Triptolide exposure triggers testicular vacuolization injury by disrupting the Sertoli cell junction and cytoskeletal organization via the AKT/mTOR signaling pathway.

BACKGROUND: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure. METHODS: Male mice were subjected to TP at doses of 15, 30, and 60 µg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining. RESULTS: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells. CONCLUSION: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes.

The Planar Cell Polarity Protein Fat1 in Sertoli Cell Function.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interfaces create an important intercellular bridge whose adhesive function is in turn supported by Fjx1, a nonreceptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila, which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood-testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor the Sertoli cell tight junction (TJ) permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with ß-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP core protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis.

Antihypertensive effects of the combined extract of Sorghum bicolor, Vigna angularis, and Eleusine coracana in spontaneously hypertensive rats.

This study investigated the antihypertensive effects of the combined extract of sorghum, adzuki bean, and finger millet (SAFE) on spontaneously hypertensive rats. The rats were divided into four groups (n = 8): WKY, SHR, SAFE (500 mg/kg SAFE), and CAP (50 mg/kg captopril). SAFE significantly decreased the lean-to-fat mass ratio with no notable changes in body weight, food intake, or food efficiency ratio, and it effectively lowered both systolic and diastolic blood pressures, comparable to CAP. Moreover, it significantly reduced the cardiac mass index and alleviated cardiac fibrosis. SAFE did not induce hepatotoxicity, as indicated by the maintenance of aspartate aminotransferase and alanine aminotransferase levels in the normal range, confirming its safety. Taken together, these findings suggested that SAFE can be used as a dietary supplement for blood pressure regulation and cardiovascular disease prevention.

Long-read single-cell sequencing reveals the transcriptional landscape of spermatogenesis in obstructive azoospermia and sertoli cell-only patients.

BACKGROUND：: High-throughput single-cell RNA sequencing (scRNA-seq) is widely used in spermatogenesis. However, it only reveals short reads in germ and somatic cells, limiting the discovery of novel transcripts and genes. AIM: This study shows the long-read transcriptional landscape of spermatogenesis in obstructive azoospermia (OA) and Sertoli cell-only (SCO) patients. DESIGN: Single cells were isolated from testicular biopsies of OA and NOA patients. Cell culture was identified by comparing Pacbio long-read single-cell sequencing (OA n = 3, NOA n = 3) with short-read scRNA-seq (OA n = 6, NOA n = 6). Ten germ cell types and eight somatic cell types were classified based on known markers. METHODS: Pacbio long read single-cell sequencing, short-read scRNA-seq, Polymerase Chain Reaction. RESULTS: A total of 130,426 long-read transcripts (100,517 novel transcripts and 29,909 known transcripts) and 49,508 long-read transcripts (26,002 novel transcripts, and 23,506 known transcripts) have been detected in OA and NOA patients, respectively. Moreover, 36,373 and 1642 new genes are identified in OA and NOA patients, respectively. Importantly, specific expressions of long-read transcripts were detected in germ and stomatic cells during normal spermatogenesis. CONCLUSION: We have identified total full-length transcripts in OA and NOA, and new genes were found. Furthermore, specific expressed full-length transcripts were detected, and the genomic structure of transcripts was mapped in different cell types. These findings may provide valuable information on human spermatogenesis and the treatment of male infertility.

Sperm immotility is associated with epididymis metabolism disorder in mice under obstructive azoospermia.

Obstructive azoospermia (OA) accounts for approximately 40% of males who suffer from azoospermia of male infertility. Currently, available treatment for OA consists of reproductive tract surgical reconstruction and sperm retrieval from the testis. However, both treatments result in low fertility compared to normal pregnancy, and the main reason remains largely unknown. Previous studies have shown that the quality of sperm retrieved from OA patients is poor compared with normal adult males but without an in-depth study. Herein, we generated a mouse OA model with vasectomy to evaluate sperm quality systematically. Our results showed that the testis had normal spermatogenesis but increased apoptotic activity in both OA patients and mice. More importantly, epididymal morphology was abnormal, with swollen epididymal tubules and vacuole-like principal cells. Especially, sperm retrieved from the epididymis of OA mice showed poor motility and low fertilization ability in vitro. Using mass spectrometry in epididymal fluid, we found differences in the expression of key proteins for sperm maturation, such as Angiotensinogen (AGT), rhophilin-associated tail protein 1 (ROPN1), NPC intracellular cholesterol transporter 2 (NPC2), and prominin 1 (PROM1). Furthermore, our results demonstrated that AGT, secreted by epididymal principal cells, could regulate sperm motility by managing PKCα expression to modify sperm phosphorylation. In conclusion, our data evaluate sperm quality systematically in OA mice and contribute to the understanding between the sperm and epididymis, which may provide novel insight into treating male infertility.

