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Global coverage of living coral has declined by half since 1950s. Reef-building species have been severely impacted in this climate crisis scenario, compromising the future of coral reefs. Despite their importance, there is a lack of knowledge regarding the reproductive biology of scleractinian corals. In the present study, we evaluated through electron microscopy approaches, the gametes of the endemic Southwestern Atlantic coral Mussismilia harttii. We observed spherical oocytes with microvilli throughout the outer membrane. Fine granular material dispersed in cytoplasm, lipid granules, numerous yolk bodies, and mitochondria were identified in the oocytes. In addition, small Symbiodinium-like cells were observed, suggesting a vertical transmission from parental coral to oocytes. The spherical-head sperm presents a 9.3 ± 2.1 µm flagellum. The nucleus is located centrally in the head, and the centrioles are positioned between the nuclear base and the flagellar insertion, which is connected to the axoneme. This axoneme has a microtubular arrangement (9+2). Vesicles, underlining the inner plasma membrane, presented the same electron-dense pattern as the Golgi complex, and mitochondria positioned surrounding the axoneme. The vesicles present in the sperm may have a role as an acrosome since the oocytes do not develop any cell specialization for fertilization.

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Sperm quality is critical to predict reproductive alterations caused by immunological factors or toxicant agents. Yet, no detailed protocol has been published focusing on analyses of sperm parameters in mice. Our aim was to evaluate the most efficient diluent for mice sperm analyses and to optimize the sperm morphology classification, through the comparison of different staining methods. The diluents assessed were PBS (baseline), HTF, DMEM, 1 % BSA in PBS and 9 % skimmed powdered milk diluted in PBS. Spermatozoa were evaluated for vitality, motility, and morphology, smears were stained with Papanicolaou, HE, Giemsa, and Rapid staining. Sperm vitality and total motility reached better scores in milk based and DMEM diluents. HE raised up as an effective option since its combination with any of the diluents we tested, resulted in a fair staining, which was appropriated to evaluate mice spermatozoa. Finally, based on WHO manual, we have updated the current morphological classification for mice sperm, since we have detailed the head defects as well as included midpiece and tail defects on it. Taken together, we presented a useful, low cost, and reliable method to assess sperm morphology that could be employed worldwide by laboratories dedicated to study reproductive biology on mice model.

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Although the order Rodentia does not present a high risk of extinction compared to mammals as a whole, several families demonstrate high levels of threat and/or data deficiency, therefore highlighting the need for targeted research and the application of ecological and reproductive data to the development of conservation actions. The order Rodentia, the largest among mammals, includes 9 families, and the family Cricetidae is the most diverse of the Brazilian rodents. In Brazil, 12 of the 16 genera of Oecomys are found. Oecomys bicolor is known in Brazil as the 'arboreal rat' and is, found in dry, deciduous and tropical forests. The mean body weight of Oecomys bicolor was 35.8 g and the gonadal, tubular and epithelial somatic indexes were, 0.53%, 0.47% and 0.37%, respectively. Seminiferous tubules volume density was 89.72% and the mitotic and meiotic indexes corresponded to 8.59 and 2.45 cells, respectively, and the yield of spermatogenesis was 23.83 cells. The intertubular compartment represented 10.28% of the testis parenchyma and around 5% of the interstitial space was occupied by Leydig cells, whose number per gram of testis was 11.10 × 107 cells. By evaluating the biometric and histomorphometric characteristics of the testis, there is evidence that this species has a high investment in reproduction. Due to the high contribution of the seminiferous epithelium and the intertubular compartment in this species, compared to the others of the same family, it is possible to infer that the species Oecomys bicolor has a promiscuous reproductive behaviour.

