Myosin IIB and F-actin control apical vacuolar morphology and histamine-induced trafficking of H-K-ATPase-containing tubulovesicles in gastric parietal cells.

Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle.

Ex vivo human testes as a practical model to simulate ultrasound-guided testicular cell transplantation for human fertility restoration.

OBJECTIVE: To develop an ex vivo model to practice ultrasound-guided injection of cellular material into human seminiferous tubules to simulate testicular cell transplantation (TCT). DESIGN: Simulated TCT injections were performed in human testes removed during orchiectomy. The rete testis was the target site of injection. Successful retrograde infiltration of injected material into the lumen of the seminiferous tubules was detected using ultrasound and confirmed with histology. SETTING: Single academic surgical center. PATIENT(S): Adult patients undergoing orchiectomy for nononcologic indications. INTERVENTION(S): The testes were injected with sonographic contrast (Optison), methylene blue, and fluorescent-labeled cells. MAIN OUTCOME MEASURE(S): A characteristic streaming pattern of sonographic contrast in the testis was used to define sonographic success, and the presence of methylene blue and fluorescent-labeled cells within the seminiferous tubules confirmed histologic success. RESULT(S): We performed simulated TCT injections in 30 testes obtained from 16 patients undergoing orchiectomy. We were able to achieve sonographic success in 57% of injections and confirmed that sonographic success is correlated with histologic success. CONCLUSION(S): Testicular cell transplantation injections can be practiced using human testes. As there appears to be a learning curve associated with this procedure, developing this infrastructure to practice these skills is critical before implementation in patients.

Five-Year Surveillance of Vitamin D Levels in NCAA Division I Football Players: Risk Factors for Failed Supplementation.

BACKGROUND: Monitoring vitamin D levels in athletes and determining their response to supplementation in cases of deficiency is thought to be necessary to modulate the risks associated with vitamin D deficiency. HYPOTHESIS/PURPOSE: To report the results of a 5-year-long surveillance program of vitamin D in the serum of football players on a National Collegiate Athletic Association (NCAA) Division I team and to examine whether factors including age, body mass index (BMI), race, position played, and supplement type would affect the response to 12-month oral vitamin D replacement therapy in athletes with deficiency. We hypothesized that yearly measurements would decrease the proportion of athletes with vitamin D insufficiency over the years and that the aforementioned factors would affect the response to the supplementation therapy. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: We measured serum 25(OH)D levels (25-hydroxyvitamin D) in 272 NCAA Division I football players from our institution annually between 2012 and 2017. Athletes with insufficient vitamin D levels (<32 ng/mL) received supplementation with vitamin D3 alone or combined vitamin D3/D2. The percentage of insufficient cases between the first 2 years and last 2 years of the program was compared, and yearly team averages of vitamin D levels were calculated. Associations between player parameters (age, BMI, race, team position, supplement type) and failed supplementation were evaluated. RESULTS: The prevalence of vitamin D insufficiency decreased significantly during the study period, from 55.5% in 2012-2013 to 30.7% in 2016-2017 (P = .033). The mean 25(OH)D level in 2012 was 36.3 ng/mL, and this increased to 40.5 ng/mL in 2017 (P < .001); however, this increase was not steady over the study period. Non-Hispanic athletes and quarterbacks had the highest average 25(OH)D levels, and Black players and running backs had the lowest overall levels. There were no significant differences in age, BMI, race, or playing position between athletes with and without failed vitamin D supplementation. Athletes receiving vitamin D3 alone had a more successful rate of conversion (48.15%) than those receiving combined vitamin D3/D2 (22.22%; P = .034). CONCLUSION: To decrease the prevalence of vitamin D deficiency in football players, serum vitamin D measurements should be performed at least once a year, and oral supplementation therapy should be provided in cases of deficiency. Black players might be at increased risk of vitamin D insufficiency. Oral vitamin D3 may be more effective in restoring vitamin D levels than combined vitamin D3/D2 therapy.

Semen amyloids participate in spermatozoa selection and clearance.

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens.