Protein In Testis Research Articles

Mevalonate pathway and male reproductive aging.

Male fertility declines with age. The mevalonate pathway, through which cholesterol and nonsteroidal isoprenoids are synthesized, plays key role in metabolic processes and is an essential pathway for cholesterol production and protein prenylation. Male reproductive aging is accompanied by dramatic changes in the metabolic microenvironment of the testis. Since the mevalonate pathway has an important role in spermatogenesis, we attempted to explore the association between male reproductive aging and the mevalonate pathway to explain the mechanism of male reproductive aging. Alterations in the mevalonate pathway may affect male reproductive aging by decreasing cholesterol synthesis and altering testis protein prenylation. Decreased cholesterol levels affect cholesterol modification, testosterone production, and remodeling of germ cell membranes. Aging-related metabolic disorders also affect the metabolic coupling between somatic cells and spermatogenic cells, leading to male fertility decline. Therefore, we hypothesized that alterations in the mevalonate pathway represent one of the metabolic causes of reproductive aging.

Loss of Atg5 in Sertoli cells enhances the susceptibility of cadmium-impaired testicular spermatogenesis in mice.

Cadmium (Cd), one of the most common contaminants in diet and drinking water, impairs testicular germ cell development and spermatogenesis. Autophagy is essential for maintaining Sertoli cell function and Sertoli-germ cell communication. However, the role of Sertoli cell autophagy in Cd-caused spermatogenesis disorder remains unclear. Here, the mice of autophagy-related gene 5 (Atg5) knockouts in Sertoli cells were used to investigate the effect of autophagy deficiency on Cd-impaired spermatogenesis and its underlying mechanisms. Results showed that Sertoli cell-specific knockout of Atg5 exacerbated Cd-reduced sperm count and MVH (a specific marker for testicular germ cells) level in mice. Additionally, Sertoli cell Atg5 deficiency reduced the number of spermatocytes and decreased the level of meiosis-related proteins (SYCP3 and STRA8) in Cd-treated mouse testes. Loss of Atg5 in Sertoli cell exacerbated Cd-reduced the level of retinoic acid (RA) and retinal dehydrogenase (ALDH1A1 and ALDH1A) in mouse testes. Meanwhile, we found that the level of transcription factor WT1 was significantly downregulated in Atg5-/- plus Cd-treated testes. Further experiments showed that Wt1 overexpression restored Cd-decreased the levels of ALDH1A1 in Sertoli cells. Collectively, the above data suggest that knockout of Atg5 in Sertoli cell enhances the susceptibility of Cd-impaired testicular spermatogenesis. These findings provide new insights into autophagy of Sertoli cell preventing environmental toxicants-impaired testicular spermatogenesis.

Clinicopathological analysis of nuclear protein in testis midline carcinoma

Objective: To investigate the clinicopathological features, immunophenotype and prognosis of nuclear protein in testis (NUT) midline carcinoma. Methods: Twenty-four resection cases of NUT midline carcinoma diagnosed at the Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China from January 2018 to September 2022, were collected, and retrospectively analyzed for their clinicopathological characteristics. Relevant literature was reviewed. Results: All 24 cases of NUT midline carcinoma occurred in the chest or head and neck, including 14 men and 10 women, with a median age of 40 years. Histological examination showed that the tumors were poorly differentiated, with solid nested or sheet-like arrangement, small to medium-sized cells, sparse cytoplasm and coarse granular chromatin, including 5 cases with abrupt squamous epithelial differentiation. Immunohistochemistry showed that all 24 cases were positive for NUT protein, while 16 cases were p63 positive, 19 cases were p40 positive, 15 out of 18 cases were CK5/6 positive. Follow-up data were obtained for 21 patients (follow-up time range, 1-21 months), of which 11 survived, 10 died, and 3 were lost to follow-up. Conclusions: NUT midline carcinoma is a rare and highly aggressive malignancy with unique histological, immunophenotypic and molecular features. It has a poor prognosis.

