Expression of the umami taste receptor T1R1/T1R3 in porcine testis of: Function in regulating testosterone synthesis and autophagy in Leydig cells.

Testosterone is a vital male hormone responsible for male sexual characteristics. The taste receptor family 1 subunit 3 (T1R3) regulates testosterone synthesis and autophagy in non-taste cells, and the links with the taste receptor family 1 subunit 1 (T1R1) for umami perception. However, little is known about these mechanisms. Thus, we aimed to determine the relationship between the umami taste receptor (T1R1/T1R3) and testosterone synthesis or autophagy in testicular Leydig cells of the Xiang pig. There was a certain proportion of spermatogenic tubular dysplasia in the Xiang pig at puberty, in which autophagy was enhanced, and the testosterone level was increased with a weak expression of T1R3. Silenced T1R3 decreased testosterone level and intracellular cyclic adenosine monophosphate (cAMP) content and inhibited the messenger RNA (mRNA) expression levels of testosterone synthesis enzyme genes [steroidogenic acute regulatory protein (StAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3ß-HSD1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and hydroxysteroid 17-beta dehydrogenase 3 (17ß-HSD3)]. In addition, T1R3 increased the number of acidic autophagy bubbles and upregulated the expression levels of autophagy markers [Microtubule-associated protein 1 A/1B-light chain 3 (LC3) and Beclin-1] in testicular Leydig cells of the Xiang pig. Using an umami tasting agonist (10 mM L-glutamate for 6 h), the activation of T1R1/T1R3 enhanced the testosterone synthesis ability by increasing the intracellular cAMP level and upregulated the expression levels of StAR, 3ß-HSD1, CYP17A1 and 17ß-HSD3 in Leydig cells. Furthermore, the number of acidic autophagy bubbles decreased in the T1R1/T1R3-activated group with the downregulation of the expression levels of the autophagy markers, including LC3 and Beclin-1. These data suggest that the function of T1R1/T1R3 expressed in testicular Leydig cells of the Xiang pig is related to testosterone synthesis and autophagy.

Pretreatment of 3-hydroxyacetophenone in pharmaceutical wastewater using combined salting-out crystallization+ Fenton system and subsequent impact analysis of effluent water.

Selecting a suitable pretreatment process for pharmaceutical wastewater that is difficult to treat biochemically so that it can enter the subsequent biochemical treatment. In this study, pharmaceutical wastewater consisting of 45 g/L sodium bisulfate, 9 g/L 3-hydroxyacetophenone (3-HAP), and 36.75 g/L sulfuric acids,which is a kind of typical pharmaceutical wastewater, was used for the pretreatment case study, and the process was screened by technology. A salting-out crystallization+Fenton system(SC-F) was developed for the treatment of this wastewater. The salting-out agent is formed by the pH adjustment process without additional additions and the salting-out crystallization effect is significant for the precipitation of 3-HAP from the wastewater. Subsequently, the optimal operating conditions for SC-F were derived from experiments as H2O2 of 0.4692 mol/L, n(H2O2):n(Fe2+)=30:1, pH=3. Under optimal conditions, the reaction time of 2 h achieved a COD removal rate of 90% and a BOD/COD value of 0.56, confirming the effectiveness of the technology in treating this wastewater. Additionally, it was discovered that the Fenton treatment was not significantly impacted by the inorganic components of the effluent. Analysis of effluent properties and possible effects on subsequent treatment by LC-MS and toxicity analysis. The results show that the biodegradability are enhanced by the pretreatment technology. However, the effluent still suffers from high acidity and high salt content, and this study proposes a solution to this problem. Furthermore, research on the treatment of 3-HAP wastewater has not been reported and this study provides a new case study in the field of wastewater treatment.

Different fixatives influence morphology, antigen preservation, and TUNEL staining in chicken (Gallus gallus) testis.

The optimized fixative for testis is still controversial. This study investigated the effects of Modified Davidson's Fluid (mDF), 4% Paraformaldehyde (4% PFA), and Bouin's Fluid (BF) fixatives on chicken testes in normal/cadmium (Cd) feeding groups using hematoxylin and eosin (HE), immunohistochemistry (IHC), and Terminal Transferase dUTP Nick End Labeling (TUNEL) staining. Compared to the mDF, we established that the testes fixed with 4% PFA and BF in the normal group had severe shrinkage in tubular and interstitial compartments. Moreover, compared with 4% PFA, the number of GATA4-positive Sertoli cells/mm2 reduced by 67.61% in mDF and 80.57% in BF for one seminiferous tubule. The TUNEL assay illustrated that more positive cells/mm2 in mDF group (28.47 ± 11.38) than in 4% PFA (10.49 ± 7.89). In Cd-treated testes, mDF showed more morphological details than 4% PFA and BF. In contrast, the number of GATA4-positive Sertoli cells/mm2 of 4% PFA was higher than that of mDF by 65.78% and BF by 64.80% in a seminiferous tubule. The number of TUNEL positive cells/mm2 in mDF (272.60 ± 34.41) were higher than in 4% PFA (175.91 ± 19.87). These results suggest that mDF fixative is suitable for normal and Cd-treated testis fixation for HE and TUNEL staining in chicken, whereas 4% PFA fixative is better for IHC examination.

