Trafficking Protein Particle Complex Subunit 4 (TRAPPC4) is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus. It plays a crucial role in tethering COPII vesicles and assembling SNARE complexes, ensuring precise protein delivery, which is vital for cellular communication and growth. Mutations in TRAPPC4 and other TRAPP subunits are associated with a group of human diseases collectively known as TRAPPopathies, often manifesting as neurological disorders. This study investigates the role of TRAPPC4 during early embryonic development by analysing the phenotypic consequences when the gene is knocked out in the mouse. Our results show that TRAPPC4 knockout leads to early embryonic lethality, where TRAPPC4 homozygous mutant embryos form morphologically normal blastocysts but fail to implant in the uterus and are not present at embryonic day 7.5. We show that mutant embryos can hatch from the zona pellucida, but do not form proper inner cell mass (ICM) outgrowths. Unlike the tightly compressed ICM colonies in control littermates, the mutant ICM appears necrotic with cells that are not adherent. This dispersion suggests that ICM cells may begin deteriorating at peri-implantation stages, underscoring TRAPPC4's pivotal role in maintaining cellular viability during early embryonic development.
INTRODUCTION:Male fertility is declining, and synthetic chemicals are a major candidate for causing it. This study investigated the effect of oral consumption of perfluorooctanoic acid on sperm quality and in vitro fertilization and the therapeutic role of alpha-lipoic acid in improving its possible effects. METHODS:Twenty adults male NMRI mice weighing 24-20 g were used. The male mice were divided into four groups. The control group received no drug. The sham group received only DMSO. The model group received PFOA (10 mg/kg), and the treatment group received PFOA (10 mg/kg) and ALA (dissolved in distilled water, 100 mg /kg). After the treatment period, blood, sperm and testicular tissue samples were collected from male mice. Serum testosterone level, testicular weight and histology, sperm analysis and sperm DFI were assayed. Then, using the IVF technique, fertilization rates at the 2PN, two-cell and cleavage stages were recorded. RESULTS:Exposure of male mice to PFOA significantly reduced sperm parameters (concentration, motility, morphology, viability), testicular weight, testosterone levels, IVF rates at the 2PN, two-cell and cleavage stages and increased sperm DFI. Simultaneous administration of ALA significantly reduced these adverse effects. P < 0.05 was considered significant. CONCLUSIONS:Our findings showed that ALA can improve the harmful effects of PFOA on sperm and fertilization rate. Therefore, the use of ALA to improve IVF outcomes deserves further attention. Given the persistent presence of PFOA in the environment and its associative (not causal) link with reduced sperm quality, further research and larger studies in this area seem necessary.
We evaluated the efficiency of two-dimensional (2D) and three-dimensional (3D) systems for in vitro culture of preantral follicles from the bovine species. Ovarian cortex fragments were obtained from a local slaughterhouse and randomly distributed into four groups: i) standard culture, directly on a culture plate (2D), ii) supported culture, on a gel monolayer (2D), iii) immersed culture on agarose gel (plating coated) covered in a medium (3D) and iv) Millicell-Biopore culture (3D), tested at 6 or 14 days. The following parameters were evaluated: follicular morphology and morphometry, reactive oxygen species production and granulosa cell proliferation. After 6 days of culture, follicles cultured on a gel monolayer (2D) showed a higher proportion of intact follicles than those cultured using other methods (P < 0.05). After 14 days of growth, standard culture on a culture plate (2D) and supported culture on a gel monolayer (2D) had a higher percentage of morphologically intact follicles (P < 0.05) than the 3D culture. An assessment of the redox status revealed that superoxide anion levels and antioxidant capacity of the samples cultured using the four methods were lower than those of the control group (non-cultured; P < 0.05). Taken together, these findings suggest that the agarose gel support system offers distinct advantages for bovine preantral follicle culture by maintaining morphological integrity and stabilising the redox environment, although further studies incorporating functional endpoints are warranted to fully confirm these observations.
This study aimed to investigate the involvement of the Wnt signalling pathway in the pathogenesis of non-obstructive azoospermia (NOA) and to identify potential diagnostic and therapeutic targets. We obtained the gene expression profiles of NOA patients from the GEO database, screened for differentially expressed genes (DEGs) and NOA-associated co-expressing gene modules, then intersected the DEGs with related genes in the Wnt signalling pathway and identified key genes. LASSO regression analysis was used to find hub genes, and qRT-PCR and WB methods were used to verify the expression of key genes in NOA patients. The immune infiltration and GSEA were conducted in order to investigate the relationship between hub genes and immune cells infiltration and NOA-associated pathways; furthermore, we built the miRNA-mRNA-TF regulatory network and predicted possible small molecules drug targets of the hub gene. Four candidate genes were related to NOA: CSNK1G2, GNG3, H2AFB1 and PARD6A. The key hub gene was CSNK1G2 based on the results of the LASSO regression analysis, and its expression level was significantly down-regulated in NOA patients. The immune infiltration analysis and GSEA results showed that the expression level of CSNK1G2 had a close relationship with immune cells infiltration and NOA-related pathways. Furthermore, we built a miRNA-mRNA-TF regulatory network and predicted the possible small molecule drugs of CSNK1G2. Our findings suggest that the Wnt signalling pathway, particularly CSNK1G2, plays a crucial role in the pathogenesis of NOA. CSNK1G2 may be a novel target for the diagnosis and treatment of NOA.
Sperm capacitation involves biochemical and functional changes necessary for fertilization. The ubiquitin-proteasome system (UPS) regulates protein turnover, but the roles of proteasomal receptors during capacitation remain unclear. This study investigates the regulation and functional impact of proteasomal subunits RPN13 (ADRM1) and RPN2 (PSMD1) during in vitro capacitation of boar sperm. Semen samples (n = 6) were assigned to four groups: non-capacitating control (Non-Cap, 4 h in medium lacking bovine serum albumin (BSA)), capacitating control (Cap, 4 h with BSA) and groups treated with vehicle (CM + dimethyl sulfoxide (DMSO)) or the RPN13 inhibitor RA190. We measured RPN2/RPN13 levels, RPN13-associated ubiquitin signal and RPN13-RPN2 interactions (Western blot, Co-IP) and assessed sperm kinematics, intracellular Ca2+, protein tyrosine phosphorylation (pTyr), mitochondrial membrane potential (ΔΨm), ATP, ZP binding and early embryo cleavage after IVF. The Cap group showed successful capacitation, confirmed by increases in pTyr, sperm hyperactivation and Ca2+-associated Fluo-4 fluorescence. During capacitation, RPN2 and RPN13 abundance decreased, while RPN2-RPN13 association increased. RA190 treatment increased RPN13 levels, inhibited RPN13 turnover and decreased its interaction with RPN2 and the associated ubiquitin signal. Functionally, RA190 attenuated sperm hyperactivation, Ca2+-associated Fluo-4 fluorescence, protein tyrosine phosphorylation, mitochondrial membrane potential and ATP levels and was associated with reduced fertilization-related outcomes, including ZP binding and early embryo cleavage after IVF. These findings highlight the role of RPN13 in sperm capacitation and fertilization.