Expression of Zonula Occludens-1 and Claudin-1 Proteins in Japanese Quails Testis

Expression of Zonula Occludens-1 and Claudin-1 Proteins in Japanese Quails Testis

NUT carcinoma in head and neck region: Case report with literature review

Abstract Squamous cell carcinoma (SCC) has several variants based on its histopathological features. Nuclear protein in testis (NUT) carcinoma (NC) is a rare and aggressive variant of SCC, previously described exclusively in midline sites. The histopathological features of NC are similar to poorly differentiated carcinoma or undifferentiated carcinoma. Abrupt keratinization in an otherwise undifferentiated carcinoma is an important diagnostic clue. The confirmatory diagnosis is dependent on molecular techniques such as Immunohistochemistry, Fluorescent in situ hybridization technique or RT-PCR to detect mutations in NUT gene. It is most commonly found in middle aged, in lungs and head and neck regions. Since NC features overlap with poorly differentiated and undifferentiated carcinomas, these cases need to be suspected and evaluated for NUT gene mutations thoroughly. Due to their rarity and less known facts, NC cases are required to be reported on large scale. Here, we report a middle-aged woman with a mass on the forehead diagnosed as NC based on molecular evaluation, with a review of the literature emphasizing the rarity of NUT carcinoma and the importance of careful histopathology as well as immunohistochemistry evaluation.

Immunolocalization of Desmin, Vimentin and S-100 proteins in testis and epididymis of African striped ground squirrel (Xerus erythropus)

The aim of the study was to immunolocalize desmin, vimentin and S-100 proteins in the testes and epididymis of African striped ground squirrel (Xerus erythropus) and establish their spatial distribution in the two organs. Formalin fixed-paraffin-embedded sections of the testis and epididymis obtained from ten apparently healthy adult male African striped ground squirrels were processed routinely for immunohistochemistry, using primary antibodies specific to desmin, vimentin and S-100. S-100The results showed that desmin reacted intensely in the myoid cells of the seminiferous tubules and smooth muscle cells of the epididymal ducts. It showed a moderate positive immunoreaction in the interstitial cells of Leydig, and the epididymal inter-ductal loose connective tissue. However, there was no immunoreaction of desmin in the spermatogonia or any other spermatogenic cell of the seminiferous tubule. Vimentin reacted intensely in the Leydig cells and spermatogonia. It showed moderate positive reaction in the myoid cells and the epididymal inter-ductal loose connective tissue. There was no immunoreaction of vimentin in the other spermatogenic cells (other than the spermatogonia). The S-100 proteins expressed a mild positive immunoreaction at the interstitial cells of Leydig, but negative immunoreaction in all other parts of testis and epididymis. Also, there was intense immunoreaction of desmin and vimentin and moderate immunoreaction of S-100 in the vascular endothelium in the testis and epididymis. In conclusion, the spatial distribution of desmin, vimentin and S-100 proteins in the testes and epididymis in the African striped ground squirrel showed some similarities and contrast with other mammals, giving insight into the functions of the proteins in these organs of the rodent.

Loss-of-function variants in KCTD19 cause non-obstructive azoospermia in humans

Azoospermia is a significant cause of male infertility, with non-obstructive azoospermia (NOA) being the most severe type of spermatogenic failure. NOA is mostly caused by congenital factors, but our understanding of its genetic causes is very limited. Here, we identified a frameshift variant (c.201_202insAC, p.Tyr68Thrfs∗17) and two nonsense variants (c.1897C>T, p.Gln633∗; c.2005C>T, p.Gln669∗) in KCTD19 (potassium channel tetramerization domain containing 19) from two unrelated infertile Chinese men and a consanguineous Pakistani family with three infertile brothers. Testicular histological analyses revealed meiotic metaphase I (MMI) arrest in the affected individuals. Mice modeling KCTD19 variants recapitulated the same MMI arrest phenotype due to severe disrupted individualization of MMI chromosomes. Further analysis showed a complete loss of KCTD19 protein in both Kctd19 mutant mouse testes and affected individual testes. Collectively, our findings demonstrate the pathogenicity of the identified KCTD19 variants and highlight an essential role of KCTD19 in MMI chromosome individualization.