Regulation of Sertoli cell function by planar cell polarity (PCP) protein Fjx1.

Four-jointed box kinase 1 (Fjx1) is a planar cell polarity (PCP) protein and a member of the Fat (FAT atypical cadherin 1)/Dchs (Dachsous cadherin-related protein)/Fjx1 PCP complex. Fjx1 is also a non-receptor Ser/Thr protein kinase capable of phosphorylating Fat1 at is extracellular cadherin domains when it is being transported across the Golgi system. As such, Fjx1 is a Golgi-based regulator of Fat1 function by determining its extracellular deposition. Herein, Fjx1 was found to localize across the Sertoli cell cytoplasm, partially co-localized with the microtubules (MTs) across the seminiferous epithelium. It was most notable at the apical ES (ectoplasmic specialization) and basal ES, displaying distinctive stage-specific expression. The apical ES and basal ES are the corresponding testis-specific cell adhesion ultrastructures at the Sertoli-elongated spermatid interface and the Sertoli cell-cell interface, respectively, consistent with the role of Fjx1 as a Golgi-associated Ser/Thr kinase that modulates the Fat (and/or Dchs) integral membrane proteins. Its knockdown (KD) by RNAi using specific Fjx1 siRNA duplexes versus non-targeting negative control siRNA duplexes was found to perturb the Sertoli cell tight junction function, as well as perturbing the function and organization of MT and actin. While Fjx1 KD did not affect the steady-state levels of almost two dozens of BTB-associated Sertoli cell proteins, including structural and regulatory proteins, its KD was found to down-regulate Fat1 (but not Fat2, 3, and 4) and to up-regulate Dchs1 (but not Dchs2) expression. Based on results of biochemical analysis, Fjx1 KD was found to be capable of abolishing phosphorylation of its putative substrate Fat1 at its Ser/Thr sites, but not at its Tyr site, illustrating an intimate functional relationship of Fjx1 and Fat1 in Sertoli cells.

Cadmium-induced Sertoli Cell Injury Through p38-MAPK and Related Signaling Proteins-A Study by RNA Sequencing.

Environmental toxicants, such as cadmium, found in foods, water, and consumer products are known to induce male reproductive dysfunction. However, the underlying molecular mechanism(s) by which cadmium-induced Sertoli cell injury as manifested by a disruption of the blood-testis barrier (BTB) remains unknown. Interestingly, one of the primary targets of cadmium toxicity in the testis is the cytoskeletons of the Sertoli cells, which, in turn, impedes cell junctions in the seminiferous epithelium. In order to expand these earlier observations and to provide a roadmap for future studies, we embarked a study using RNA sequencing to identify the pertinent genes involved in cadmium-induced Sertoli cell injury. Using bioinformatics analyses, multiple gene sets that regulated actin and microtubule (MT) cytoskeletons were identified along with components of the mitogen-activated protein kinase (MAPK) signaling protein and several signaling pathways. More important, we have also discovered that while the gene expression of p38-MAPK (also JNK or c-Jun) was considerably up- or downregulated during cadmium-induced Sertoli cell injury, the activated (phosphorylated) form was upregulated. Importantly, doramapimod (also known as BIRB 796), a specific p38-MARK inhibitor, that was shown to selectively block cadmium-induced p-p38 MAPK activation via phosphorylation in Sertoli cells, was indeed capable of blocking cadmium-induced Sertoli cell injury including disruption of the Sertoli cell-permeability barrier function, disruptive distribution of BTB-associated proteins, and disruptive organization of the actin and MT cytoskeletons. These data provide a helpful source of information for investigators to probe the role of signaling proteins and/or their signaling cascades, besides MAPKs, that likely utilized by cadmium to induce reproductive dysfunction.

Absence of MerTK disrupts spermatogenesis in an age-dependent manner.