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Natural products derived from plants can be used as photosensitizers for antimicrobial photodynamic therapy (aPDT) combining key therapeutic strategies for tissue repair while controlling microorganisms' growth. We investigated a standardized extract of pequi peels (Caryocar brasiliense Cambess) as a brownish natural photosensitizer for aPDT using blue light. Three concentrations of the pequi extract (PE; 10, 30, or 90 µg/mL) were tested solely or associated with blue laser (445 nm, 100 mW, 138 J/cm2 , 6 J, 60 s). In vitro, we quantified reactive oxygen species (ROS), assessed skin keratinocytes (HaCat) viability and migration, and aPDT antimicrobial activity on Streptococcus or Staphylococcus strains. In vivo, we assessed wound closure for the most active concentration disclosed by the in vitro assay (30 µg/mL). Upon aPDT treatments, ROS were significantly increased in cell monolayers regardless of PE concentration. PE at low doses stimulates epithelial cells. Although PE stimulated cellular migration, aPDT was moderately cytotoxic to skin keratinocytes, particularly at the highest concentration. The antimicrobial activity was observed for PE at the lowest concentration (10 µg/mL) and mostly at PE 10 µg/mL and 30 µg/mL when used as aPDT photosensitizers. aPDT with PE 30 µg/mL presents antimicrobial activity without compromising the initial phases of skin repair.

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Previously, by employing 3D organotypic tissue culture and patient-derived xenograft (PDX) model, oral myxoma response to a MAPK/MEK inhibitor was observed. Gross examination of the tumour fragments obtained after 55 days of PDX grafting revealed increased capsule vascularization. Microscopic analyses showed blood capillaries intermixed with myxoma cells, but the origin of these capillaries, from mice or humans, was not established. This study aimed to investigate whether the endothelial cells observed in the myxoma PDX model are derived from the mouse or from the primary human tumour. Immunohistochemistry was performed on five tumour fragments from the PDX of myxoma after 55 days of implantation in mice. Immunopositivity for antibodies against human (HLA-ABC) and mouse (H2 Db/H2-D1) major histocompatibility complex class I (MHCI) was assessed in the endothelial cells. The endothelial cells in the PDX fragments revealed a membrane staining for the human MHCI protein in the PDX tumour and adjacent connective tissue capsule, indicating that capillaries were derived from the human tumour fragment. Considering the probable human origin of the endothelial cells from capillary blood vessels in the myxoma PDX, we conclude that this PDX model is an interesting model to study myxoma angiogenesis.

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BACKGROUND: Central giant cell granulomas (CGCG) of the jaws are osteolytic lesions that may behave aggressively and respond poorly to surgery. Microscopically, in addition to giant cells, there is a mononuclear cell population composed of macrophage/monocytic cells and spindle-shaped cells of mesenchymal origin. Seventy two percent of these tumours harbour mutually exclusive TRPV4, KRAS and FGFR1 mutations. We aimed to assess the mutational status of mononuclear and giant cells and the osteogenic potential of stromal cells in vitro and in vivo. METHODS AND RESULTS: We screened CGCG for signature mutations and used laser-capture microdissection to demonstrate that the mutations are restricted to the mononuclear cells. Additionally, we established CGCG primary cell culture and observed that the cells retained the mutations throughout passages. By flow cytometry, we observed predominance of CD14- CD51- CD61- cells, consistent with the expected profile for stromal cells. Considering the mesenchymal origin of stromal cells, we assessed the osteogenic differentiation potential of CGCG cells in culture by cytochemistry (von Kossa and alizarin red staining), alkaline phosphatase (ALP) activity assay and gene expression of osteogenic markers. CGCG cells presented self-capacity to increase ALP levels in a time-dependent manner and under osteogenic induction presented increasing number of calcium deposits, and overall higher expression of osteocalcin, RUNX2, ALPL and osteopontin than cells without osteogenic induction. A patient-derived xenograft model for CGCG was established, and osteoid material deposition was observed. CONCLUSION: Collectively, the results confirm that the signature mutations are restricted to stromal cells in CGCG, and the in vitro and in vivo results support that these cells have the capacity to differentiate into osteoblasts, in line with the bone formation often observed in the stroma of these lesions.