Altered expression of necroptotic, angiogenic and spermatogenic proteins in degenerate testes from aged stallions

We utilized RNA sequencing to compare the testis transcriptomes of fertile, adult stallions to those of adult stallions with age-related testicular degeneration (TD). Large-scale gene ontology analysis identified pathway-wide upregulation of inflammation and alterations in apoptotic pathways that likely play roles in the disease process (Woodward et al., Eq Vet J. 2022; 1-14). In humans and mice, inflammation, together with necroptosis, and alterations in capillary density, all contribute to age-related degenerative changes (Li et. al, ELife. 2017; 6:e27692 DOI: 10.7554/eLife.27692; Das et al, Cell. 2018;(173)74-89). One objective of the current study was to identify differentially expressed genes (DEGs) that may contribute similarly to TD in the horse. We also searched for DEGs that play roles in spermatogenesis. A second objective was to use immunohistochemistry (IHC) to determine if the protein products of these genes are differentially expressed between normal and diseased testes and to determine their patterns of expression. The hypothesis is that aberrations in necroptotic, angiogenic, and spermatogenic signaling pathways contribute to the pathophysiology of TD in stallions. RNA isolation and sequencing were performed on testicular tissue samples from 3 fertile adult stallions and from 3 adult stallions with confirmed TD as previously described (Woodward et al., Eq Vet J. 2022; 1-14). Transcript expression was compared between the 2 populations using either EdgeA or DESeq2, and a minimum of >1.5x fold change and Padj<0.05 asthe cutoffs for significance. Protein expression from selected DEGs was compared between normal adult and TD tissue using IHC. In degenerate testes, significant increases in gene expression were found in testicular-origin growth factors (BMP4 (9x increase), FGF2 (9x increase), and CSF1 (7x increase)). IHC confirmed increased expression of BMP4 and FGF2 proteins in germ cells from degenerate testes. Expression of CSF1R protein also was altered, with loss of expression of the protein in early germ cells, and increased expression in later stages of spermatogenesis. Upregulation of RIPK3 (3x increase) and VEGF (17x increase) transcripts also were confirmed in degenerate testes. These genes are central to necroptotic and angiogenic pathways, respectively. IHC confirmed diffuse upregulation of the RIPK3 protein in germ cells from degenerate testes. IHC for the VEGF protein is pending. In conclusion, alterations in expression of proteins responsible for stimulation of mitosis and meiosis in male germ cells were identified. Additionally, we provide evidence that chronic inflammation, necroptosis and aberrant angiogenesis may cause age-related degenerative changes in equine testes. In mice, inhibitors of the RIPK3 pathway and stimulation of angiogenesis can ameliorate clinical signs of aging in testes and muscle respectively (Li et. al, ELife. 2017; Das et al, Cell. 2018). Financial support: Grayson Jockey Club Research Foundation.

