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.

Virus analog decreases estradiol secretion in FSH-treated human ovarian granulosa cells.

The aim of this study was to evaluate the effect of virus infection on estradiol (E2) production in human ovarian granulosa cells. Polyriboinosinic polyribocytidylic acid [Poly (I: C)], a synthetic analog of viral double stranded RNA that can be recognized by Toll like receptor 3 (TLR3), was used to imitate virus infection. Granulosa cells (GCs) obtained from patients undergoing in vitro fertilization and embryo transfer (IVF-ET) were cultured in vitro and treated with Poly (I: C), FSH, or both. Concentration of E2 was assayed by electrochemiluminescence. The mRNA and protein expression of TLR3 and aromatase were determined by real-time quantitative PCR (qPCR) and Western blot, respectively. The results showed that expression of TLR3 mRNA was significantly increased after Poly (I: C) stimulation. Poly (I: C) decreased E2 synthesis in FSH-treated GCs. Poly (I: C) inhibited the expression of aromatase in FSH-treated GCs. This study demonstrated that Poly (I: C) inhibits the synthesis of estradiol by granulosa cells under the stimulation of FSH, which might contribute to disturbance of follicular development and ovulation.

Mumps virus induces innate immune responses in mouse ovarian granulosa cells through the activation of Toll-like receptor 2 and retinoic acid-inducible gene I.

Mumps virus (MuV) infection may lead to oophoritis and perturb ovarian function. However, the mechanisms underlying the activation of innate immune responses to MuV infection in the ovary have not been investigated. This study showed that Toll-like receptor 2 (TLR2) and retinoic acid-inducible gene I (RIG-I) cooperatively initiate innate immune responses to MuV infection in mouse ovarian granulosa cells. Ovarian granulosa cells infected with MuV significantly produced pro-inflammatory cytokines and chemokines, including interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and type 1 interferons (IFN-α and IFN-ß). Knockdown of RIG-I significantly decreased MuV-induced cytokine expression. TLR2 deficiency reduced the expression of IL-1ß, TNF-α, and MCP-1 but did not affect the expression of IFN-α and IFN-ß in granulosa cells after infection with MuV. Intraperitoneal injection of MuV induced the ovarian innate immune responses in vivo, which suppressed estradiol synthesis and induced granulosa cell apoptosis. The results provide novel insights into the mechanisms underlying MuV-induced innate immune responses in the mouse ovary.

Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4) Strains Isolated from In Vitro Bovine Embryos production in Argentina.

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.

Coxsackievirus B3 infection reduces female mouse fertility.

Previously we demonstrated coxsackievirus B3 (CVB3) infection during early gestation as a cause of pregnancy loss. Here, we investigated the impacts of CVB3 infection on female mouse fertility. Coxsackievirus-adenovirus receptor (CAR) expression and CVB3 replication in the ovary were evaluated by immunohistochemistry or reverse transcription-polymerase chain reaction (RT-PCR). CAR was highly expressed in granulosa cells (GCs) and CVB3 replicated in the ovary. Histological analysis showed a significant increase in the number of atretic follicles in the ovaries of CVB3-infected mice (CVBM). Estrous cycle evaluation demonstrated that a higher number of CVBM were in proestrus compared to mock mice (CVBM vs. mock; 61.5%, 28.5%, respectively). Estradiol concentration in GC culture supernatant and serum were measured by an enzyme-linked immunosorbent assay. Baseline and stimulated levels of estradiol in GC were decreased in CVBM, consistent with significantly reduced serum levels in these animals. In addition, aromatase transcript levels in GCs from CVBM were also decreased by 40% relative to the mock. Bone mineral density evaluated by micro-computed tomography was significantly decreased in the CVBM. Moreover, the fertility rate was also significantly decreased for the CVBM compared to the mock (CVBM vs. mock; 20%, 94.7%, respectively). This study suggests that CVB3 infection could interfere with reproduction by disturbing ovarian function and cyclic changes of the uterus.

Hepatitis B virus infection and replication in primary cultured human granulosa cells.

To investigate the infection and replication of hepatitis B virus (HBV) in primary cultured human granulosa cells. Human granulosa cells were cultured with HBV-positive serum. Media were collected and assayed for HBsAg and HBeAg by ELISA, and HBV DNA by quantitative PCR. HBsAg and HBcAg were detected by immunocytochemistry in cultured cells. HBV DNA and RNA were extracted and amplified by nested PCR. Intracellular HBV DNA was localized by in situ hybridization. By co-cultivation of human GCs with HBV-positive serum, a system was established to study HBV infection and replication in GCs. HBsAg in medium could be detected from 4 to 96 h, and HBV DNA could be detected from 12 to 96 h after exposure. HBsAg and HBcAg showed positive signals by immunocytochemistry. A 206-bp fragment was amplified by nested PCR to detect HBV DNA and RNA in granulosa cells. HBV DNA was detected in GC nuclei by in situ hybridization. HBV can infect and replicate in human primary granulosa cells. This culture system could enable us to study infection of ova by HBV.

Positive and negative regulation of chicken anemia virus transcription.