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We aimed to investigate the combined effects of aerobic exercise (EXE) and cocoa flavanol (COCOA) supplementation on performance, metabolic parameters, and inflammatory and lipid profiles in obese insulin-resistant rats. Therefore, 32 male Wistar rats (230-250 g) were fed a high-fat diet and a fructose-rich beverage for 30 days to induce insulin resistance. Next, the rats were randomized into four groups, orally administered placebo solution or COCOA supplementation (45 mg·kg-1), and either remained sedentary or were subjected to EXE on a treadmill at 60% peak velocity for 30 min, for 8 weeks. Blood samples and peripheral tissues were collected and processed to analyze metabolic and inflammatory parameters, lipid profiles, and morphological parameters. Supplementation with COCOA and EXE improved physical performance and attenuated body mass gain, adipose index, and adipocyte area. When analyzed as individual interventions, supplementation with COCOA and EXE improved glucose intolerance and the lipid profile reduced the concentrations of leptin, glucose, and insulin, and reduced homeostasis assessment index (all effects were p < .001 for both interventions), while ameliorated some inflammatory mediators in examined tissues. In skeletal muscles, both COCOA supplementation and EXE increased the expression of glucose transporter (p < .001 and p < .001), and combined intervention showed additive effects (p < .001 vs. COCOA alone or EXE alone). Thus, combining COCOA with EXE represents an effective nonpharmacological strategy to treat insulin resistance; it could prevent Type 2 diabetes mellitus by improving physical performance, glucose metabolism, neuroendocrine control, and lipid and inflammatory mediators in the liver, pancreas, adipose tissue, and skeletal muscle in obese male insulin-resistant rats.

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Yellowish myotis is a Neotropical vespertilionid bat that presents a seasonal reproduction. The sperm is produced in the Mature stage, stored in the Regressed stage and released in the Rest stage (mating period). Aiming to understand, for the first time, the relationship between testis and epididymis physiology in yellowish myotis reproduction, the spermatogenesis length, sperm production, and seminal parameters were herein evaluated. Fifty-one adult male bats were captured in Santuário do Caraça, Minas Gerais, Brazil. The gonads were collected in the Maturing and Mature stages for histomorphometric and immunohistochemical analyses, whereas the epididymis was evaluated in all reproductive stages for seminal studies. Our results demonstrated that the yellowish myotis spermatogenic process is fast, lasting 31.70 ± 0.15 days. Despite the low Sertoli cell efficiency (6.60 ± 1.23), the high numbers of Sertoli cells per testis enable an elevated sperm production in the Mature stage. The sperm concentration, vitality, and motility presented the highest values in the Regressed stage; however, in this period, an increased incidence of sperm morphological defects was detected. In the following period (Rest stage), a drastic reduction of defective sperm was observed, suggesting quality control of sperm before the mating period. Furthermore, the epididymis ability to maintain a long-term sperm-storage was observed in 26.7% of the bats in the Maturing stage. In summary, yellowish myotis presented a fast and high sperm production during the Mature stage. These sperms are stored and selected before mating period.

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OBJECTIVE: The aim of this study was to verify the effects of consumption of a high-fat diet (HFD) combined with fructose-rich beverages (FRT) in promoting metabolic and physiologic changes associated with insulin resistance. METHODS: Thirty-two male Wistar rats (250 ± 10 g) were randomly allocated into four groups (n = 8) that received either a standard diet (CON), HFD, FRT, or HFD + FRT for 30 d. Insulin sensitivity and glucose tolerance were evaluated using the insulin tolerance test (ITT) and oral glucose tolerance test (OGTT). Serum samples were used to analyze the metabolic parameters and hormone levels. Interleukin (IL)-6, IL-10, IL-1ß, and tumor necrosis factor-α assays were performed in the liver, pancreas, gastrocnemius muscle, and epididymal adipose tissue by enzyme-linked immunosorbent assay. Histologic and morphometric analyses were performed on the liver, pancreas, and adipose tissues. RESULTS: Consumption of HFD + FRT promoted a significant increase (P < 0.05) in body weight, index adiposity, and in the area under the curve of ITT (P < 0.001) and OGTT (P < 0.001) when compared with the CON group. Consumption of FRT alone increased fasting glucose (P = 0.015), insulin (P = 0.035), and homeostasis model assessment index (P = 0.018), and these changes were of greater magnitude when FRT was combined with HFD. Moreover, the rats fed an HFD + FRT demonstrated a significant increase in lipid droplets in the liver (P < 0.001), an increase in adipocyte area, and an increase in inflammatory cytokines in the liver, pancreas, skeletal muscle, and adipose tissue. CONCLUSION: Consumption of an HFD + FRT promotes insulin resistance, increases inflammatory cytokines, and modulates histomorphometric parameters of the liver, pancreas, and adipose tissue, typical of insulin resistance in humans.

