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Viral infections of the ovary may perturb ovarian functions. However, the mechanisms underlying innate immune responses in the ovary are poorly understood. The present study demonstrates that cytosolic viral DNA sensor signaling initiates the innate immune response in mouse ovarian granulosa cells and affects endocrine function. The cytosolic DNA sensors p204 and cGAS and their common signaling adaptor stimulator of interferon (IFN) genes (STING) were constitutively expressed in granulosa cells. Transfection with VACV70, a synthetic vaccinia virus (VACV) DNA analog, induced the expression of type I interferons (IFNA/B) and major inflammatory cytokines (TNFA and IL6) through IRF3 and NF-κB activation respectively. Moreover, several IFN-inducible antiviral proteins, including 2′,5′-oligoadenylate synthetase, IFN-stimulating gene 15 and Mx GTPase 1, were also induced by VACV70 transfection. The innate immune responses in granulosa cells were significantly reduced by the transfection of specific small-interfering RNAs targeting p204, cGas or Sting. Notably, the VACV70-triggered innate immune responses affected steroidogenesis in vivo and in vitro. The data presented in this study describe the mechanism underlying ovarian immune responses to viral infection.
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Transforming growth factor β (TGFβ) has been shown to be a multifunctional cytokine required for embryonic development and regulation of trophoblast cell behaviors. In the present study, a non-transformed cell-line representative of normal human trophoblast (NPC) was used to examine the effect of TGFβ1 on trophoblast cell adhesion and invasion. In vitro assay showed that TGFβ1 could significantly promote intercellular adhesion, while inhibiting cell invasion across the collagen I-coated filter. Reverse transcription (RT)-PCR and gelatin zymography demonstrated that TGFβ1 evidently repressed the mRNA expression and proenzyme production of matrix metalloproteinase (MMP)-9, but exerted no effect on mRNA expression and secretion of MMP-2. On the other hand, both the mRNA and protein expression of epithelial-cadherin and β-catenin were obviously upregulated by TGFβ1 in dose-dependent fashion, as revealed by RT-PCR and western-blot analysis. What is more, one of the critical TGFβ signaling molecules – Smad2 was notably phosphorylated in TGFβ1-treated NPC cells. The data indicates that cell invasion and adhesion are coordinated processes in human trophoblasts and that there exists paracrine regulation on adhesion molecules and invasion-associated enzymes in human placenta.
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In brief
PLCZ1 mutations are related to total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), characterised by abnormal oocyte oscillations. The novel PLCZ1 compound heterozygous mutations reported by this study were associated with TFF after ICSI, with one of the mutations indicating a gene dosage effect.
Abstract
Oocyte activation failure is thought to be one of the main factors for total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), which could be induced by abnormal calcium oscillations. Phospholipase C zeta (PLCZ), a sperm factor, is associated with Ca2+ oscillations in mammalian oocytes. To date, some mutations in PLCZ1 (the gene that encodes PLCZ) have been linked to TFF, as demonstrated by the observed reduction in protein levels or activity to induce Ca2+ oscillations. In this study, normozoospermic males whose sperms exhibited TFF after ICSI and their families were recruited. First, mutations in the PLCZ1 sequence were identified by whole exome sequencing and validated using Sanger sequencing. Then, the locations of PLCZ1/PLCZ and the transcript and protein levels in the sperm of the patients were studied. Subsequently, in vitro function analysis and in silico analysis were performed to investigate the function–structure correlation of mutations identified in PLCZ1 using western blotting, immunofluorescence, RT-qPCR, and molecular simulation. Ca2+ oscillations were detected after cRNA microinjection into MII mouse oocytes to investigate calcium oscillations induced by abnormal PLCZ. Five variants with compound heterozygosity were identified, consisting of five new mutations and three previously reported mutations distributed across the main domains of PLCZ, except the EF hands domain. The transcript and protein levels decreased to varying degrees among all detected mutations in PLCZ1 when transfected in HEK293T cells. Among these, mutations in M138V and R391* of PLCZ were unable to trigger typical Ca2+ oscillations. In case 5, aberrant localisation of PLCZ in the sperm head and an increased expression of PLCZ in the sperm were observed. In conclusion, this study enhances the potential for genetic diagnosis of TFF in clinics and elucidates the possible relationship between the function and structure of PLCZ in novel mutations.