(088) The Essential Role of O-GlcNAcylation in the Testis Aging

Abstract Introduction With the delay of reproductive age, reproductive disorders related to advanced age have gradually attracted widespread attention. Studies have shown that the decline in male fertility due to aging is manifested as a decrease in semen parameters and spermatogenetic cells histologically. O-GlcNAc (O-acetylglucosamine) glycosylation is a post-translational modification that exists widely in cells. The level of UDP-GlcNAc, which is the substrate of O-GlcNAcylation, changes rapidly with various nutrients and external factors. Therefore, O-GlcNAcylation can respond to glucose, amino acid, fatty acid, and nucleoside metabolism, and is considered to be an important sensor of cellular metabolism. The previous study of our project found that the level of O-GlcNAc modification in aged testis tissue was significantly increased, so this study aimed to explore the specific mechanism of O-GlcNAc modification involved in testicular aging. Objective 1.Explore the modification pattern of O-GlcNAcylation in testis 2.Mimic the high O-GlcNAcylation state of aging mice in young mice, to study the effect of elevated high O-GlcNAcylation level on testis. 3.Reveal the role of key proteins modified by O-GlcNAc in testicular aging Methods Mice were injected intraperitoneally with OGA inhibitor Thiamet-G for 35 days to increase the level of O-GlcNAc to simulate aging. CASA system was used to detect semen parameters, and chromosome spreading technology was used to study mouse testis meiosis. Using protein profiling technology, the differentially modified O-GlcNAc proteins in aged testis and young testis were screened, and point mutations were made at their O-GlcNAc sites to study the specific mechanism of key O-GlcNAc proteins involved in testis aging. Results The O-GlcNAc modification level in the testis of aged mice was significantly increased. After intraperitoneally injecting Thiamet-G into mice for 35 days, the level of O-GlcNAcylation in the testis tissue was significantly increased. High level of O-GlcNAcylation in testis damaged the sperm parameter, decreased the thickness of seminiferous epithelium and increased apoptosis in testis. Chromosome spreading experiments showed that, γH2Ax, which is the marker of DNA double strand breaks, stained unusually at autosomes in pachytene spermatocytes. The elevation of γH2Ax affected the transition of pachytene spermatocytes to diplotene. Importantly, we found that the O-GlcNAcylation of Hyou1 protein was significantly increased in the testis of aged mice. O-GlcNAcylation of Hyou1 promotes the degradation of Hyou1. An O-GlcNAc site of Hyou1 was identified at S634 and mutation of this site decreased the O-GlcNAcylation, increased the protein stability and exerts protective roles in aging testis. Conclusions In the testis of aged mice, the level of O-GlcNAcylation is increased, which leads to the arrest of spermatocyte meiosis by affecting the DNA damage repair pathway. Hyou1 is the key O-GlcNAc protein in testicular aging and mutation of this site exerts protective roles. This study revealed for the first time the specific mechanism of O-GlcNAc involved in testicular aging, and provided a new therapeutic target for clinically delaying testicular aging and treating male infertility. Disclosure No

Cypermethrin induced liver oxidative DNA damage via the JNK/c-Jun pathway

To clarify the adverse effect of cypermethrin(CYP) on the liver and explore the underlying role of the MAPK pathway. Twenty-four Sprague-Dawley(SD) rats were exposed to 0, 5, 10 and 20 mg/(kg·d) β-CYP by gavage for 31 days. Histomorphological and ultrastructural changes were evaluated by the hematoxylin &amp; eosin(HE) staining and transmission electron microscope(TEM). Levels of MDA and 8-OHdG were detected by ELISA. Expressions of p-JNK and γ-H2A. X were assessed by IHC and IF respectively. RT-PCR was performed to examine mRNA levels of GPx1, GPx4, SOD1, and SOD2 in rat testes. Western blot was conducted to determine protein expressions of GPx1, SOD2, CAT, γ-H2A. X, and the MAPK pathway-associated proteins in rat testes. After β-CYP exposure, the histomorphology and ultrastructures of rat livers were abnormally altered, as evidenced by hepatic sinusoidal dilation, hepatic plate space formation, mitochondrial crest fracture, etc. Moreover, β-CYP induced mRNA levels of GPx1, GPx4, SOD1 and SOD2, as well as protein expressions of GPx1 and SOD2 in the liver. Compared to the control, GPx1 and SOD2 protein expressions were decreased by 57.9% and 50.0%(P&lt;0.05), whereas the MDA level was increased by 56.2%(P&lt;0.05) in the high-dose group. Additionally, the JNK/c-Jun pathway, one of MAPK pathways, in the liver was activated by β-CYP, as shown by the increase of JNK and c-Jun phosphorylation, and protein expressions of p-JNK and p-c-Jun in the high-dose group were elevated by 47.7% and 46.5%(P&lt;0.05) in comparison to the control, but the ERK and p38 pathways were not affected after β-CYP exposure. Furthermore, β-CYP promoted 8-OHdG and γ-H2A. X expressions in the liver. Compared to the control, γ-H2A. X protein expression in the mid-and high-dose group was upregulated by 16.9% and 33.9%(P&lt;0.05), respectively. Cypermethrin had detrimental effects on the liver. CYP not only directly altered liver histomorphology and ultrastructures, but also caused oxidative stress, which activated the JNK/c-Jun pathway, finally inducing the DNA damage.