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The sexual segment of the kidney (SSK) is a hypertrophied region of the nephron, which occurs in males of most squamate species that have been investigated, at least, during the active season. Many studies have shown that the SSK has a seasonal secretory cycle that could be correlated to the mating season, testicular activity, and androgen synthesis. However, to date, no study has investigated the presence of androgen receptors (AR) in cells of the SSK, nor the relation between the expression of AR, testosterone levels, and testicular condition. The SSK in Crotalus durissus corresponds to the distal segment of the nephron and presents a peak of hypertrophy during the period of testicular activity (spermatogenesis) and high testosterone levels, suggesting that seasonal variation of the SSK might be under the control of androgens. Testosterone concentrations and expression of AR varied seasonally with increased values for both parameters directly correlated to hypertrophy of the SSK. This study is, therefore, the first to target the SSK of a tropical snake and to establish a relationship between the secretory cycle of the SSK, testicular cycle, and levels of androgens. Furthermore, this study is the first to identify the presence of AR in the nucleus of the SSK cells.

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In this study we prepared annatto-loaded cellulose acetate nanofiber scaffolds and evaluated both in vitro cytotoxicity and potential for wound healing in a rat model. Annatto extract, which has been used to accelerate wound healing, was added to cellulose acetate polymer and the resulting material was used to produce nanofiber scaffolds via electrospinning. Physicochemical, and thermal evaluation of the resulting nanofiber mats showed that incorporating annatto did not significantly affect the thermal or chemical stability of the polymer. Annatto extract did not demonstrate cytotoxicity in the HET-CAM assay or MTT assay for fibroblast culture. Scanning electron microscopy of the fibroblasts confirmed that cells spread and penetrated into the nanofiber. In vivo experiments confirmed that cellulose acetate retained its biocompatibility when associated with crude annatto extract, and suggested that dose/response modulation occurs between the annatto-functionalized nanofibers and mast cells, indicating the potential of this material for wound healing applications.

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Mice are widely used as experimental models due to several positive characteristics and in particular their suitability for studies involving molecular biology and transgenesis. Despite the large number of mice strains currently available, the literature regarding their basic reproductive biology is still relatively scarce. Herein, we comparatively evaluated several important and correlated parameters related to testis structure and function in sexually mature male mice of inbred (C57BL/6, n = 19; BALB/c, n = 17) and outbred (Swiss, n = 17) strains, frequently utilized in research. Swiss mice presented significant variation for many parameters evaluated, including higher sperm production, mainly when compared to the C57BL/6 strain. However, some key parameters such as the duration of spermatogenesis, the Sertoli cell number per testis, and the spermatogenic efficiency were similar among the different strains. Although presenting significantly higher Leydig cell (LC) proportion and numbers per testis gram and per testis, the anogenital index was smaller in Swiss mice. Estradiol levels were lower in C57BL/6, whereas testosterone levels and 3ß-HSD expression were similar among strains. Regarding the LC/macrophages relationship, in comparison to the literature, we reported a much higher contribution of macrophages to the mouse intertubule. Thus, we estimated that there are around 1.6 macrophages per LC in BALB/c mice and this intriguing finding could be relevant to testis function in overall and spermatogonial biology in particular. Taken together, our results highlight the importance of knowing more accurately the testis structure and function in the different mice strains available for research, particularly when a specific testis parameter is being investigated.

