Ursonic acid attenuates spermatogenesis in oligozoospermia mice through inhibiting ferroptosis.

Ursonic acid (UNA) is a natural pentacyclic triterpene found in some medicinal plants and foods. The reproductive protective effect of UNA was evaluated in a mouse model of oligozoospermia induced by busulfan (BUS) at 30 mg/kg b.w.. The mice were initially divided into groups with UNA concentrations of 10, 30, 50, 100 mg/kg. Subsequently, based on sperm parameters, the optimal concentration of 50 mg/kg was identified. As a control, an additional group was supplemented with ursolic acid at a concentration of 50 mg/kg. The results indicated that BUS caused the loss of spermatogenic cells in testis, the decrease of sperm in epididymis, the disorder of testicular cytoskeleton, the decrease of serum sex hormones such as testosterone which induced an increase in feedback of androgen receptor and other testosterone-related proteins, the increase of malondialdehyde and reactive oxygen species levels and the increase of ferroptosis in testis while UNA successfully reversed these injuries. High-throughput sequencing revealed that UNA administration significantly upregulated the expression of genes associated with spermatogenesis, such as Tnp1, Tnp2, Prm1, among others. These proteins are crucial in the histone to protamine transition during sperm chromatin remodeling. Network pharmacology analysis reveals a close association between UNA and proteins related to the transformation of histones to protamine. Molecular docking studies reveal that UNA can interact with the ferroptosis-inhibiting gene SLC7A11, thereby modulating ferroptosis. Taken together, UNA alleviated BUS-induced oligozoospermia by regulating hormone secretion, mitigating oxidative stress and promoting recovery of spermatogenesis by inhibiting the ferroptosis.

Tandem mass tag-based quantitative proteomics analyses of the spermatogenesis-ameliorating effect of Youjing granule on rats.

RATIONALE: Male infertility is a common reproductive system disease manifested as aberrant spermatogenesis and identified as "kidney deficiency and dampness" in Chinese traditional medicine. Youjing granule (YG) is a Chinese material medica based on tonifying kidneys and removing dampness. It has proven to be able to regulate semen quality in clinical application, but the underlying mechanism has not been clarified. METHODS: Using serum containing YG to treat primarily cultured spermatogonial stem cells (SSCs), the apoptotic rate and mitosis phase ratio of SSCs were measured. The liquid chromatography-tandem mass spectrometry with tandem mass tags method was applied for analyzing the serum of rats treated with YG/distilled water, and proteomic analyses were performed to clarify the mechanisms of YG. RESULTS: Totally, 111 proteins in YG-treated serum samples were differentially expressed compared with control groups, and 43 of them were identified as potential target proteins, which were further annotated based on their enrichment in Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. Proteomic analyses showed that the mechanisms of YG may involve regulation of glycolysis, gluconeogenesis and nucleotide-binding and oligomerization domain-like receptor signaling pathway. In addition, RhoA and Lamp2 were found to be possible responders of YG through reviewing the literature. CONCLUSIONS: The results demonstrate that our serum proteomics platform is clinically useful in understanding the mechanisms of YG.

The anti-spermatogenic activity of nanoliposomes loaded with Heracleum persicum phenolic compounds in Balb/C mice.

There are various types of bioactivities that have been reported for Heracleum persicum species, such as antioxidant, anti-inflammatory, and cytotoxicity properties. In the current study, the bio-accessibility of H. persicum bioactive compounds was improved by purifying its phenolic-enriched fractions (PEF) and encapsulating them into nanoliposomes to analyze its cytotoxic impacts on mice testicular tissue and their fertility status. Nano liposomal H. persicum PEF (NL-HPEF) was prepared by ultrasound-based encapsulation of HPEF and L-agranular lecithin mixture. The size, morphology, and stability of NL-HPEF were characterized by dynamic light scattering, field emission scanning electron microscopy, and zeta potential analysis. The 18 white male Balb/c mice (20-25 g) at 3 treatment groups were provided to study the NL-HPPF cytotoxicity by measuring the mice liver enzyme including aspartate aminotransferase (AST), ALP and alanine aminotransferase (ALT), testis lipid peroxidation, and testicular tissue destruction levels. Moreover, the mice's fertility was evaluated by studying the Adam3, Prm1, Spata19, and Tnp2 gene expression in the testicular tissues. The obtained results manifested that the synthesized NL-HPEF was stable (193.7 nm) and exhibited a notable cytotoxic impact on the mice's liver (ALT and AST enhancement levels) and testicular tissues. Moreover, their increasing treatment doses impaired the male mice's fertility by decreasing the sperm count, viability, and motility. In addition, fertility suppression was verified by decreasing serum testosterone and downregulating the Adam3, Prm1, Spata19, and Tnp2 gene expression in their testicular tissues. The male mice's fertility was significantly (p < 0.05) suppressed by increasing treatment doses of NL-HPEF. Hence, the NL-HPEF could be considered a promising alternative to replace the male chemical contraceptives drugs.