Deletion of Gpatch2 does not alter Tnf expression in mice

The cytokine TNF has essential roles in immune defence against diverse pathogens and, when its expression is deregulated, it can drive severe inflammatory disease. The control of TNF levels is therefore critical for normal functioning of the immune system and health. We have identified GPATCH2 as a putative repressor of Tnf expression acting post-transcriptionally through the TNF 3’ UTR in a CRISPR screen for novel regulators of TNF. GPATCH2 is a proposed cancer-testis antigen with roles reported in proliferation in cell lines. However, its role in vivo has not been established. We have generated Gpatch2−/− mice on a C57BL/6 background to assess the potential of GPATCH2 as a regulator of Tnf expression. Here we provide the first insights into Gpatch2−/− animals and show that loss of GPATCH2 affects neither basal Tnf expression in mice, nor Tnf expression in intraperitoneal LPS and subcutaneous SMAC-mimetic injection models of inflammation. We detected GPATCH2 protein in mouse testis and at lower levels in several other tissues, however, the morphology of the testis and these other tissues appears normal in Gpatch2−/− animals. Gpatch2−/− mice are viable, appear grossly normal, and we did not detect notable aberrations in lymphoid tissues or blood cell composition. Collectively, our results suggest no discernible role of GPATCH2 in Tnf expression, and the absence of an overt phenotype in Gpatch2−/− mice warrants further investigation of the role of GPATCH2.

Sinonasal NUT carcinoma: A retrospective case series from a single institution.

Nuclear protein in testis (NUT) carcinoma is a rare, aggressive tumor defined by the presence of NUT gene rearrangement. The aim of this study was to describe the clinical, radiologic, and biological features of sinonasal NUT carcinoma. We retrospectively investigated NUT expression with clinicopathologic features in 145 cases with sinonasal malignancies diagnosed from January 2017 to December 2021 and reviewed the reported cases. Three (3/145, 2.07%) cases showed strong nuclear expression for NUT immunohistochemical, including one male and two females with ages from 37 to 57 years (mean, 45.33 years). All three cases involved the nasal cavity and sinuses; one of them involved the orbit and intracranial area. Histologically, all subjects showed poorly differentiated, small round cell morphology with distinct nuclei. All patients received surgery and chemoradiotherapy. One patient died of the disease 13 months after diagnosis, and two survived 12 and 15 months, respectively, without evidence of tumor recurrence. 51 cases of sinonasal NUT carcinoma (mean age 40.96 years) have been described to date. Among them, 28 are male, and 23 are female. Most cases expressed p63, AE1/AE3, as well as p40. NUT carcinoma is a rare and aggressive disease with a poor prognosis. It is crucial to perform NUT rearrangement-related tests for differential diagnosis of poorly differentiated/undifferentiated tumors in the nasal cavity and sinuses.

ADAD2 functions in spermiogenesis and piRNA biogenesis in mice.

Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive. This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2. Adad2 homozygous mutant mouse lines, Adad2-/- and Adad2Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes. Adad2-/- and Adad2Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs. In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Clinical and molecular features of pulmonary NUT carcinoma characterizes diverse responses to immunotherapy, with a pathologic complete response case.