Longitudinal Expression of Testicular TAS1R3 from Prepuberty to Sexual Maturity in Congjiang Xiang Pigs.

Testicular expression of taste receptor type 1 subunit 3 (T1R3), a sweet/umami taste receptor, has been implicated in spermatogenesis and steroidogenesis in mice. We explored the role of testicular T1R3 in porcine postnatal development using the Congjiang Xiang pig, a rare Chinese miniature pig breed. Based on testicular weights, morphology, and testosterone levels, four key developmental stages were identified in the pig at postnatal days 15-180 (prepuberty: 30 day; early puberty: 60 day; late puberty: 90 day; sexual maturity: 120 day). During development, testicular T1R3 exhibited stage-dependent and cell-specific expression patterns. In particular, T1R3 levels increased significantly from prepuberty to puberty (p < 0.05), and expression remained high until sexual maturity (p < 0.05), similar to results for phospholipase Cß2 (PLCß2). The strong expressions of T1R3/PLCß2 were observed at the cytoplasm of elongating/elongated spermatids and Leydig cells. In the eight-stage cycle of the seminiferous epithelium in pigs, T1R3/PLCß2 levels were higher in the spermatogenic epithelium at stages II-VI than at the other stages, and the strong expressions were detected in elongating/elongated spermatids and residual bodies. The message RNA (mRNA) levels of taste receptor type 1 subunit 1 (T1R1) in the testis showed a similar trend to levels of T1R3. These data indicate a possible role of T1R3 in the regulation of spermatid differentiation and Leydig cell function.

Carbonic anhydrase inhibition, antioxidant activity against alveolar epithelial cells and antibacterial effect against Klebsiella pneumoniae enabled by synthesized silica nanoparticles through laser ablation technique.

Silica (SiO2) nanoparticles (NPs) were synthesized by laser ablation method and were characterized by TEM and DLS techniques. Afterwards, their inhibition activity against carbonic anhydrase (CA) isoforms (CA I and CA II) was explored by experimental and theoretical analysis. Also, the protective effect of SiO2 NPs against H2O2-induced oxidative stress in alveolar epithelial cells (A549) were assessed by measurement of MTT, ROS level, CAT and SOD activity and GSH content. Finally, the NPs were screened for their antimicrobial activity using the MICs method against the Klebsiella pneumoniae. The result showed that the synthesized NPs have a size of around 40 nm. The inhibition activity by comparing IC50 values with acetazolamide as a positive control revealed that SiO2 NPs in comparison with acetazolamide served as potent inhibitors against CA isoforms which was also confirmed by docking studies. The cellular assays indicated that the SiO2 NPs with a concentration of 20 µg/mL stimulated a significant antioxidant activity against H2O2-induced oxidative cell damage through activation of CAT and SOD, an increase in the GSH content and reducing the level of ROS. The synthesize NPs also showed a good inhibition effect against Klebsiella pneumoniae as compared to Sulfamethoxazole as a positive control. In conclusion, this data may provide some useful information on the development of some platforms for pneumonia treatment and management.

Postnatal differentiation and regional histological variations in the ductus epididymidis of the Congjiang Xiang pig.

The Congjiang Xiang pig is a rare Chinese miniature breed whose epididymal histologic features through the postnatal development are poorly understood. To clarify the histomorphological differences between each region of epididymis during postnatal development, 24 male Congjiang Xiang pigs aged from neonatal (15 d), peri-puberty (30 d), puberty (60 d) to adult (180 d) stages, were examined. Postnatal differentiation of the different regions (I-V) of the epididymis started from birth and continued until maturity that showed regional variations. Developmental progression was disto-proximal. At the neonatal stage, Wolffian duct differentiation starts in the distal region, then ascends to the middle region which forms regions V, IV and III, respectively. A simple lined cuboidal in the epidydimal epithelial, which gradually differentiated into a pseudostratified columnar with stereocilia from neonatal to post-pubertal. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV-V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV-V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility.

Effects of 4% paraformaldehyde and modified Davidson's fluid on the morphology and immunohistochemistry of Xiang pig testes.

Modified Davidson's fluid (mDF) is a good fixative for morphological and antigen preservation. However, recent studies have shown that 4% paraformaldehyde (PFA) can better preserve the actin structure in rodent testes. It remains controversial which of these fixatives is best for testicular tissue. This study investigated the effects of both mDF and 4% PFA on the morphology and antigen preservation of Xiang pig testes using hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). The stronger testis penetration of mDF compared with that of 4% PFA was primarily manifested as testicular color change and decrease in tissue weight loss. Testes fixed with 4% PFA displayed a severe shrinkage of both the tubular and interstitial compartments and the seminiferous tubule area decreased by 12.02% compared with that in mDF-fixed tissues. In contrast, IHC results showed that 4% PFA fixation achieved better IHC-positive performance than mDF fixation for antigens specifically expressed in germ cells, Leydig cells and Sertoli cells. Due to this improved antigen preservation by 4% PFA fixation, the relative immunoreactions intensity significantly increased by 39.8%, 27.8%, and 76.4%, respectively, compared with that in mDF fixation. In summary, fixation of Xiang pig testes with mDF was suitable for HE staining, while fixation with 4% PFA was more suitable for IHC.