Spermatogenesis is a highly specialized cell differentiation process regulated by the testicular microenvironment. During the process of spermatogenesis, phagocytosis performs an essential role in male germ cell development, and its dysfunction in the testis can cause reproduction defects. MerTK, as a critical protein of phagocytosis, facilitates the removal of apoptotic substrates from the retina and ovaries through cooperation with several phagocytosis receptors. However, its role in mammalian spermatogenesis remains undefined. Here, we found that 30-week-old MerTK-/- male mice developed oligoasthenospermia due to abnormal spermatogenesis. These mice showed damaged seminiferous tubule structure, as well as altered spermatogonia proliferation and differentiation. We also found that Sertoli cells from MerTK-/- mice had decreased phagocytic activity on apoptotic germ cells in vitro. Moreover, a transcriptomic analysis demonstrated that the pivotal genes involved in spermatid differentiation and development changed expression. These results indicate that MerTK is crucial for spermatogenesis, as it regulates the crosstalk between germ cells and Sertoli cells. This provides us insight into the molecular mechanism of MerTK on spermatogenesis and its implications for the diagnosis and treatment of human male infertility.

Defects of microtubule cytoskeletal organization in NOA human testes.

The importance of actin and microtubule (MT) cytoskeletons in testis function in rodents is known to some extent, but its role in the etiology of azoospermia in humans remains unexplored. Here, we examined if MT cytoskeleton was defective in NOA (non-obstructive azoospermia) testes versus normal human testes based on histopathological, immunofluorescence (IF), and scRNA-Seq transcriptome profiling. Testis biopsy samples from n = 6 normal men versus n = 3 Sertoli cell only (SCO) and n = 3 MA (meiotic arrest) of NOA patients were used for histopathological analysis. IF analysis was also used to examine MT organization across the seminiferous epithelium, investigating the likely involvement of microtubule-associated proteins (MAPs). scRNA-Seq transcriptome profiling datasets from testes of 3 SCO patients versus 3 normal men in public domain in Gene Expression Omnibus (GEO) Sample (GSM) with identifiers were analyzed to examine relevant genes that regulate MT dynamics. NOA testes of MA and SCO patients displayed notable defects in MT organization across the epithelium with extensive truncation, mis-alignments and appeared as collapsed structures near the base of the tubules. These changes are in contrast to MTs in testes of normal men. scRNA-Seq analyses revealed considerable loss of spermatogenesis capacity in SCO testes of NOA patients versus normal men. An array of genes that support MT dynamics displayed considerable changes in expression and in spatial distribution. In summary, defects in MT cytoskeleton were noted in testes of NOA (SCO) patients, possibly mediated by defective spatial expression and/or distribution of MAPs. These changes, in turn, may impede spermatogenesis in SCO testes of NOA patients.

G3BP2, a stress granule assembly factor, is dispensable for spermatogenesis in mice.

Background: Spermatogenesis is a complex process that includes mitosis, meiosis, and spermiogenesis. During spermatogenesis, genetic factors play a vital role inthe formation of properly functioning sperm. GTPase-activating protein (SH3 domain)-binding protein 2 (G3BP2) is known to take part in immune responses, mRNA transport, and stress-granule assembly. However, its role in male fertility is unclear. Here, we generated a G3bp2 conditional knockout (cKO) mouse model to explore the function of G3BP2 in male fertility. Methods: Polymerase chain reaction (PCR) and western blotting (WB) were used to confirm testis-specific G3bp2 knockout. Hematoxylin-eosin (HE) staining to observe testicular morphology and epididymal structure. Computer-aided sperm analysis (CASA) to detect sperm concentration and motility. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay was used to detect apoptotic cells. Results: We found that cKO male mice are fertile with the normal morphology of the testis and sperm. Additionally, CASA of the semen from cKO mice showed that they all had a similar sperm concentration and motility. In addition, sperm from these mice exhibited a similar morphology. But the tunnel assay revealed increased apoptosis in their testes relative to the level in the wild type (WT). Conclusion: Together, our data demonstrate that G3BP2 is dispensable for spermatogenesis and male fertility in mice albeit with the increased germ-cell apoptosis.

NBMA Promotes Spermatogenesis by Mediating Oct4 Pathway.