Nuclear protein in testis (NUT) carcinoma is an uncommon malignant cancer characterized by NUTM1 rearrangement. We aimed to investigate the clinicopathological and molecular features and immunotherapy of pulmonary NUT carcinoma. Immunohistochemistry (IHC) for NUT (C52B1) and programmed cell death ligand 1 (PD-L1: 22C3) and fluorescence in situ hybridization (FISH) for NUTM1 break and BRD4-NUTM1 fusion were performed on six pulmonary NUT carcinoma samples. The 6 pulmonary NUT carcinoma samples were obtained from 5 males and 1 female, with ages ranging from 31 to 73years (average, 46years). Five tumors occurred in the lobes, with one in the trachea. Pathologically, all cases showed primitive-appearing round to epithelioid cells growing in nests and sheets. Squamous differentiation and abrupt keratinization were observed. All tumors expressed the NUT protein and p63, and 4 tumors showed focal synaptophysin, but PD-L1 expression was not observed. All cases displayed NUTM1 rearrangement, 5 had BRD4-NUTM1 fusion, and one had an unknown partner. Three patients presented regional lymph node involvement at diagnosis. Five patients underwent intensive radiation and/or chemotherapy. Furthermore, 2 patients (1 and 2) received a combination of PD-L1 inhibitor and chemotherapy. Patient 1 exhibited a poor response and soon showed tumor progression and metastasis; however, patient 2 responded remarkably and achieved pathologic complete response (pCR) without uncontrollable adverse events. The overall survival time was 2.9months. Pulmonary NUT carcinoma exhibits poorly differentiated morphological features with diffuse NUT staining, low PD-L1 expression, and NUTM1 rearrangement. Despite its poor prognosis, it presents a diverse response to immunotherapy. Immune checkpoint inhibitors (ICIs) need to be further explored in NUT carcinoma.

P-Element-Induced Wimpy Testis Proteins and P-Element-Induced Wimpy Testis-Interacting RNAs Expression in Ovarian Cancer Stem Cells.

Background: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNA and are predominantly expressed in germline cells. piRNAs function as gene regulators and potential biomarkers for the development of a number of malignancies. The biological importance of piRNAs in ovarian cancer is still unknown. In this study, we investigated the expression of piRNAs in ovarian cancer stem cells and compared it with that in adherent cells. Methods: To assess changes in the expression levels of PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2, we used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis. Changes in piRNA expression levels in ovarian cancer stem cells were analyzed using Arraystar piRNA microarray screening. Gene Ontology (GO) enrichment analysis was conducted to determine the potential functions of piRNAs. Results: Using microarray analysis, we identified a cohort of differentially expressed piRNAs. Fifteen piRNAs, including DQ570763 and DQ597396, were downregulated, and 58 piRNAs were upregulated when compared with those in adherent A2780 and SKOV3 cells (p > 0.05, >2.0, respectively). GO functions of the downregulated piRNAs (DQ570763 and DQ570797) suggest that their roles are commonly associated with the Golgi apparatus. In addition, A2780-SP and SKOV3-SP cells had higher PIWIL3 and PIWIL4 mRNA levels than adherent cells (A2780 and SKOV3). Moreover, we determined, using receiver operating characteristic plot, that the expression level of PIWIL4 was lower in responders than in nonresponders after treatment with platins in patients with ovarian cancer. Finally, in ovarian cancer, PIWIL4 expression was associated with somatic mutations of dynein axonemal heavy chain 2, signal induced proliferation associated 1 like 2, YTH N6-methyladenosine RNA-binding protein 1, TBC1 domain family member 8, and LPS responsive Beige-like anchor protein. Conclusion: Our study showed that PIWI proteins and piRNAs are potential diagnostic and prognostic biomarkers for ovarian cancer.