L-Arginine abrogates maternal and pre-pubertal codeine exposure-induced impaired spermatogenesis and sperm quality by modulating the levels of mRNA encoding spermatogenic genes.

Introduction: Although, codeine has been demonstrated to lower sperm quality; the effects of maternal and prepubertal codeine exposure on male offspring is yet to be reported. In addition, the effect of arginine on codeine-induced decline in sperm quality has not been explored. This study investigated the impact of maternal and prepubertal codeine exposure on spermatogenesis and sperm quality in F1 male Wistar rats to study the effect that codeine may have during recreational use in humans. Also, the effect of arginine supplementation on codeine-induced alteration in spermatogenesis and sperm quality was evaluated. Methods: Female rats were treated with either 0.5 ml distilled water or codeine orally for eight weeks, and then mated with male rats (female:male, 2:1). The F1 male offsprings of both cohorts were weaned at 3 weeks old and administered distilled water, codeine, arginine, or codeine with arginine orally for eight weeks. Results: Prepubertal codeine exposure in rats whose dams (female parents) were exposed to codeine delayed puberty and reduced the weight at puberty. Prepubertal codeine exposure exacerbated maternal codeine exposure-induced reduced total and daily spermatid production, sperm count, sperm motility, and normal sperm form, as well as impaired sperm plasma membrane integrity and increased not intact acrosome and damaged sperm DNA integrity. These perturbations were accompanied by a decrease in mRNA levels encoding spermatogenic genes, testicular testosterone and androgen receptor (AR) concentrations, and upregulation of sperm 8-hydroxydeoxyguanosine (8OHdG). Prepubertal arginine supplementation mitigated codeine-induced alterations. Discussion: This study provides novel experimental evidence that maternal and prepubertal codeine exposure reprogramed spermatogenesis and sperm quality of male FI generation by decreasing mRNA levels encoding spermatogenic genes and AR via oxidative stress-mediated signaling, which was abrogated by prepubertal arginine supplementation.

The three-dimensional conformation and activity of mitochondria in syncytial male germ line-cysts of medicinal leeches.

We studied the spatial conformation and activity of mitochondria in the developing syncytial male germline cysts during spermatogenesis of the medicinal leeches using light, fluorescent, transmission electron microscopy, and serial block-face scanning electron microscopy. In cysts with spermatogonia and spermatocytes, mitochondria form networks and are in a dynamic hyperfusion state, while in cysts with spermatids, a single huge mitochondrion is observed. As spermiogenesis progresses, this huge mitochondrion is finally located in the future midpiece. The highest activity, in terms of membrane potential, of the mitochondria in H. medicinalis germline cysts was observed in cysts with spermatocytes; the lowest was in cysts with late elongated spermatids.

Studying the effect of hyperoside on recovery from cyclophosphamide induced oligoasthenozoospermia.

Oligoasthenozoospermia is becoming a serious problem, but effective prevention or treatment is lacking. Hyperoside, one of the main active ingredients in traditional Chinese medicine, may be effective in the treatment of oligoasthenozoospermia. In this study, we used cyclophosphamide (CTX: 50 mg/kg) to establish a mouse model of Oligoasthenozoospermia to investigate the therapeutic effect of hyperoside (30 mg/kg) on CTX-induced oligoasthenozoospermia. All mice were divided into four groups: blank control group (Control), treatment control group (Hyp), disease group (CTX) and treatment group (CTX + H). Mice body weight, testicular weight, sperm parameters and testicular histology were used to assess the reproductive capacity of mice and to explore the underlying mechanism of hyperoside in the treatment of oligoasthenozoospermia by assessing hormone levels, protein levels of molecules related to hormone synthesis and transcript levels of important genes related to spermatogenesis. Treatment with hyperoside significantly improved sperm density, sperm viability and testicular function compared to untreated oligoasthenozoospermia mice. In mechanism, treatment with hyperoside resulted in significant improvement in pathological changes in spermatogenic tubules, with an increase in testosterone production, and upregulations of Protein Kinase CAMP-Activated Catalytic Subunit Beta (PRKACB), Steroidogenic Acute Regulatory Protein (STAR), and Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17A1) for testosterone production. Hyperoside also promoted the cell cycle of germ cells and up-regulated meiosis and spermatogenesis-related genes, including DNA Meiotic Recombinase 1 (Dmc1), Ataxia telangiectasia mutated (Atm) and RAD21 Cohesin Complex Component (Rad21). In conclusion, hyperoside exerted protective effects on oligoasthenozoospermia mice by regulating testosterone production, meiosis and sperm maturation of germ cells.

