Comparative electron microscopy study of spermatozoa in snakes (Lepidosauria, Squamata).

The ultrastructure of snake sperm has received substantial attention primarily because snakes exhibit considerable variability in reproductive characteristics between species, with a wide range of mating systems and reproductive behaviors. Variability of sperm morphology among snake species may be associated with the reproductive strategies of each taxon, such as competition or sperm storage. We provide a detailed description of the sperm ultrastructure of nine snake species (Anilius scytale, Tropidophis paucisquamis, Bothrops jararaca, Oxyrhopus guibei, Dipsas mikanii, Micrurus corallinus, Xenopholis scalaris, Acrochordus javanicus, and Cylindrophis ruffus) and compared this with sperm data from the literature for the following taxa: Liotyphlops beui, Amerotyphlops reticulatus, Trilepida koppesi, Anilios waitii, Anilios endoterus, Aspidites melanochephalus, Boa constrictor amarali, Corallus hortulana, Epicrates cenchria, Boa constrictor occidentalis, Eryx jayakari, Micrurus corallinus, Micrurus surinamensis, Micrurus frontalis, Micrurus altirostris, Oxyuranus microlepidotus, Bothrops alternatus, Bothrops diporus, Crotalus durissus, Agkistrodon contortrix, Vipera aspis, Boiga irregularis, Zamenis schrenckii, Zamenis scalaris, Stegonotus cuculatus, Nerodia sipedon, Liodytes pygaea, and Myrrophis chinensis. We found twelve polymorphic characters in the ultrastructure of sperm among the described snakes. Our work supports the importance of ultrastructural analysis of sperm morphology to understand snake reproduction, and provides sperm-derived morphological characters for phylogenetic analysis.

Colchicine reduces the activation of NLRP3 inflammasome in COVID-19 patients.

OBJECTIVE: To evaluate whether colchicine treatment was associated with the inhibition of NLRP3 inflammasome activation in patients with COVID-19. METHODS: We present a post hoc analysis from a double-blinded placebo-controlled randomized clinical trial (RCT) on the effect of colchicine for the treatment of COVID-19. Serum levels of NOD-like receptor protein 3 (NLRP3) inflammasome products-active caspase-1 (Casp1p20), IL-1ß, and IL-18-were assessed at enrollment and after 48-72 h of treatment in patients receiving standard-of-care (SOC) plus placebo vs. those receiving SOC plus colchicine. The colchicine regimen was 0.5 mg tid for 5 days, followed by 0.5 mg bid for another 5 days. RESULTS: Thirty-six patients received SOC plus colchicine, and thirty-six received SOC plus placebo. Colchicine reduced the need for supplemental oxygen and the length of hospitalization. On Days 2-3, colchicine lowered the serum levels of Casp1p20 and IL-18, but not IL-1ß. CONCLUSION: Treatment with colchicine inhibited the activation of the NLRP3 inflammasome, an event triggering the 'cytokine storm' in COVID-19. TRIAL REGISTRATION NUMBERS: RBR-8jyhxh.

Effect of glucosamine sulphate on the temporomandibular joint of ovariectomised rats.

Glycosamine is an amino-monosaccharide present in connective and cartilage tissues that contribute to the maintenance, resistance, flexibility, and elasticity of these tissues. This study aimed to determine the in vivo effects of glucosamine sulphate (GS) on the temporomandibular joint (TMJ) of ovariectomised rats (OVX).Thirty-two rats were distributed into four groups as follows: G1, sham-OVX+saline solution; G2, sham-OVX+glucosamine sulphate (80mg/kg) - oral administration; G3, OVX+saline solution; G4, OVX+glucosamine sulphate (80mg/kg) - oral administration. Animals were treated for seven days. The TMJ was removed and stained with toluidine blue. The thickness of the cartilage layers and cytokines IL-1ß, IL-6, and TNF-α levels were determined by histomorphometry and immunoassay, respectively. The administration of GS to OVX females did not change the thickness of condylar cartilage when compared with the other groups (p>0.05). There was an increase in the total cartilage thickness in sham-OVX females. IL-1ß and TNF-α levels were significantly lower in sham-OVX females than in OVX females, indicating that ovariectomy acts as potent cytokine inducer. IL-6 levels were significantly higher in sham-OVX females. GS did not affect cytokine production in OVX females (p>0.05). In conclusion, the administration of GS did not affect cytokine levels, but did induce an increase in the total thickness of the TMJ condylar cartilage in sham-OVX rats.