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PURPOSE: Testicular torsion is an urological emergency and an important cause of male infertility. Recent studies have shown that not only ischemia, but also the restoration of blood flow creates a deleterious situation for the testis. The goal of this work was to evaluate the role of systemic epithelial growth factor application and its association with decompressive fasciotomy in tissue recovery of the testes undergoing the torsion of the spermatic cord. METHOD: Torsion of the spermatic cord was induced in 40 Wistar pubescent testes for a period of 4 h. At the time of detorsion systemic epithelial growth factor and decompressive fasciotomy were applied as treatment alternatives. After 21 days, the testicles were examined macro and microscopically. RESULTS: The variables evaluated were weight, testicular volume and the degree of histological alterations by optical microscopy. The rate of healing of testicular torsion showed significant difference between the groups. It was noted that in terms of testicular weight and volume the animals treated with systemic epithelial growth factor resembled the control group and had a better histological classification than the other groups. The mean Johnsen score was significantly higher in the group treated with systemic growth factor and decompressive testicular fasciotomy than in the group without fasciotomy. No significant changes were noted in the contralateral testes. CONCLUSION: Although decompressive testicular fasciotomy improved spermatogenesis, recovery was limited. Systemic epithelial growth factor administered in combination with decompressive testicular fasciotomy was more effective for spermatogenesis than fasciotomy alone. Administering epithelial growth factor after reperfusion might have the potential to decrease long-term histologic damage after testicular torsion. The combination of decompressive testicular fasciotomy and epithelial growth factor had a synergistic effect on the healing of these testes.

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Mutations in Foxn1 and Prkdc genes lead to nude and severe combined immunodeficiency (scid) phenotypes, respectively. Besides being immunodeficient, previous reports have shown that nude mice have lower gonadotropins and testosterone levels, while scid mice present increased pachytene spermatocyte (PS) apoptosis. Therefore, these specific features make them important experimental models for understanding Foxn1 and Prkdc roles in reproduction. Hence, we conducted an investigation of the testicular function in nude and scid BALB/c adult male mice and significant differences were observed, especially in Leydig cell (LC) parameters. Although the differences were more pronounced in nude mice, both immunodeficient strains presented a larger number of LC, whereas its cellular volume was smaller in comparison to the wild type. Besides these alterations in LC, we also observed differences in androgen receptor and steroidogenic enzyme expression in nude and scid mice, suggesting the importance of Foxn1 and Prkdc genes in androgen synthesis. Specifically in scid mice, we found a smaller meiotic index, which represents the number of round spermatids per PS, indicating a greater cell loss during meiosis, as previously described in the literature. In addition and for the first time, Foxn1 was identified in the testis, being expressed in LC, whereas DNA-PKc (the protein produced by Prkdc) was observed in LC and Sertoli cells. Taken together, our results show that the changes in LC composition added to the higher expression of steroidogenesis-related genes in nude mice and imply that Foxn1 transcription factor may be associated to androgen production regulation, while Prkdc expression is also important for the meiotic process.

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OBJECTIVE: Odontogenic myxoma (OM) occasionally responds poorly to surgical treatment. The MAPK pathway is constitutively activated in several neoplasms and we aimed to test if the MAPK pathway is activated in OM, in order to pave the way for an alternative therapy for aggressive and recurrent cases. MATERIALS AND METHODS: The immunoexpression of phosphorylated ERK1/2 (pERK1/2) was assessed in OM. We established a 3D organotypic culture model for the in vitro study and patient-derived xenografts (PDX) in mice for the in vivo study. The MEK inhibitor U0126 was used to inhibit phosphorylation of ERK1/2 in the in vitro and in vivo models. RESULTS: All OM showed strong pERK1/2 immunoexpression, consistent with MAPK pathway activation. Treatment of the 3D culture with U0126 resulted in a reduced pERK1/2/ERK1/2 ratio. Consistent with the in vitro results, all PDX of animals treated with U0126 showed a decreased volume fold change compared with controls. CONCLUSIONS: The MAPK pathway is activated in OM and its inhibition leads to tumor shrinkage in PDX and cell culture models. CLINICAL RELEVANCE: Our results offer a pre-clinical frame for OM-targeted therapy. Further work is needed to determine if this initial finding holds clinical promise.

