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Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

ABSTRACT: Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The oocytes recovered were divided into two groups. Group I comprised COCs (n = 312) and denuded oocytes (n = 296), which were experimentally infected with BHV1 and incubated for 1 h at 38.5°C and 5% CO2. Group II comprised COCs (n = 425) and denuded oocytes (n = 405), which were co-incubated with BHV1 under the same conditions for 24 h. The negative control of these two groups was respectively subjected to the same protocol, except for exposure to BHV1. To our knowledge, this study provides the first evidence of BHV1 detection within COCs and denuded oocytes exhibiting intact zona pellucida when co-incubated with the virus for 24 h. Immunolocalization also confirmed the presence of BHV1 in the cytoplasm of the cumulus cells of all COCs exposed to the virus after both incubation periods. In conclusion, detection of BHV1 inside oocytes has a great meaning for the field of animal reproduction. The detection of BHV1 in different layers of cumulus cells also demonstrates that these cells are sources of viral infection.

SUBMITTER: Queiroz-Castro VLD

PROVIDER: S-EPMC6638837 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

Queiroz-Castro Vanessa Lopes Dias VLD da Costa Eduardo Paulino EP Alves Saullo Vinicius Pereira SVP Machado-Neves Mariana M Guimarães José Domingos JD Gomes Lidiany Lopes LL Domingos Stella Vieira SV Ribeiro Caroline Gomides CG Caldas Rebeca Toledo RT Silva-Júnior Abelardo A

PloS one 20190718 7

Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The ...[more]

PMID: 31318892

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Project description:BackgroundAgar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP.MethodsHuman metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software.ResultsWe identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP.ConclusionsOur findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest.

Project description:The zona pellucida (ZP) surrounding the oocyte is an extracellular fibrillar matrix that plays critical roles during fertilization including species-specific gamete recognition and protection from polyspermy. The mouse ZP is composed of three proteins, ZP1, ZP2, and ZP3, all of which have a ZP polymerization domain that directs protein fibril formation and assembly into the three-dimensional ZP matrix. Egg coats surrounding oocytes in nonmammalian vertebrates and in invertebrates are also fibrillar matrices and are composed of ZP domain-containing proteins suggesting the basic structure and function of the ZP/egg coat is highly conserved. However, sequence similarity between ZP domains is low across species and thus the mechanism for the conservation of ZP/egg coat structure and its function is not known. Using approaches classically used to identify amyloid including conformation-dependent antibodies and dyes, X-ray diffraction, and negative stain electron microscopy, our studies suggest the mouse ZP is a functional amyloid. Amyloids are cross-β sheet fibrillar structures that, while typically associated with neurodegenerative and prion diseases in mammals, can also carry out functional roles in normal cells without resulting pathology. An analysis of the ZP domain from mouse ZP3 and ZP3 homologs from five additional taxa using the algorithm AmylPred 2 to identify amyloidogenic sites, revealed in all taxa a remarkable conservation of regions that were predicted to form amyloid. This included a conserved amyloidogenic region that localized to a stretch of hydrophobic amino acids previously shown in mouse ZP3 to be essential for fibril assembly. Similarly, a domain in the yeast protein α-agglutinin/Sag 1p, that possesses ZP domain-like features and which is essential for mating, also had sites that were predicted to be amyloidogenic including a hydrophobic stretch that appeared analogous to the critical site in mouse ZP3. Together, these studies suggest that amyloidogenesis may be a conserved mechanism for ZP structure and function across billions of years of evolution.

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Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

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Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

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