Adenylyl cyclase 3 deficiency results in dysfunction of blood-testis barrier during mouse spermiogenesis.

Human infertility has become a global medical and social health problem. Mice deficient in type 3 adenylyl cyclase (AC3), a key enzyme that synthesizes cyclic adenosine monophosphate (cAMP), develop male infertility, although the underlying molecular mechanisms remain unknown. We performed a label-free quantitative (LFQ) proteomics analyses to identify testicular differentially expressed proteins (DEPs) and their respective biological processes. Furthermore, histological examination demonstrated that AC3 deficiency in mice led to mild impairment of spermatogenesis, including the thinning of seminiferous epithelium and local lesions in the testis. We further identified that the integrity of the blood-testis barrier (BTB) was impaired in AC3 knockout (AC3-/-) mice accompanied with the reduction in the expression of tight junctions (TJs) and ectoplasmic specialization (ESs)-related proteins. In addition, the deletion of AC3 in mice also reduced the germ cell proliferation, increased apoptosis, and decreased lipid deposition in the seminiferous tubules. Collectively, our results revealed a role of AC3 in regulating the BTB integrity during spermatogenesis. Thus, our findings provide new perspectives for future research in male infertility.

Dynamics of hyperacetylated histone H4 (H4Kac) during spermatogenesis in four decapod crustaceans.

During spermatogenesis, the transition from histone to protamine is highly conserved in most invertebrates and vertebrates. Thus far, a large and growing body of literature has demonstrated that histones and histone modifications still exist in the sperm nucleus of decapod crustaceans. H4Kac is believed to play an important role in the process of sperm chromatin condensation. However, the dynamics of hyperacetylated histone H4 (H4Kac) during spermatogenesis in decapoda are still unknown. In this paper, the distribution of H4Kac in four decapod crustaceans (Eriocheir sinensis, Charybdis japonica, Procambarus clarkii, and Macrobrachium nipponense) were investigated via immunofluorescence. Our results indicated that H4Kac was visible in the mature sperm nucleus of E. sinensis, C. japonica, and M. nipponense. Unlike the other three species, H4Kac was translocated from the nuclei to cytoplasm in mid-spermatids of P. clarkii. Eventually, H4Kac were not present in mature spermatozoa of P. clarkii. Importantly, we observed for the first time that H4Kac was distributed outside the nucleus, which reminds us that H4Kac may participate in the formation of acrosome structure in decapod crustaceans and may be a prerequisite for proper chromatin decondensation.

Automated Radiology Alert System for Pneumothorax Detection on Chest Radiographs Improves Efficiency and Diagnostic Performance.

We aimed to set up an Automated Radiology Alert System (ARAS) for the detection of pneumothorax in chest radiographs by a deep learning model, and to compare its efficiency and diagnostic performance with the existing Manual Radiology Alert System (MRAS) at the tertiary medical center. This study retrospectively collected 1235 chest radiographs with pneumothorax labeling from 2013 to 2019, and 337 chest radiographs with negative findings in 2019 were separated into training and validation datasets for the deep learning model of ARAS. The efficiency before and after using the model was compared in terms of alert time and report time. During parallel running of the two systems from September to October 2020, chest radiographs prospectively acquired in the emergency department with age more than 6 years served as the testing dataset for comparison of diagnostic performance. The efficiency was improved after using the model, with mean alert time improving from 8.45 min to 0.69 min and the mean report time from 2.81 days to 1.59 days. The comparison of the diagnostic performance of both systems using 3739 chest radiographs acquired during parallel running showed that the ARAS was better than the MRAS as assessed in terms of sensitivity (recall), area under receiver operating characteristic curve, and F1 score (0.837 vs. 0.256, 0.914 vs. 0.628, and 0.754 vs. 0.407, respectively), but worse in terms of positive predictive value (PPV) (precision) (0.686 vs. 1.000). This study had successfully designed a deep learning model for pneumothorax detection on chest radiographs and set up an ARAS with improved efficiency and overall diagnostic performance.

The type 3 adenylyl cyclase is crucial for intestinal mucosal neural network in the gut lamina propria.