Chicken anemia virus (CAV) is a small circular single-stranded DNA virus with a single promoter-enhancer region containing four consensus cyclic AMP response element sequences (AGCTCA), which are similar to the estrogen response element (ERE) consensus half-sites (A)GGTCA. These sequences are arranged as direct repeats, an arrangement that can be recognized by members of the nuclear receptor superfamily. Transient-transfection assays which use a short CAV promoter construct that ended at the transcription start site and drive expression of enhanced green fluorescent protein (EGFP) showed high basal activity in DF-1, LMH, LMH/2A, and primary theca and granulosa cells. The estrogen receptor-enhanced cell line, LMH/2A, had significantly greater expression than LMH cells, and this expression was significantly increased with estrogen treatment. A long promoter construct which included GGTCA-like sequences downstream of the first CAV protein translation start site was found to have significantly less EGFP expression in DF-1 cells than the short promoter, which was largely due to decreased RNA transcription. DNA-protein binding assays indicated that proteins recognizing a consensus ERE palindrome also bind GGTCA-like sequences in the CAV promoter. Estrogen receptor and other members of the nuclear receptor superfamily may provide a mechanism to regulate CAV activity in situations of low virus copy number.

Efficient replication of caprine arthritis-encephalitis virus in goat granulosa cells.

Recent reports demonstrated the susceptibility of epithelial cells from different organs to caprine arthritis-encephalitis virus (CAEV) both in vitro and in vivo. Since granulosa cells (GC) are of epithelial origin and currently used for in vitro oocyte maturation, we addressed the question whether these cells are susceptible or resistant to CAEV infection. GC were isolated from goats from certified CAEV-free herds. PCR analysis on GC DNA using CAEV specific primers confirmed the absence of CAEV infection and immunocytochemistry using specific K813 anti-cytokeratin monoclonal antibodies confirmed the epithelial nature of GC. These cells were then inoculated with CAEV using two strains: the CAEV-pBSCA molecular clone and the CAEV-3112 French field isolate. Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using an hyperimmune serum. Supernatant of infected cells were shown to contain high titers (ranging 10(5) tissue culture infectious doses 50 per ml: TCID(50) per ml) of infectious cytopathic viruses when assayed onto the indicator goat synovial membrane (GSM) cells. Our findings demonstrate the large cell tropism of CAEV and suggest that GC could serve as a reservoir for the virus during the sub-clinical phase of infection. Furthermore, given the high seroprevalence of CAEV in the all industrialised countries and the large number of ovaries derived from unknown serological status animals used for in vitro goat embryo production, one can conclude that these feeder cell cultures might be a potential source of early transmission of CAEV to goat embryos.

Identification of a novel retrovirus expressed in rat Sertoli cells and granulosa cells.

Differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to identify a novel retrovirus, designated SC1, that is expressed at high levels in rat granulosa cells and prepubertal Sertoli cells. The initial DDRT-PCR screen was performed using RNA from cultured prepubertal rat Sertoli cell, liver, and brain samples. SC1 was detected in the prepubertal rat Sertoli cell samples but not in those from liver and brain. SC1 cDNA was 6 kilobases in length and contained regions encoding for the gag, pol, and env retroviral proteins. Northern blot analysis failed to detect expression of the SC1 gene in total RNA isolated from adult brain, heart, spleen, lung, liver, skeletal muscle, kidney, prostate, and epididymis. Similarly, Northern blot analysis of testes from rats at various ages of development showed that high-level expression of the SC1 gene was limited to prepubertal testis samples. In situ hybridization analysis localized the SC1 mRNA to the seminiferous tubules of prepubertal testes and at a much lower level in Sertoli cells of adult testes. Northern blot analysis of total RNA isolated from Sertoli cells from 20-, 27-, and 35-day-old rat Sertoli cells and type A spermatogonia, pachytene spermatocytes, and round spermatids showed expression of the SC1 gene to be restricted to 20- and 27-day-old Sertoli cells, with no expression detected in germ cells. Furthermore, Northern blot analysis also showed expression of the SC1 gene in rat ovaries, and the level of expression was affected during eCG/hCG-induced ovulation. Expression of SC1 mRNA was localized by in situ hybridization of eCG-treated ovaries to the granulosa cell layer in developing follicles. Southern blot analysis showed SC1 to be endogenous in the rat and absent in mouse and human cell genomes. Transient transfection assays using the SC1 promoter region showed high promoter activity in MSC-1 and cultured prepubertal rat Sertoli cells, and no activity in 3T3 or MCF-7 cell lines.

Evidence for the localization of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and RNA in ovarian follicles in gilts.

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos.

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.

In vitro attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis phage type 8.

The attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis was examined in vitro. The attachment was inhibited by preincubation of granulosa cells with anti-chicken fibronectin antibody (approximately 70% reduction in attachment) or preincubation with a 14-kDa fimbrial protein isolated from S. enteritidis (68% reduction in attachment). Treatment of bacterial cells with the tetrapeptide RGDS before addition to granulosa cells resulted in inhibition of attachment (60% inhibition when 2 x 10(7) CFU of bacteria was treated with 500 microg of peptide). Treatment with the peptide GRGD resulted in similar magnitude of inhibition, indicating that extracellular matrix proteins play significant roles in the interaction of S. enteritidis with granulosa cells. In contrast, treatment of the bacterial cells with the peptide GRAD did not result in significant inhibition of attachment to the granulosa cells. S. enteritidis was found to attach specifically to fibronectin, collagen IV, and laminin-coated microtiter plate wells, with the rank order of attachment as follows: fibronectin > laminin > collagen IV. Light and transmission electron micrographs of S. enteritidis invasion of granulosa cells showed organisms with or without a surrounding membrane in the cytoplasm of granulosa cells. In some instances, dividing bacterial cells were observed in the cytoplasm. Results of this study demonstrated that S. enteritidis interacts with granulosa cells in a specific manner and can invade and multiply in these cells. The granulosa cell layer of the preovulatory follicles may be a preferred site for the colonization of the chicken ovaries by invasive strains of S. enteritidis.

Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle.

Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus-oocyte complexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)