded blastocyst stages were appropriately collected for analyses on day seven and day 8 of incubation. 365 Nuclear progression, mitochondrial activity, and intracellular ROS in oocytes and embryos A combined fluorescence staining technique using Hoechst 33342, MitoTracker® Red CMXRos, and CellROX™ Green Reagent (Invitrogen, São Paulo, Brazil) was used to estimate nuclear progression, indirect mitochondrial inner membrane potential (MMP), and oxidative stress (ROS), respectively, in the oocytes. For the embryos, blastocysts were used on the 370 expanded stage, and only MMP and ROS were analyzed. Matured oocytes were denuded by vortex and washed in Phosphate-Buffered Saline pH 7.2 with 1 mg/ml polyvinylpyrrolidone (PBS-PVP). Denuded oocytes were incubated in drops with Hoechst (1µg/ml), CellROX Green (5µM), and MitoTracker Red (0.5µM), while embryos 52 were incubated in drops with only CellROX and MitoTracker, for 30 minutes at 5.5% CO2 and 375 38.5 °C. Then, both samples were washed thrice in PBS-PVP and fixed in 4% paraformaldehyde for 15 min. After they were washed three times in PBS-PVP again and placed in glycerol micro drops on a blade covered by a coverslip and analyzed using Leica DM4500 epifluorescence microscope to detect DNA nuclear progression, ROS, and MMP. Images were acquired by LAS software using a camera attached to the microscope. The quantification of 380 average pixel intensity was performed using Image J (NIH, MD, USA) software for ROS and MMP analysis. Zona Pellucida solubillity The zona pellucida (ZP) hardening was estimated according to (P. Coy et al., 2008). 385 Matured COCs (n= 20 oocytes per group in 4 replicated) were denuded in vortex (13,000 rpm for 1 min), washed in PBS-PVP. To assess the hardening, the oocytes were added in 25 µl of pronase (0.5% v/v in PBS; Sigma-Aldrich Co., St Louis, MO), and the ZP was continuously observed using a stereomicroscope and the time digestion was clocked and registered as the time between the placement of samples in pronase solution and time at which the ZP was no 390 longer visible. Detection of oocyte penetration: polyspermatic assay The polyspermy was assessed as demonstrated by (Ferraz et al., 2017). Semen samples from the same Nelore bull previously described were thawed at 37 °C for 30 seconds and 395 washed in a discontinuous gradient of BotuFIV Select SPERM (Botupharma, Botucatu-SP) at 3,000 rpm for 5 min. The spermatozoa pellet was recovered and was added 350 µl of MitoTracker™ Green FM solution (Invitrogen, São Paulo, Brazil) – 200 nM in IVF medium, and incubated for 30 min at 37 °C. The mitotracker stained spermatozoa were washed three 53 times in IVF medium at 700 × g for 5 min. The spermatozoa pellet was resuspended in an IVF 400 medium and used for in vitro fertilization in the same conditions previously described. After 18 hours of in vitro fertilization with mitotracker stained spermatozoa, presumptive zygotes were fixed in 4% paraformaldehyde for 15 min, washed three times in PBS-PVP, then incubated with Hoechst (5µg/ml) for 30 min. Later, the presumptive zygotes were washed three times in PBS-PVP again and then mounted into glycerol micro drops in a 405 blade covered by a coverslip and analyzed using laser scanning confocal microscopy (Leica TCS SP8) attached to an inverted microscope with a 40× using LAS X software. Polyspermy was identified by the detection of 2 or more sperm mid-pieces into the ooplasm. GSH content determination 410 Total glutathione (GSH) content was assessed according to (Anderson, 1985). Denuded oocytes (n= 20 oocytes per pool in 5 replicates), their cumulus cells, and were kept in trichloroacetic acid (TCA) at -80 °C. The MIV medium used in the cultured (20 µl) was also collected and stored at -80 °C, and TCA was added immediately before the GSH assay. The samples were mixed in vortex (except medium samples) for 20 sec at 12,000 rpm. Standards 415 containing 0 to 200 pmol of GSH in 50 ml were prepared in water, simultaneously with the samples, and both were kept on ice until be allocated in a microtiter plate. Each sample and standard was added in a volume of 20 µl in a 96-well microtiter plate with 0.1 ml of reaction mixture newly-prepared (6 mM of DTNB, 0.2 mM of NADPH, and 1.0 IU of GSH reductase/ml (final concentrations) in 0.1M phosphate buffer supplemented with 1 mM of EDTA, pH 7.8). 420 Immediately the plate was analyzed at 405 nm (SpectraCount, Packard, Meriden, CT, USA), first only with the mixing reaction, and then repeated-reads functions at 2 min intervals for 30 min. 54 Transcript profile of blastocysts 425 Embryos that reach the expanded blastocyst stage at day 7 of development were collected (n= 5 blastocysts per pool in 4 replicates) and stored at -80 °C until the RNA extraction. Total RNA from samples was extracted using PicoPure™ RNA Isolation Kit (Applied Biosystem®, Foster City, CA, USA) according to the manufacturer’s protocol. After the purification, the RNA samples were eluted in 12μL of RNAse free water, and all volume of 430 sample was used in the analyze. The total RNA was incubated with DNAse I (1IU/µg; Invitrogen, São Paulo, Brazil) and then reverse-transcribed using random primers according to the protocol provided with the High Capacity kit (Applied Biosystem®, Foster City, CA, USA). Then, the mRNA abundance of 96 genes was determined to analyze the transcripts 435 profile of blastocysts produced. All analyses were performed using Biomark HD assay (Fluidigm, South San Francisco, CA, USA) as previously described (Fontes, Castilho, Razza, & Nogueira, 2020; Franchi et al., 2019; Giroto et al., 2019). The relative expression values for each gene were calculated with the ΔCt method. Data were normalized using geometric means of the most stable reference genes for each cellular type (NormFinder software tested). The 440 mRNA abundance geometric mean of HMBS, PPIA, and RPL30 was used to normalize the blastocysts from the IVM treatment experiment (Fontes et al., 2020). 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