Effects of moxibustion on acupoints at "opening" time on testicular tissue apoptosis in aging rats

To observe the effect of "lingguibafa" moxibustion at "opening" time on the level of superoxide dismutase(SOD), malondialdehyde(MDA), the protein expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in aging model rats. SD rats were randomly divided into blank control, model and lingguibafa groups, with 8 rats in each group. The aging model was established by daily intraperitoneal injection of D-galactose (500 mg/kg) for 42 consecutive days. After successful modeling, moxibustion intervention was applied at"lingguibafa" acupoints, 3 moxa-cone for each acupoint, once a day for 28 consecutive days. The contents of SOD and MDA in serum were detected by ELISA. Morphological changes of testicular tissue and the number of Leydig cells were observed after HE staining. Apoptosis rate of testicular cells was detected by TUNEL staining. The protein expression levels of Bcl-2 and Bax in testis were detected by Western blot. Compared with the blank control group, the SOD content in the serum, the number of testicular Leydig cells, the expression level of Bcl-2 protein in testis were significantly decreased (P<0.01) in the model group, while the MDA content in the serum, the apoptosis rate of testicular cells and the expression level of Bax protein in testis were significantly increased (P<0.01). Compared with the model group, the SOD content in serum, the number of testicular Leydig cells, the Bcl-2 protein expression in testis were significantly increased (P<0.01), while the MDA content in serum, the apoptosis rate of testicular cells, the expression level of Bax protein in testis were significantly decreased(P<0.01) in the lingguibafa group. Moxibustion on acupoints at "opening" time can delay testicular aging, and the mechanism may be related to balancing the metabolism of free radicals, reducing oxidative damage, and inhibiting the apoptosis of testicular cells.

Differential protein expression of GABA A receptor alpha 1 subunit and calbindin in rat spermatozoa associated with proteomic analysis in testis following methamphetamine administration.

Methamphetamine (METH) can induce spermatogenesis impairment, testicular apoptosis, and abnormal sperm quality. It also promotes changes in the expression of receptors for sex hormones and neurotransmitters, including GABA receptors in the testis. Proteomic assessment focusing on proteins involved in the calcium signalling pathway in the testis can facilitate diagnostic factors contributing to testicular and sperm functions, especially those related to spermatogenesis and fertilisation. In this study, we proposed to determine the localisation and differential expression of GABA A receptor alpha 1 subunit (GABA A-α1) in the spermatozoa of METH-administered rats. The differential proteomic profile of the testis was also observed by focusing on proteins in the KEGG pathways belonging to the calcium signalling pathway. There were 212 differentially expressed proteins in the rat testis, based on the cut-off value of 1.2-fold change. Most of those proteins, 13 proteins, were classified in the calcium signalling pathway, including 4 down-regulated and 9 up-regulated proteins. An immunolocalisation study of the GABA A-α1 receptor and calbindin revealed their localisation in the equatorial segment of the head in the rat spermatozoa. The expression of calbindin is also found in the middle piece of sperm. An increase in GABA A-α1 receptor in rat spermatozoa was correlated with an increase in abnormal sperm motility and morphology after methamphetamine exposure. Moreover, calbindin expression in sperm decreased in METH-administered rats. All our findings demonstrate that METH influences intracellular calcium homeostasis by acting through the calcium signalling pathway-associated proteins. Moreover, it might disrupt ion homeostasis in sperm through the GABA A-α1 receptor and calbindin, triggering a change in intracellular calcium and chloride ions. These changes may cause abnormalities in spermatogenesis, testicular apoptosis, and sperm quality impairment.

Genomically Driven NUTM1-NSD3 Carcinoma of the Lung: Opportunity for Chemotherapy Response