Non-obstructive azoospermia is one of the most common causes of male infertility, but there is still no specific treatment drug. Given that the Oct4 (Octamer-binding transcription factor 4) has an important regulatory effect on spermatogenesis, activating it can effectively promote spermatogenesis, so it is of great value to develop Oct4-targeted drug design and elucidating its mechanism of action. Here, we screened out the Oct4-targeted drug molecule NBMA (N-benzyl-4-methoxy-2-(1-(4-(trifluoromethyl)phenyl)vinyl)aniline) by computer-assisted technology, and found that it has a significant promoting effect on spermatogenesis in the established mouse azoospermia model. Subsequently, through transcriptome sequencing and enrichment analysis, real-time fluorescent quantitative PCR (qPCR) and western blot experiments revealed that NBMA promotes the differentiation of spermatogonial stem cells by activating the Oct4 pathway, thereby promoting spermatogenesis. This study proves that NBMA is a molecule with great potential to be developed as a therapeutic drug for azoospermia. It also shows that computer-assisted, chemical and biological multidisciplinary methods play a very important role in innovative drug discovery.

Single-cell ATAC-Seq reveals cell type-specific transcriptional regulation and unique chromatin accessibility in human spermatogenesis.

During human spermatogenesis, germ cells undergo dynamic changes in chromatin organization/re-packaging and in transcriptomes. In order to better understand the underlying mechanism(s), scATAC-Seq of 5376 testicular cells from 3 normal men were performed. Data were analyzed in parallel with the scRNA-Seq data of human testicular cells. In all, 10 germ cell types associated with spermatogenesis and 6 testicular somatic cell types were identified, along with 142 024 peaks located in promoter, genebody and CpG Island. We had examined chromatin accessibility of all chromosomes, with chromosomes 19 and 17 emerged as the leading chromosomes that displayed high chromatin accessibility. In accessible chromatin regions, transcription factor-binding sites were identified and specific motifs with high frequencies at different spermatogenesis stages were detected, including CTCF, BORIS, NFY, DMRT6, EN1, ISL1 and GLI3. Two most remarkable observations were noted. First, TLE3 was specifically expressed in differentiating spermatogonia. Second, PFN4 was found to be involved in actin cytoskeletal organization during meiosis. More important, unique regions upstream of PFN4 and TLE3 were shown to display high accessibility, illustrating their significance in supporting human spermatogenesis.

Black Soybean and Adzuki Bean Extracts Lower Blood Pressure by Modulating the Renin-Angiotensin System in Spontaneously Hypertensive Rats.

Hypertension, causing cardiovascular disease, stroke, and heart failure, has been a rising health issue worldwide. Black soybeans and adzuki beans have been widely consumed throughout history due to various bioactive components. We evaluated the antihypertensive effects of black soybean and adzuki bean ethanol extracts on blood pressure, renin-angiotensin system (RAS), and aortic lesion in spontaneously hypertensive rats. A group of WKY (normal) and six groups of spontaneously hypertensive rats were administered with saline (SHR), 50 mg/kg of captopril (CAP), 250 and 500 mg/kg of black soybean extracts (BE250 and BE500), 250 and 500 mg/kg of adzuki bean extracts (AE250 and AE500) for eight weeks. BE250, BE500, AE250, and AE500 significantly (p < 0.05) reduced relative liver weight, AST, ALT, triglyceride, total cholesterol, systolic blood pressure, and angiotensin-converting-enzyme level compared to SHR. The angiotensin II level in AE500 and renin mRNA expression in BE500 and AE500 were significantly (p < 0.05) decreased compared to SHR. The lumen diameter was significantly (p < 0.05) reduced in only CAP. Furthermore, systolic and diastolic blood pressure and angiotensin II level in AE500 were lower than those of BE500. These results suggest that AE exhibit more antihypertensive potential than BE in spontaneously hypertensive rats.

Dopamine-Modified AuCu Bimetallic Nanoclusters as Charge Transfer-Based Biosensors for Highly Sensitive Glycine Detection.

Glycine is the simplest amino acid in living organisms and plays important roles in biology and medicine. However, few biosensors for glycine sensing have been reported. Herein, we present a facile strategy to construct dopamine-modified AuCu bimetallic nanoclusters (denoted as AuCu NC-DA) as charge transfer-based biosensors for highly sensitive glycine sensing. The AuCu NCs stabilized by bovine serum albumin (BSA) exhibited a fluorescence maximum at 400 nm. Because of the high affinity of BSA for dopamine (DA), the surface of the AuCu NCs was modified with DA without any complicated chemical reactions, resulting in fluorescence quenching through a charge transfer process. Among 20 amino acids, AuCu NC-DA exhibited an off/on fluorescence switching response specifically toward glycine through the formation of hydrogen bonds with oxidized DA, which inhibited the charge transfer process, leading to the emergence of a new emission peak at 475 nm. Spectroscopic and thermodynamic results combined with molecular docking analyses provided comprehensive understanding of the sensing mechanism. Furthermore, we showed that AuCu NC-DA was able to sense glycine in cells by imaging. Finally, the practicability of AuCu NC-DA for glycine detection was validated in milk drink samples. This study presents a promising type of a charge transfer-based sensor.