Does isotretinoin affect spermatogenesis in the long term? A rat model.

OBJECTIVES: Spermatogenesis, in which cell regeneration continues, can be affected by environmental, chemical, psychological factors or various diseases. There is conflicting information in the literature about the effect of isotretinoin, which is widely used in acne treatment, on testes and spermatogenesis. Therefore, we planned a rat study to evaluate the long-term efficacy of oral isotretinoin on testicular tissues and spermatogenesis. MATERIALS AND METHODS: The Group 1 (n = 6) 7.5 mg/kg/day and the Group 2 (n = 6) received isotretinoin at a dose of 30 mg/kg/day dissolved in sunflower oil, the Sham Group (n = 6) received only sunflower oil by gavage, and the control group (n = 6) received standard feed and water for four weeks. After the 4th week, all animals were fed with standard feed and water and followed for the next four weeks. At the end of the 8th week, all animals were sacrificed under deep anesthesia. Seminiferous tubule diameters, epithelial thickness, apoptotic index, sperm number and motility recorded Results: Sperm count, motility, vitality, diameter of seminiferous tubule and germinal epithelium thickness were decreased and apoptotic index increased in the groups received isotretinoin. There was no significant difference between the groups in terms of testosterone levels. CONCLUSIONS: We consider that further comprehensive studies, including human clinical trials, should be conducted to examine the negative effects of isotretinoin on spermatogenesis in the long term especially when there is a need using isotretinoin in men for various reasons and to eliminate the contradictions in the literature in this regard.

Melatonin alleviated fluoride-induced impairment of spermatogenesis and sperm maturation process via Interleukin-17A.

Fluoride-induced male reproductive failure is a major environmental and human health concern, but interventions are still lacking. Melatonin (MLT) has potential functions in regulating testicular damage and interleukin-17 (IL-17) production. This study aims to explore whether MLT can mitigate fluoride-induced male reproductive toxicity through IL-17A, and screen the potential targets. So the wild type and IL-17A knockout mice were employed and treated with sodium fluoride (100 mg/L) by drinking water and MLT (10 mg/kg.BW, intraperitoneal injection per two days starting from week 16) for 18 weeks. Bone F- concentrations, grade of dental damage, sperm quality, spermatogenic cells counts, histological morphology of testis and epididymis, and the mRNA expression of spermatogenesis and maturation, classical pyroptosis related and immune factor genes were detected respectively. The results revealed that MLT supplementations alleviated fluoride-induced impairment of spermatogenesis and maturation process, protecting the morphology of testis and epididymis through IL-17A pathway, and Tesk1 and Pten were identified as candidate targets from 29 regulation genes. Taken together, this study demonstrated a new physiological role for MLT in the protection against fluoride-induced reproductive injury and possible regulation mechanisms, which providing a useful therapeutic strategy for male reproductive function failure caused by fluoride or other environmental pollutants.

