Feline microRNAome in ovary and testis: Exploration of in-silico miRNA-mRNA networks involved in gonadal function and cellular stress response.

The aim of the study was to perform the first in-depth analysis of miRNAs in ovarian and testicular tissues of the domestic cat, a critical biomedical model. Specifically, potential miRNA involvement was explored in gonadal function, testis development, and cellular stress response to preservation protocols. We performed miRNA-sequencing on 20 ovarian and 20 testicular samples from 15 cats, including different ages and tissue treatments. Using fresh tissues (n = 15), we confirmed gonadal expression of 183 miRNA precursors and discovered additional 52 novel feline candidate precursors. We integrated the mRNA data from our previous study on the same age and treatment groups to create in-silico miRNA-mRNA networks and their functional enrichment, which allows comprehensive exploration into possible miRNA functions in cat gonads. Clusters of miRNAs united by shared differentially expressed mRNA targets are potentially involved in testicular development and spermatogenesis. MicroRNAs could play a significant role in ovarian tissue response to stress from microwave-assisted dehydration, with smaller roles in cellular response to vitrification in both ovary and testis. This new list of miRNAs with potential function in cat gonads is a major step towards understanding the gonadal biology, as well as optimizing fertility preservation protocols.

Morphological, morphometric, ultrastructural, and functional evaluation of red-rumped agouti (Dasyprocta leporina) sperm during epididymal transit.

The red-rumped agouti (Dasyprocta leporina) is a hystricognath rodent with reproductive anatomical peculiarities presenting as an intra-abdominal testes-epididymis complex. This study was carried out to describe, for the first time, details related to the morphological and functional changes in sperm along the epididymal transit in agoutis. The testes-epididymal complexes were sampled from seven sexually mature agoutis. Sperm from different epididymal regions (caput, corpus, and cauda) were collected using the floating technique, and their morphology, morphometry, ultrastructure, mitochondrial activity, membrane structural integrity, and kinetic parameters were determined. The number of sperm collected (823.5 ×106 sperm) was higher in the epididymis cauda. No significant differences in normal sperm morphology among the different epididymal regions (caput, 82.42%; corpus, 86.71%; and cauda, 88.86 %) were observed. The mean head length, head width, and tail length were highest in the caput (5.15 µm, 3.44 µm, and 32.04 µm, respectively), decreasing along the epididymal transit. Ultrastructure by scanning electron microscopy (SEM) revealed agglomeration of spermatozoa from caput and corpus, thus, enabling analysis of the gametes from only the epididymal cauda with clarity. Sperm from epididymis cauda showed the greatest proportion of membrane integrity and mitochondrial activity, followed by those from corpus and caput (79.71 %, 58.9 %, 47.7 %, respectively). Significant increase in total motility, progressive motility, velocity average pathway -VAP, velocity straightline - VSL, velocity curvilinear - VCL, and rapid sperm in the caput-corpus-cauda direction were observed. These novel data contribute to the knowledge of sperm maturation in the red-rumped agouti.

Comparison of different intracellular cryoprotectants on the solid surface vitrification of red-rumped agouti (Dasyprocta Leporina Lichtenstein, 1823) ovarian tissue.

In contributing to the conservation of wild rodents, the aim of this study was to evaluate the use of distinct cryoprotectants, separately or in combination, for solid surface vitrification (SSV) of red-rumped agouti ovarian tissue. Ovarian cortex from nine females was recovered and fragmented. Fresh fragments (control) were used to analyse the pre-antral follicle (PF) morphology using a histologic procedure, viability using the Trypan blue test, cell proliferation by counting the argyrophilic nucleolar organizing regions (Ag-NORs technique) and DNA integrity using the TUNEL assay. The remaining fragments were vitrified using SSV method with 3 M or 6 M ethylene glycol (EG) or dimethyl sulfoxide (DMSO), or in combination (3 M EG/3 M DMSO), and further evaluated as reported for the fresh samples. All cryoprotectants were effective at preserving PFs morphology compared to the control group (80.7 ± 5.21%), except 6 M EG and 3 M DMSO that provoked a significant (p < .05) decrease on the values of morphologically normal primary (60.0 ± 19.0%) and primordial (44 ± 4.5%) follicles, respectively. Regarding viability, all cryoprotectants provided values similar to that verified for the control group (79.0%), but a significant decrease (p < .05) was observed with EG/DMSO combination (59%). Using Ag-NORs technique, the highest (p < .05) cell proliferative capacity was detected when using EG at each tested concentration. The TUNEL proved the preservation of DNA integrity regardless of the cryoprotectant. In summary, we suggest the use of 3 M EG for the solid surface vitrification of red-rumped agouti ovarian tissue.

Regioselective synthesis, biological evaluation, and molecular docking of dihydropyrimidin-4-ols as acetylcholinesterase inhibitors.

A new series of 3,6-disubstituted 2-(methylthio)-4-(trifluoromethyl)-3,4-dihydropyrimidin-4-ols displaying methyl, phenyl, aryl, and heteroaryl groups at the 6-position; and methyl, ethyl, allyl, and phenyl groups at the 3-position of the dihydropyrimidine ring, were synthesized and evaluated in vitro for acetylcholinesterase inhibitory activity. Seven compounds showed activity with IC50 values in the lower micromolar range. The compound 4-trifluoromethyl-6-(4-fluorophenyl)-3-methyl-2-methylthio-3,4-dihydropyrimidin-4-ol (6e) had the best inhibitory activity (IC50 2.2 ± 0.9 µm) and this inhibition was characterized as competitive. The molecular docking study showed that the acetylcholinesterase enzyme accommodates compound 6e in its catalytic site. The enantiomers of compound 6e, present similar interactions: π-π stacking interactions between the aromatic ring of the ligand's 4-fluorophenyl moiety and the aromatic rings of the electron-rich Trp84; and H-bonds between the hydroxyl group of Tyr121 and the hydroxyl moiety from 6e. The antioxidant effect of the dihydropyrimidin-4-ols was also investigated.

Regioselectively Controlled Synthesis of N-Substituted (Trifluoromethyl)pyrimidin-2(1H)-ones.

A simple and regioselectively controlled method for the preparation of both 1,4- and 1,6-regioisomers of 1-substituted 4(6)-trifluoromethyl-pyrimidin-2(1H)-ones is described. Both regioisomers were synthesized from the cyclocondensation reaction of 4-substituted 1,1,1-trifluoro-4-methoxybut-3-en-2-ones: with nonsymmetric ureas for the 1-substituted 4-(trifluoromethyl)pyrimidin-2(1H)-ones (1,4-isomer) and with nonsymmetric 1-substituted 2-methylisothiourea sulfates for the synthesis of 1-substituted 6-(trifluoromethyl)pyrimidin-2(1H)-ones (1,6-isomer). Each method furnished only the respective isomer in very good yields. The structure of the products was assigned based on the (1)H and (13)C NMR as well as 2D HMBC spectral analysis.