Tethering of Telomeres to the Nuclear Envelope Is Mediated by SUN1-MAJIN and Possibly Promoted by SPDYA-CDK2 During Meiosis.

During meiosis, telomeres attach to the nuclear envelope (NE) to promote homologous chromosome moving, pairing, synapsis, and recombination. The telomere-NE attachment is mediated by SUN1, TERB1-TERB2-MAJIN (TTM complex), and TRF1. The interaction of the TTM complex with shelterin is mediated by TERB1 and TRF1, but how SUN1 interacts with the TTM complex is not yet fully understood. In this study, we found that SUN1 not only interacted with TERB1 but also interacted with MAJIN, and the interaction of SUN1 with MAJIN is stronger than TERB1. We also found that SUN1 interacted with SPDYA, an activator of CDK2. The binding sites of MAJIN and SPDYA at SUN1 were mapped, and both MAJIN and SPDYA bound to the N-terminal domain of SUN1 and the two binding sites were close to each other. Furthermore, SPDYA bound to SUN1 via the Ringo domain and recruited CDK2 to SUN1. Then, we found that the interaction of SUN1 with MAJIN was decreased by the CDK2 inhibitors. Taken together, our results provide the possible mechanism of SUN1, MAJIN, and SPDYA-CDK2 in promoting the telomere-NE attachment during meiosis.

The testis-specific gene 1700102P08Rik is essential for male fertility.

Male infertility is a rising problem around the world. Often the cause of male infertility is unclear, and this hampers diagnosis and treatment. Spermatogenesis is a complex process under sophisticated regulation by many testis-specific genes. Here, we report the testis-specific gene 1700102P08Rik is conserved in both the human and mouse and highly expressed in spermatocytes. To investigate the role of 1700102P08Rik in male fertility, knockout mice were generated by CRISPR-Cas9. 1700102P08Rik knockout male mice were infertile with smaller testis and epididymis, but female knockout mice retained normal fertility. Spermatogenesis in the 1700102P08Rik knockout male mouse was arrested at the spermatocyte stage, and no sperm were found in the epididymis. The deletion of 1700102P08Rik causes apoptosis in the testis but did not affect the serum concentration of testosterone, luteinizing hormone, and follicle-stimulating hormone or the synapsis and recombination of homologous chromosomes. We also found that 1700102P08Rik is downregulated in spermatocyte arrest in men. Together, these results indicate that the 1700102P08Rik gene is essential for spermatogenesis and its dysfunction leads to male infertility.

Aluminum-Enhanced Fluorescence of Cu8 Nanoclusters: An Effective Method for Sensitive Detection of Fluoride in Aqueous and Bioimaging.

Here, we report glutathione-protected water-soluble copper nanoclusters (Cu8 NCs) with excellent properties of high stability, large Stokes shift, and low toxicity. The molecular formula and structure of the Cu8 NCs were confirmed through good agreement between spectroscopic results and theoretical calculations. The origin of optical properties and chemical stability of the Cu8 NCs was determined through natural bond orbital analysis combined with time-dependent density functional theory calculations. It was found that the fluorescence emission and quantum yields of Cu8 NCs could be effectively enhanced by aluminum(III) ions (Al3+) through the aggregation-induced enhanced emission mechanism. On the basis of the strong reaction of Al3+ with F-, the enhanced fluorescence of the obtained Cu8 NC-Al3+ ensemble could be selectively turned off by fluoride ions (F-), achieving highly sensitive detection of F- in aqueous with a detection limit of 0.16 µM. Also, the fluorescence of Cu8 NC-Al3+ ensemble quenched by F- could be enhanced again upon binding additional Al3+. In addition, the proposed Cu8 NC-Al3+ ensemble with enhanced fluorescence was used for bioimaging in vitro and in vivo. These results indicated that Cu8 NCs are promising materials for sequential sensing and bioimaging in the future.