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Rodrigo Garcia Barros Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Veterinary Medicine and Animal Science, University of Milan, Milan, Italy

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Valentina Lodde Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Veterinary Medicine and Animal Science, University of Milan, Milan, Italy

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Federica Franciosi Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Veterinary Medicine and Animal Science, University of Milan, Milan, Italy

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Alberto Maria Luciano Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Veterinary Medicine and Animal Science, University of Milan, Milan, Italy

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In brief

The proposed culture system improves the current state of in vitro culture of growing oocytes in the bovine species and allows access to the untapped gamete reserve, thus improving reproductive efficiency.

Abstract

The present study aimed to improve the in vitro culture of bovine oocytes collected from early antral follicles (EAFs) to support the progressive acquisition of meiotic and developmental competence. The rationale that drove the development of such a culture system was to maintain as much as possible the physiological conditions that support the oocyte growth and differentiation in vivo. To this extent, oocytes were cultured for 5 days, which parallels the transition from early to medium antral follicles (MAFs) in the bovine, and supports promoting a 3D-like structure were provided. Additionally, the main hormones (follicle-stimulating hormone, estradiol, progesterone, and testosterone) were added in concentrations similar to the ones previously observed in bovine EAFs. The meiotic arrest was imposed using cilostamide. The cultured cumulus–oocyte complexes (COCs) reached a mean diameter of 113.4 ± 0.75 µm and showed a progressive condensation of the chromatin enclosed in the germinal vesicle (GV), together with a gradual decrease in the global transcriptional activity, measured by 5-ethynyl uridine incorporation. The described morpho-functional changes were accompanied by an increased ability to mature and develop to the blastocyst stage in vitro, although not matching the rates obtained by MAF-retrieved oocytes. The described system improves the current state of in vitro culture of growing oocytes in the bovine species, and it can be used to increase the number of gametes usable for in vitro embryo production in animals of high genetic merit or with specific desirable traits.

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Laura Terzaghi Department of Health, Animal Science and Food Safety, Reproductive and Developmental Biology Laboratory, University of Milan, Milan, Italy

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Alberto Maria Luciano Department of Health, Animal Science and Food Safety, Reproductive and Developmental Biology Laboratory, University of Milan, Milan, Italy

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Priscila C Dall’Acqua School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, Brazil

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Silvia C Modina Department of Health, Animal Science and Food Safety, Reproductive and Developmental Biology Laboratory, University of Milan, Milan, Italy

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John J Peluso Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut, USA

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Valentina Lodde Department of Health, Animal Science and Food Safety, Reproductive and Developmental Biology Laboratory, University of Milan, Milan, Italy

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Progesterone receptor membrane component-1 (PGRMC1) is a highly conserved multifunctional protein that is found in numerous systems, including reproductive system. Interestingly, PGRMC1 is expressed at several intracellular locations, including the nucleolus. The aim of this study is to investigate the functional relationship between PGRMC1 and nucleolus. Immunofluorescence experiments confirmed PGRMC1’s nucleolar localization in cultured bovine granulosa cells (bGC) and oocytes. Additional experiments conducted on bGC revealed that PGRMC1 co-localizes with nucleolin (NCL), a major nucleolar protein. Furthermore, small interfering RNA (RNAi)-mediated gene silencing experiments showed that when PGRMC1 expression was depleted, NCL translocated from the nucleolus to the nucleoplasm. Similarly, oxidative stress induced by hydrogen peroxide (H2O2) treatment, reduced PGRMC1 immunofluorescent signal in the nucleolus and increased NCL nucleoplasmic signal, when compared to non-treated cells. Although PGRMC1 influenced NCL localization, a direct interaction between these two proteins was not detected using in situ proximity ligation assay. This suggests the involvement of additional molecules in mediating the co-localization of PGRMC1 and nucleolin. Since nucleolin translocates into the nucleoplasm in response to various cellular stressors, PGRMC1’s ability to regulate its localization within the nucleolus is likely an important component of mechanism by which cells response to stress. This concept is consistent with PGRMC1’s well-described ability to promote ovarian cell survival and provides a rationale for future studies on PGRMC1, NCL and the molecular mechanism by which these two proteins protect against the adverse effect of cellular stressors, including oxidative stress.

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Isa Mohammed Alkali Department of Veterinary Medicine and Animal Sciences, University of Milan, Italy
Department of Theriogenology, University of Maiduguri, Maiduguri, Nigeria

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Martina Colombo Department of Veterinary Medicine and Animal Sciences, University of Milan, Italy

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Alberto Maria Luciano Department of Veterinary Medicine and Animal Sciences, University of Milan, Italy

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Wojciech Nizanski Department of Reproduction and Clinic for Farm Animals, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland

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Hiba Ali Hassan Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium

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Piotr Dziegiel Department of Histology and Embryology, Wroclaw Medical University, Wroclaw, Poland

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Gaia Cecilia Luvoni Department of Veterinary Medicine and Animal Sciences, University of Milan, Italy

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In brief

Ovarian tissue cryopreservation and culture provide an option for fertility preservation without tissue grafting, but need optimization. This study reveals that vitrified bovine ovarian tissue can be cultured on agarose gel and maintain follicle morphology, low activation, and low apoptosis.

