Transcriptomic responses of cumulus granulosa cells to SARS-CoV-2 infection during controlled ovarian stimulation.

Cumulus granulosa cells (CGCs) play a crucial role in follicular development, but so far, no research has explored the impact of SARS-CoV-2 infection on ovarian function from the perspective of CGCs. In the present study, we compared the cycle outcomes between infected and uninfected female patients undergoing controlled ovarian stimulation, performed bulk RNA-sequencing of collected CGCs, and used bioinformatic methods to explore transcriptomic changes. The results showed that women with SARS-CoV-2 infection during stimulation had significantly lower number of oocytes retrieved and follicle-oocyte index, while subsequent fertilization and embryo development were similar. CGCs were not directly infected by SARS-CoV-2, but exhibited dramatic differences in gene expression (156 up-regulated and 65 down-regulated). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated a high enrichment in antiviral, immune and inflammatory responses with necroptosis. In addition, the pathways related to telomere organization and double strand break repair were significantly affected by infection in gene set enrichment analysis. Further weighted gene co-expression network analysis identified a key module associated with ovarian response traits, which was mainly enriched as a decrease of leukocyte chemotaxis and migration in CGCs. For the first time, our study describes how SARS-CoV-2 infection indirectly affects CGCs at the transcriptional level, which may impair oocyte-CGC crosstalk and consequently lead to poor ovarian response during fertility treatment.

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

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.

Detection of bovine herpesvirus 1 in cumulus-oocyte complexes of cows.

Bovine herpesvirus 1 (BoHV-1) is the causative agent of infectious bovine rhinotracheitis (IBR) and is also associated with reproductive failure. This study investigated the presence of BoHV-1 in cumulus-oocyte complexes (COCs) of naturally-infected cows without clinical signs of IBR. The presence of BoHV-1 in COCs was evaluated by immunofluorescence using confocal laser scanning microscopy. Blood samples and ovaries from 82 cows that had not been vaccinated against BoHV-1 were collected for serological analysis. COCs were divided into two pools: COCs derivate from seropositive cows and from seronegative cows. Then, the samples were processed for confocal microscopy analysis. The results indicated that 61% (50/82) of cows were seropositive for BoHV-1. A total of 719 COCs were obtained from the cows and processed. None of 276 COCs from the 32 seronegative cows presented BoHV-1. However, BoHV-1 was present in the cytoplasm of cumulus cells from 158 out of 443 COCs aspirated from the seropositive cows. The detection of BoHV-1 in the COCs of seropositive cows suggests that the COCs of naturally-infected, asymptomatic cows may be infected with BoHV-1.

Interaction of bovine viral diarrhea virus with bovine cumulus-oocyte complex during IVM: Detection in permissive cells.

Structural changes in the zona pellucida (ZP) of bovine oocytes seem to modulate their interaction with various viral agents, facilitating the viral infection in in vitro production systems. To evaluate the susceptibility of bovine oocytes to noncytopathogenic bovine viral diarrhea virus (ncp-BVDV), cumulus-oocyte complexes were exposed to 10(7) ​tissue culture-infective doses (TCID50)/mL of an ncp-BVDV strain during IVM (in vitro maturation). After that, cumulus cells and the ZP were removed by hyaluronidase and pronase treatment, respectively, and the percentages of oocytes with polar body were analyzed as a sign of nuclear maturation. After passage through cell culture, the virus was isolated from granulosa cells, ZP-free mature oocytes, and ZP-intact mature oocytes. These results were confirmed by reverse transcription-polymerase chain reaction. After consecutive washes, the virus remained associated with ZP-free oocytes, maintaining its replication and infectivity in permissive cells. Based on these findings, it is concluded that the classical viral isolation procedure has a predictive value to detect BVDV associated with ZP-free oocytes and that it was novelty demonstrated that both washing and trypsin treatment of oocytes were ineffective to remove BVDV infection.

[Study of HBV-DNA in ovarian granulosa cells of hepatitis B carriers].

OBJECTIVE: To study the infection status of hepatitis B virus in ovarian granulosa cells of female hepatitis B carriers. METHODS: The follicular fluid of 6 female carriers of hepatitis B (HBsAg+, HBeAg+, HBcAb+) was used to extract granule cells and prepare smears. The samples were detected via in situ hybridization by digoxin-labeled HBV-DNA probe. The color reaction system of alkaline phosphates was used. And the positive hybridization signal was purple blue. RESULTS: Three granulosa cell samples, located at the nuclei of granulosa cells, were HBV-DNA positive. CONCLUSION: HBV DNA exists in the granulosa cells of hepatitis B patients. Thus HBV infection may be vertically transmitted to the offspring via the infected reproductive cells.

Lack of transmission of mouse minute virus (MMV) from in vitro-produced embryos to recipients and pups due to the presence of cumulus cells during the in vitro fertilization process.

The risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of cumulus-enclosed oocytes (CEOs) or without cumulus cells (CDOs) in the presence of MMV-exposed (10(4) TCID(50) [mean tissue culture infective dose]/ml MMVp [prototype strain of MMV]) spermatozoa was evaluated. Also, the time after embryo transfer to detection of MMV antibody and the presence of MMV DNA in the mesenteric lymph nodes of recipients and pups were investigated. All mice were MMV free, but two seropositive recipients and four seropositive pups were found in the group with CDOs. With regard to the CEOs, two of 11 holding drops and five of 11 groups of embryos were MMV positive using PCR, while neither holding drops nor embryos carried infectious MMVp, as evidenced by the in vitro infectivity assay. From IVF with CDOs, five of 14 holding drops and four of nine groups of embryos were MMV positive, while one of 14 holding drops and no embryos carried infectious MMVp. When 10(5) cumulus cells were analyzed 5 h after exposure to 10(4) TCID(50)/ml MMVp, cells had an average titer of 10(4) TCID(50)/ml MMVp. The present data show that, in contrast to CDOs, 2-cell embryos from CEOs did not transmit infectious MMVp to the holding drops and to recipients. This observation is due to the presence of cumulus cells during the IVF process that reduce entry of MMV into the zona pellucida and absorb some of the virus. These data further confirm the efficacy of the IVF procedure in producing embryos that are free of infectious virus, leading to virus-free seronegative recipients and rederived pups.