ABSTRACT Nuclear protein in testis (NUT) carcinoma is a rare, genetically-driven carcinoma defined by NUTM1 gene rearrangements. It frequently presents as an aggressive, poorly differentiated squamous cell carcinoma of the midline. NUT carcinoma originating in the lung is extremely uncommon. We present a unique case of a 23-year-old female diagnosed mucoepidermoid carcinoma of the lung found to harbor a rare NSD3-NUTM1 gene fusion on targeted sequencing. Despite multiple metastases at diagnosis, she exhibited dramatic response to first-line carboplatin and paclitaxel doublet chemotherapy. Interim PET-CT after three cycles showed near complete resolution of the left hilar mass and metastases. This response was sustained after six total cycles and continues after six months on maintenance chemotherapy. To our knowledge, this represents the first reported case of NUTM1-NSD3 fusion positive lung carcinoma displaying chemosensitivity, unlike the typically aggressive course of NUT cancers. Our case reveals the emerging molecular heterogeneity of these rare malignancies and suggests platinum doublets may be an effective treatment option warranting further study. Overall, it highlights the potential for precision diagnostics to uncover actionable targets and prognostic insights even in elusive cancers.

NUT Carcinoma: A Diagnostically Challenging Rare Entity with an Aggressive Course: A Case Series

Nuclear Protein in Testis (NUT) carcinomas are rare, extremely aggressive, poorly differentiated squamous carcinoma, characterised by the presence of NUT gene (NUTM1) rearrangement. NUT carcinoma most commonly arises in head and neck region and thorax however, they can occur in other parts of the body also. Here, we are presenting a case series of three cases of NUT carcinoma.All patients were females with disease involving head and neck region. The first case was an eight-year-old girl. The second case was a 36-year-old woman. The third case was a 61-year-old woman. Histopathology of all cases showed poorly differentiated neoplasm, comprising of sheets of monotonous cells with minimal indistinct, clear cytoplasm, round to oval nuclei with prominent nucleoli. High mitotic rate with areas of tumour necrosis were also present. Areas of abrupt keratinisation classically described in this entity were seen in two of the cases. Speckled nuclear positivity for NUT1 monoclonal antibody by immunohistochemistry, which is sensitive and specific for diagnosis was observed in all three cases. There is no definite treatment for this disease, and most patients respond poorly to conventional chemotherapy and radiation. Targeted therapy targeting the BRD4 portion of BRD4-NUT, termed BET bromodomain inhibitors, are in clinical trials. Two of our patients succumbed to disease within one year of diagnosis. Third patient is on follow-up for eleven months after diagnosis.

The Effects of Prenatal and Postnatal Exposure to 50-Hz and 3 mT Electromagnetic Field on Rat Testicular Development.

Background and objectives: It has been shown that electromagnetic fields (EMFs) have negative effects on the reproductive system. The biological effects of EMF on the male reproductive system are controversial and vary depending on the frequency and exposure time. Although a limited number of studies have focused on the structural and functional effects of EMF, the effects of prenatal and postnatal EMF exposure on testes are not clear. We aimed to investigate the effects of 50-Hz, 3-mT EMF exposure (5 days/wk, 4 h/day) during pre- and postnatal periods on testis development. Materials and Methods: Pups from three groups of Sprague-Dawley pregnant rats were used: Sham, EMF-28 (EMF-exposure applied during pregnancy and until postnatal day 28), EMF-42 (EMF-exposure applied during pregnancy and until postnatal day 42). The testis tissues and blood samples of male offspring were collected on the postnatal day 42. Results: Morphometric analyses showed a decrease in seminiferous tubule diameter as a result of testicular degeneration in the EMF-42 group. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were decreased in the EMF-42 group. Lipid peroxidation levels were increased in both EMF groups, while antioxidant levels were decreased only in the EMF-28 group. We found decreased levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF1) in the EMF-42 group, and decreased levels of the SRC homology 3 (SH3) and multiple ankyrin repeat domain (SHANK3) in the EMF-28 group in the testis tissue. Conclusions: EMF exposure during pre- and postnatal periods may cause deterioration in the structure and function of testis and decrease in growing factors that would affect testicular functions in male rat pups. In addition to the oxidative stress observed in testis, decreased SHANK3, VEGF, and IGF1 protein levels suggests that these proteins may be mediators in testis affected by EMF exposure. This study shows that EMF exposure during embryonic development and adolescence can cause apoptosis and structural changes in the testis.