BACKGROUND: The type 3 adenylyl cyclase (AC3) enzyme is involved in the synthesis of cyclic adenosine monophosphate (cAMP). It is primarily expressed in the central nervous system (CNS) and plays a crucial role in neurogenesis and neural dendritic arborization. However, the AC3's functional role in the gastrointestinal tract remains ambiguous. METHODS: AC3 expression in enteric tissue of AC3+/+ mice was investigated using immunohistochemistry and RT-PCR. AC3 knock-out mice (AC3-/- ) were used to examine the effect of AC3 on the enteric nervous system (ENS) function and the number of cilia and apoptotic cells. Additionally, total gastrointestinal transit time and colonic motility were compared between the AC3-/- and AC3+/+ groups of mice. KEY RESULTS: AC3 was predominately expressed in the myenteric plexus of the large intestine. Colonic-bead expulsion analysis showed accelerated propulsion in the large intestine of the AC3-/- mice. The AC3-/- mice demonstrated reduced nerve fibers and enteric glial cells count in colonic mucosa compared to the AC3+/+ mice. Furthermore, AC3-/- mice exhibited increased cellular apoptosis and reduced ARL13B+ cilium cells in the colonic lamina propria compared to the AC3+/+ mice. CONCLUSIONS: In AC3-/- mice, innervation of the lamina propria in the colonic mucosa was reduced and colonic propulsion was accelerated. AC3 is crucial for the development and function of the adult neural network of ENS. AC3 deficiency caused atrophy in the colonic mucosal neural network of mice.

Spermiogenic histone transitions and chromatin decondensation in Decapoda.

Decapoda are among of the most diverse groups of Crustacea with an important economic value, and have thus been the focus of various reproductive biology studies. Although spermatozoa are morphologically diverse, decapod spermatozoa possess common features, such as being non-motile and having uncondensed nuclear chromatin. Many scholars have studied uncondensed chromatin in decapod spermatozoa; however, the role of biologically regulated decondensation in spermatozoa remains unclear. In this study, histone changes in the spermatozoa of five commercially relevant aquatic crustacean species (Eriocheir sinensis, Scylla paramamosain, Procambarus clarkii, Fenneropenaeus chinensis, and Macrobrachium nipponense) were studied via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunofluorescence. The LC-MS/MS results confirmed that all four core histones were present in the sperm nuclei of the five Decapoda species. Positive fluorescent signals from histones H2A, H2B, H3, and H4 were detected in the spermatozoa nuclei of E. sinensis, S. paramamosain and M. nipponense via immunofluorescence. Histone H2A was first identified in the membrane sheets or cytoplasm of mature sperm in P. clarkii and F. chinensis, whereas H3 and H4 were generally distributed in the nucleus of the spermatozoa. Histone H2B gradually disappeared during spermiogenesis and was not found in the sperm of P. clarkii and F. chinensis eventually. Our data suggest that core histones are instructive and necessary for chromatin decondensation in decapods spermatozoa. Thus, our results may help resolve the complex sperm histone code and provide a reference for the study of spermatozoa evolution in Decapoda.

H4S1ph, an alternative epigenetic marker for sperm maturity.

Histone phosphorylation, an epigenetic post-translational modification, plays essential roles in male gamete chromatin compaction during spermatogenesis and sperm maturity. Previously, we studied the epigenetic marker of phosphorylated serine 1 of histone H2A and H4 (HS1ph) during spermatogenesis in mice and crabs, which was shown to be closely related to the sperm maturity. To further investigate the correlation between phosphorylated serine 1 of histone H4 (H4S1ph) and sperm maturation, a comparison study was conducted in this work between the healthy and the precocious crabs. It was discovered that the distribution of H4S1ph was similar for the two groups of crabs during spermatogenesis before maturity, but totally different in the sperm nuclei. H4S1ph vanished in the nuclei of healthy crab spermatozoa mostly, while retained in the precocious crabs just like what it was in elongated spermatid of both kinds of crabs. The results showed that a high level of H4S1ph conservation was closely associated with immaturity and might indicate inefficient fertility of male precocious crabs. Thus, H4S1ph was suggested to be an epigenetic marker of sperm maturity.

Ultrastructure of spermiogenesis and the distribution of spermatozoal nuclear histones in the Japanese mantis shrimp, Oratosquilla oratoria (Crustacea: Stomatopoda).