Interaction between estradiol and follicle-stimulating hormone promotes in vitro survival and development of caprine preantral follicles.

The aim of this study was to investigate the effects of estradiol and follicle-stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) containing estradiol (1, 5, 10, 20 or 40 pg/ml), FSH (50 ng/ml), or a combination of the two hormones. Cultured and noncultured control ovarian tissues were processed for histological and ultrastructural studies. The results showed that after 7 days of culture, the treatments that yielded the highest percentage of normal follicles relative to MEM alone were those that combined FSH with estradiol at 1, 5 or 20 pg/ml. The addition of FSH to 1-day cultures containing 1 pg/ml estradiol or to 7-day cultures with 1 or 5 pg/ml estradiol increased the percentage of normal follicles compared to estradiol alone at the same concentrations. After 7 days of culture, all treatments generated higher percentages of developing follicles as compared to control and MEM alone. The addition of either FSH or 10 pg/ml of estradiol to the culture media or estradiol (1, 5, 10 or 20 pg/ml) and FSH in combination significantly increased follicular diameter as compared with MEM alone following 7 days of culture. Ultrastructural studies confirmed follicular integrity after 7 days of culture in the presence of 1 pg/ml estradiol plus FSH. In conclusion, this study demonstrated that the interaction between estradiol and FSH maintains ultrastructural integrity and stimulates activation and further growth of cultured caprine preantral follicles.

Ultrastructural description and cytochemical study of the spermatozoon of Crotallus durissus (Squamata, Serpentes).

The present study was undertaken to elucidate some aspects about the nature of the spermatozoon ultrastructure of Crotallus durissus using cytochemical methods. We also provide for the first time the ultrastructural description of this species spermatozoon. Cytochemical studies of spermatozoa have not been performed so far in the Serpentes, and species spermatozoon may prove helpful to better understand the reproductive biology of this group. Besides the synapomorphies of the Squamata and Serpentes, the C. durissus spermatozoon possess the following: circular acrosome tip; rounded perforatorium tip with a stopper-like basal modification; bilateral stratified laminar structures; central electron-dense structure within the proximal centriole; fibrous sheath extending until the level of the second mitochondrial ring; rounded mitochondria in cross-section, but with variable shape and organization in longitudinal and oblique sections, respectively; linear annulus; developed multilaminar membranes in the nuclear region and the midpiece. The formation of membrane filipin-sterol complexes occur sparsely along the head region, specially around the nucleus; the complexes were also present in the midpiece membrane and scarcely lining the flagellum. The complexes were present in the different layers of the multilaminar membranes. The ethanol-phosphotungstic acid (E-PTA) treatment relieved the presence of basic proteins in acrosome vesicle, pericentriolar material, peripheral fibers of the axoneme and fibrous sheath. The tannic acid technique revealed the microtubules of the centrioles and the axoneme; the extracellular tubules encircling the spermatozoa and those spread in the epididymal lumen were also observed. However, the immunocytochemistry assay using antibodies against alpha-tubulin and beta-tubulin, the primary microtubule monomers, does not support the existence of composition similarity between these tubular structures, since the extracellular tubules were not labeled by the antibodies. The results obtained in this work demonstrate that the utilization of electron microscopic techniques may provide relevant information to the study of ophidian reproductive biology, particularly in analyses concerning spermatozoal ultrastructure.