Abstract

Ovarian tissue preservation is hitherto a promising fertility insurance option for precious animals. Ovarian tissue vitrification and culture combined approach would eliminate the need of transplanting ovarian tissue to obtain mature oocytes. We aimed at optimizing vitrification and in vitro culture conditions for improved bovine ovarian tissue viability. Ovaries obtained from the slaughterhouse were punched into fragments and divided into three groups. Group 1 (fresh) was divided into two and immediately placed in two-culture systems (culture inserts and agarose inserts). Group 2 was vitrified, warmed, and placed in the two-culture systems, while group 3 was only equilibrated and then placed in the two-culture systems. All cultures were maintained for 6 days and spent media were collected on alternate days for cytokine (interleukin 1β and interleukin 6) evaluation. Fragments were fixed for morphology assessment and immunohistochemistry. Higher percentages (P < 0.05) of grade 1 (morphologically intact) follicles were observed in fragments on agarose compared to those on culture inserts on days 2 and 4 of the culture. Conversely, we found higher (P < 0.05) shifts of primordial follicles to transitional follicles in fragments on culture inserts vis-à-vis agarose inserts which was consistent with a higher proportion of Ki-67 and MCM-7 and activated caspase-3-positive follicles. In conclusion, in vitro culture of bovine ovarian tissue on agarose inserts maintained follicle morphology, low follicle activation, and low apoptosis compared to culture inserts.

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Noemi Monferini Reproductive and Developmental Biology Laboratory (ReDBioLab), Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy

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Pritha Dey Reproductive and Developmental Biology Laboratory (ReDBioLab), Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy

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Ludovica Donadini Reproductive and Developmental Biology Laboratory (ReDBioLab), Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy

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Niki Katsakoglou Reproductive and Developmental Biology Laboratory (ReDBioLab), Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy

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Federica Franciosi Reproductive and Developmental Biology Laboratory (ReDBioLab), Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy

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Valentina Lodde Reproductive and Developmental Biology Laboratory (ReDBioLab), Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy

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Alberto Maria Luciano Reproductive and Developmental Biology Laboratory (ReDBioLab), Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy

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In brief

Preantral follicles constitute the largest follicle reserve in the mammalian ovary. This study assesses a mechanical isolation method to maximize the number of follicles retrieved from a defined cortex volume.

Abstract

Primordial, primary, and secondary follicles (collectively defined as preantral follicles) constitute the most abundant source of gametes inside the mammalian ovarian cortex. The massive isolation of preantral follicles and the refinement of stage-specific protocols for in vitro follicle growth would provide a powerful tool to boost the rescue and restoration of fertility in assisted reproduction interventions in human medicine, animal breeding, and vulnerable species preservation. Nevertheless, together with an efficient culture system, the most significant limitation to implementing in vitro follicle growth is the lack of an efficient method to isolate viable and homogeneous subpopulations of primordial, primary, and secondary follicles suitable for in vitro culture. Our study provides a strategy for high-yielding mechanical isolation of primordial, primary, and early secondary follicles from a limited portion of the ovarian cortex in the bovine animal model. In the first part of the study, we refined a mechanical isolation protocol of preantral follicles, adopting specific methodological strategies to separate viable and distinct subpopulations of primordial (oblate and prolate forms), primary, and early secondary follicles from 0.16 cm3 of the ovarian cortex. In the second part of the study, we tested the effectiveness of the isolation protocol, considering the individual’s age as a critical factor, bearing in mind the progressive decrease in the ovarian reserve that naturally accompanies the reproductive life span. Our study provides a way for designing quantitative and conservative fertility preservation approaches to preserve organ function and minimize the invasiveness of the interventions, also considering age-related differences.

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Valentina Lodde Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Health, Animal Science and Food Safety ‘Carlo Cantoni’, Università degli Studi di MIlano, Milan, Italy

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Rodrigo Garcia Barros Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Health, Animal Science and Food Safety ‘Carlo Cantoni’, Università degli Studi di MIlano, Milan, Italy

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Priscila Chediek Dall’Acqua Department of Preventive Medicine and Animal Reproduction, School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, Brazil
Laboratory of Reproductive Physiology, School of Veterinary Medicine, São Paulo State University (UNESP), Araçatuba, Brazil

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Cecilia Dieci Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Health, Animal Science and Food Safety ‘Carlo Cantoni’, Università degli Studi di MIlano, Milan, Italy

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Claude Robert Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI), Département des Sciences Animales, Université Laval, Québec, Canada

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Alexandre Bastien Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI), Département des Sciences Animales, Université Laval, Québec, Canada

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Marc-André Sirard Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI), Département des Sciences Animales, Université Laval, Québec, Canada

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Federica Franciosi Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Health, Animal Science and Food Safety ‘Carlo Cantoni’, Università degli Studi di MIlano, Milan, Italy

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Alberto Maria Luciano Reproductive and Developmental Biology Laboratory (Redbiolab), Department of Health, Animal Science and Food Safety ‘Carlo Cantoni’, Università degli Studi di MIlano, Milan, Italy

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In the last years, many studies focused on the understanding of the possible role of zinc in the control of mammalian oogenesis, mainly on oocyte maturation and fertilization. However, little is known about the role of zinc at earlier stages, when the growing oocyte is actively transcribing molecules that will regulate and sustain subsequent stages of oocyte and embryonic development. In this study, we used the bovine model to gain insights into the possible involvement of zinc in oocyte development. We first mined the EmbryoGENE transcriptomic dataset, which revealed that several zinc transporters and methallothionein are impacted by physiological conditions throughout the final phase of oocyte growth and differentiation. We then observed that zinc supplementation during in vitro culture of growing oocytes is beneficial to the acquisition of meiotic competence when subsequently subjected to standard in vitro maturation. Furthermore, we tested the hypothesis that zinc supplementation might support transcription in growing oocytes. This hypothesis was indirectly confirmed by the experimental evidence that the content of labile zinc in the oocyte decreases when a major drop in transcription occurs in vivo. Accordingly, we observed that zinc sequestration with a zinc chelator rapidly reduced global transcription in growing oocytes, which was reversed by zinc supplementation in the culture medium. Finally, zinc supplementation impacted the chromatin state by reducing the level of global DNA methylation, which is consistent with the increased transcription. In conclusion, our study suggests that altering zinc availability by culture-medium supplementation supports global transcription, ultimately enhancing meiotic competence.

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Elolo Karen Nenonene Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)
Réseau Québécois en Reproduction (RQR), Université Laval, Québec, Québec, Canada

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Mallorie Trottier-Lavoie Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)
Réseau Québécois en Reproduction (RQR), Université Laval, Québec, Québec, Canada

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Mathilde Marchais Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)
Réseau Québécois en Reproduction (RQR), Université Laval, Québec, Québec, Canada

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Alexandre Bastien Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)
Réseau Québécois en Reproduction (RQR), Université Laval, Québec, Québec, Canada

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Isabelle Gilbert Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)
Réseau Québécois en Reproduction (RQR), Université Laval, Québec, Québec, Canada

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Angus D Macaulay Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)

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Edouard W Khandjian Centre de recherche CERVO, Département de psychiatrie et de neurosciences, Faculté de médecine, Université Laval, Québec, Québec, Canada

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Alberto Maria Luciano Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Science, University of Milan, Milan, Italy

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Valentina Lodde Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Science, University of Milan, Milan, Italy

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Robert S Viger Département d’obstétrique, gynécologie et reproduction, Faculté de médecine, Université Laval, Québec, Québec, Canada
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)
Réseau Québécois en Reproduction (RQR), Université Laval, Québec, Québec, Canada

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Claude Robert Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI)
Réseau Québécois en Reproduction (RQR), Université Laval, Québec, Québec, Canada

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In brief

RNA granules travel through the cumulus cell network of transzonal projections which is associated with oocyte developmental competence, and RNA packaging involves RNA-binding proteins of the Fragile X protein family.

Abstract

The determinants of oocyte developmental competence have puzzled scientists for decades. It is known that follicular conditions can nurture the production of a high-quality oocyte, but the underlying mechanisms remain unknown. Somatic cumulus cells most proximal to the oocyte are known to have cellular extensions that reach across the zona pellucida and contact with the oocyte plasma membrane. Herein, it was found that transzonal projections (TZPs) network quality is associated with developmental competence. Knowing that ribonucleoparticles are abundant within TZPs, the distribution of RNA-binding proteins was studied. The Fragile X-related proteins (FXR1P and FXR2P) and two partnering protein families, namely cytoplasmic FMRP-interacting protein and nuclear FMRP-interacting protein, exhibited distinctive patterns consistent with roles in regulating mRNA packaging, transport, and translation. The expression of green fluorescent protein (GFP)–FMRP fusion protein in cumulus cells showed active granule formation and their transport and transfer through filipodia connecting with neighboring cells. Near the projections’ ends was found the cytoskeletal anchoring protein Filamin A and active protein synthesis sites. This study highlights key proteins involved in delivering mRNA to the oocyte. Thus, cumulus cells appear to indeed support the development of high-quality oocytes via the transzonal network.

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