The Japanese mantis shrimp Oratosquilla oratoria (Stomatopoda; Crustacea) is one of the most economically important aquatic species of Pacific shrimp and it is distributed from Japan to the coast of China, the Philippines, the Malay Peninsula, and the Hawaiian Islands. Early studies described certain characteristics of spermatogenesis and the sperm ultrastructure in Stomatopoda, but the composition of sperm basic nuclear proteins (SBNPs) remains completely unknown. We studied the sperm ultrastructure of O. oratoria using transmission electron microscopy and the histone composition using immunofluorescence and immunoelectron microscopy. We found that the spherical nucleus is adjacent to the electron translucent external coat, which occurs in early spermatids. The acrosomal structure begins to form at the junction of the nucleus and the external coat. At the mid-spermatid stage, part of the chromatin appears to be more electron-dense than the external coat side. The aflagellate sperm of O. oratoria, are rounded or slightly ovoid in shape and have a consistent granular nucleus, an acrosome structure of pushpin shape and a spherical vesicular body in which faintly granular material is scattered. The acrosome consists of an acrosomal vesicle, perforatorium, and subacrosomal material. The sperm contains histones H2A, H2B, H3, H4, H3.3, H2AX, and H2AZ as well as some histone modifications, that is, H3K9me3, H3K4me2, H3S10ph, H4Kac, and H2A + H4S1ph. Histones are localized not only in the nucleus of the sperm but also in other structures outside the nucleus. The results may provide new perspectives for systematic studies of crustaceans and their sperm chromatin components. These findings extend the study of the sperm structure of Stomatopoda and provide basic data to elucidate the epigenetic mechanism of fertilization.

Resected specimen size: A reliable predictor of severe Frey syndrome after parotidectomy.

BACKGROUND: Frey syndrome is a common complication after parotidectomy. This study aimed to investigate the potential predictors for developing severe Frey syndrome after parotidectomy and to identify patients who may benefit from additional preventive maneuvers. METHODS: A total of 485 patients received parotidectomy because of parotid tumors at the Otolaryngology Department of the National Cheng Kung University Hospital, from July 2009 to November 2015. Only 115 of 485 patients were included in this study and to fill in a questionnaire to determine the occurrence and severity of Frey syndrome. RESULTS: A total of 115 parotidectomies were identified. 84 (73%, 84/115) patients were aware of the discomfort and were thus considered symptomatic. 39 (34%, 39/115) patients considered the symptoms apparently affected their quality of life. MSI tests showed that 56 (49%, 56/115) patients had a positive MSI test. By combining the results from symptom questionnaire and MSI test, 23 patients (20%, 23/115) had a severe form of Frey syndrome. Among all clinicopathological variables, the resected specimen size was the only significant predictor of the severe Frey syndrome group (P = 0.04). Disease pathology, tumor size, and adjuvant radiotherapy did not correlate with the severe Frey syndrome. Using receiver operating curve analysis, the best cutoff value of the resected specimen size (in largest dimension) for predicting severe Frey syndrome was 40 mm(sensitivity: 71.7%, specificity: 42.0%; area under the curve = 0.6483). The odds ratio of severe Frey syndrome with every 10 mm increase in the largest diameter of resected specimen was 1.30 (95% confidence interval, 1.01-1.68; P = 0.04). CONCLUSIONS: Resected specimen size is the only significant predictor of developing severe Frey syndrome after parotidectomy. Preventive interventions may have to be considered in high-risk patients whose resected specimen size (in largest dimension) is greater than 40 mm.

Histone H2B gene cloning, with implication for its function during nuclear shaping in the Chinese mitten crab, Eriocheir sinensis.

Spermatogenesis in animals is the process by which male spermatogonia develop into mature spermatozoa. In most taxa, the process involves changes in the basic proteins associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines producing compact packaging of the genome. Sperm chromatin in the Chinese mitten crab (Eriocheir sinensis) has a noncompacted loosely arranged organization. However, its formation during spermatogenesis is not clear. In this study, a cDNA sequence encoding histone H2B was cloned by polymerase chain reaction amplification, and its recombinant protein was expressed and purified. Protein alignment studies demonstrated that this histone H2B had 80.80%, 95.12%, 80.16%, 91.87%, 81.75%, 77.78% and 99.19% identity with its counterparts in zebrafish, fruit fly, human, prawn, mouse, African clawed frog, and crayfish, respectively. Western blotting indicated that the recombinant protein could be recognized by an anti-H2B antibody and confirmed that histone H2B exists in sperm nuclei. Immunofluorescence demonstrated that histone H2B was present in the nuclei of spermatogonia, spermatocytes, spermatids, and mature spermatozoa. This is the first report that the mature sperm nucleus of E. sinensis contains histone H2B. This work complements a previous study of sperm histones of this species and provides a basis for further study of the noncondensed sperm nuclei of decapod crustaceans.