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AIMS: To investigate the effects of moderate aerobic physical training on cardiac function and morphology as well as on the levels of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) of animals infected with the Y strain of Trypanosoma cruzi. MAIN METHODS: Twenty-eight male C57BL/6 mice were distributed into 4 groups: sedentary control (SC), trained control (TC), sedentary infected (CHC) and trained infected (CHT). The infection was performed by intraperitoneal injection of trypomastigote forms and the animals were adapted to treadmill in the week before the beginning of the training protocol, initiated 45 days post infection. Maximal exercise test (TEM) was performed at the baseline as well as at the end of the 4th, 8th and 12th weeks of training. At the end of the 12th week, all animals were evaluated for cardiac morphology and function by echocardiography. KEY FINDINGS: CHC group showed a larger area of right ventricle (RVA), increased end-systolic volume and reduction in ejection fraction (EF), stroke volume (SV), cardiac output (CO) and fractional area change (FAC). The training reduced the RVA and improved the FAC of chagasic animals. GDNF level was higher in TC and CHC groups compared to SC in heart and BDNF levels were higher in CHC compared to SC in heart and serum. SIGNIFICANCE: Physical training ameliorated the cardiac function of infected animals and promoted adjusts in BDNF and GDNF levels. These findings evidenced these neurotrophins as possible biomarkers of cardiac function responsive to exercise stimulus.

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OBJECTIVES: The establishment of animal models of xenotransplantation can contribute to the elucidation of the molecular pathogenesis of ameloblastic fibrodentinomas (AFD) and it also provides an opportunity for drug tests. We aimed to evaluate the possibility of AFD tumour growth in a patient-derived xenograft (PDX) model. In addition, we characterized the human tumour and the PDXs. MATERIALS AND METHODS: A sample of a recurrent AFD was obtained and two fragments were contralaterally implanted subcutaneously in an 8-week old female NUDE mouse. After 250 days, the PDXs were removed and submitted to histopathological and molecular analysis. Immunohistochemical reactions for Ki67 and the phosphorylated form of ERK1/2 were carried out in both, PDXs and human tumour, and the presence of BRAFV600E was assessed. RESULTS: From day 135 onwards, the PDXs presented a growth peak and remained stable until day 250. Histopathologically, the PDXs presented the same features of the patient's tumour. Tumour cells exhibited Ki67 and pERK1/2 immunoexpression in the patient's tumour and PDX. The AFD was wild-type for BRAFV600E. CONCLUSION: The PDX model recapitulated well the human tumour after a long implantation time, representing a possible model to study the AFD and other odontogenic tumours pathobiology.

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Collared peccaries (Tayassu tajacu) present a unique testis cytoarchitecture, where Leydig cells (LC) are mainly located in cords around the seminiferous tubules (ST) lobes. This peculiar arrangement is very useful to better investigate and understand the role of LC in spermatogonial stem cells (SSCs) biology and niche. Recent studies from our laboratory using adult peccaries have shown that the undifferentiated type A spermatogonia (Aund or SSCs) are preferentially located in ST regions adjacent to the intertubular compartment without LC. Following these studies, our aims were to investigate the collared peccary postnatal testis development, from birth to adulthood, with emphasis on the establishment of LC cytoarchitecture and the SSCs niche. Our findings demonstrated that the unique LC cytoarchitecture is already present in the neonate peccary's testis, indicating that this arrangement is established during fetal development. Based on the most advanced germ cell type present at each time period evaluated, puberty (the first sperm release in the ST lumen) in this species was reached at around one year of age, being preceded by high levels of estradiol and testosterone and the end of Sertoli cell proliferation. Almost all gonocytes and SSCs expressed Nanos1, Nanos2 and GFRA1. The analysis of SSCs preferential location indicated that the establishment of SSCs niche is coincident with the occurrence of puberty. Taken together, our findings reinforced and extended the importance of the collared peccary as an animal model to investigate testis function in mammals, particularly the aspects related to testis organogenesis and the SSCs biology and niche.

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Zika virus (ZIKV) is a teratogenic mosquito-borne flavivirus that can be sexually transmitted from man to woman. The finding of high viral loads and prolonged viral shedding in semen suggests that ZIKV replicates within the human male genital tract, but its target organs are unknown. Using ex vivo infection of organotypic cultures, we demonstrated here that ZIKV replicates in human testicular tissue and infects a broad range of cell types, including germ cells, which we also identified as infected in semen from ZIKV-infected donors. ZIKV had no major deleterious effect on the morphology and hormonal production of the human testis explants. Infection induced a broad antiviral response but no IFN upregulation and minimal proinflammatory response in testis explants, with no cytopathic effect. Finally, we studied ZIKV infection in mouse testis and compared it to human infection. This study provides key insights into how ZIKV may persist in semen and alter semen parameters, as well as a valuable tool for testing antiviral agents.

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