Oral administration of Turnera diffusa willd. ex Schult. extract ameliorates steroidogenesis and spermatogenesis impairment in the testes of rats with type-2 diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: Turnera diffusa Willd. ex Schult. (T. diffusa) has traditionally been used to treat male reproductive dysfunction and have aphrodisiac properties. AIMS OF THE STUDY: This study aims to investigate the ability of T. diffusa to ameliorate the impairment in testicular steroidogenesis and spermatogenesis in DM that might help to improve testicular function, and subsequently restore male fertility. MATERIALS AND METHODS: DM-induced adult male rats were given 100 mg/kg/day and 200 mg/kg/day T. diffusa leaf extract orally for 28 consecutive days. Rats were then sacrificed; sperm and testes were harvested and sperm parameter analysis were performed. Histo-morphological changes in the testes were observed. Biochemical assays were performed to measure testosterone and testicular oxidative stress levels. Immunohistochemistry and double immunofluorescence were used to monitor oxidative stress and inflammation levels in testes as well as Sertoli and steroidogenic marker proteins' expression. RESULTS: Treatment with T. diffusa restores sperm count, motility, and viability near normal and reduces sperm morphological abnormalities and sperm DNA fragmentation in diabetic rats. T. diffusa treatment also reduces testicular NOX-2 and lipid peroxidation levels, increases testicular antioxidant enzymes (SOD, CAT, and GPx) activities, ameliorates testicular inflammation via downregulating NF-ΚB, p-Ikkß and TNF-α and upregulating IκBα expression. In diabetic rats, T. diffusa treatment increases testicular steroidogenic proteins (StAR, CYP11A1, SHBG, and ARA54, 3 and 17ß-HSD) and plasma testosterone levels. Furthermore, in diabetic rats treated with T. diffusa, Sertoli cell marker proteins including Connexin 43, N-cadherin, and occludin levels in the testes were elevated. CONCLUSION: T. diffusa treatment could help to ameliorate the detrimental effects of DM on the testes, thus this plant has potential to be used to restore male fertility.

Artemisia annua L. (Sweet wormwood) leaf extract attenuates high-fat diet-induced testicular dysfunctions and improves spermatogenesis in obese rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L., known as "sweet wormwood," is widely used in Egyptian folk medicine. Egyptians implement the aerial parts in the treatment of respiratory, digestive and sexual dysfunctions. However, the mechanism by which Artemisia annua improves testicular function is still being discovered. AIM OF THE STUDY: This study aimed to evaluate the modulatory effects of the crude leaf extract of Artemisia annua (AAE) on a high-fat diet induced testicular dysfunction in rats and compare it with the antilipolytic drug Orlistat. MATERIAL AND METHODS: Forty adult rats were randomly classified and assigned to four groups. The first group typically consumed a balanced diet and served as a negative control (GP1). A high-fat diet-induced obesity was applied to the other three groups for 12 weeks. A positive control remained on HFD for another 8 weeks, which is GP2. Other groups were administered for 8 consecutive weeks either with Orlistat (50 mg/kg body weight) or AAE (100 mg/kg body weight), which have been defined as GP3 and GP4, respectively. Testosterone (TST), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in the sera of all groups. In addition, the oxidant/antioxidant biomarkers such as protein carbonyl, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) activities, lactate dehydrogenase (LDH) and creatine kinase isoenzyme-B (CK-MB) were determined. An immunohistochemical stain with the apoptotic marker caspase-3 and the proliferating cell nuclear antigen (PCNA) were also investigated. RESULTS: In the testes of the obese group, the results showed hormonal imbalance, an increase in oxidative stress biomarkers and apoptosis. In the group treated with orlistat (GP3), noticeably more perturbations were noted. The obese rats that had been treated with AAE (GP4) showed a significantly reduced level of oxidative stress, hormonal balance restoration and reduced apoptosis. CONCLUSIONS: The crude leaf extract of A. annua is a potential herbal therapeutic for the treatment of obesity-related testicular dysfunction and the restoration of hormonal imbalance in obese rats.

Diallyl Trisulfide, a Biologically Active Component of Garlic Essential Oil, Decreases Male Fertility in Sitotroga cerealella by Impairing Dimorphic Spermatogenesis, Sperm Motility and Lipid Homeostasis.

Diallyl trisulfide (DAT) is a biologically active component of garlic essential oil and exhibits multi-targeted activity against many organisms. The current study tested the capacity of DAT to decrease the male fertility of Sitotroga cerealella. The effects on testis morphology, sperm number, motility, and lipid homeostasis were observed in adult males fumigated with DAT at a dose of 0.01 µL/L in air. The results indicated that the DAT significantly decreased the dimorphic sperm number. Meanwhile, the ultrastructural analysis of the sperm showed that the DAT caused malformed and aberrant structures of mitochondrial derivatives of dimorphic sperm. Additionally, the lipid homeostasis and ATP contents in the male adults were significantly decreased after treatment. Moreover, the total sperm motility was reduced, while the wave-propagation velocity, amplitude, frequency, and wavelength were significantly decreased compared with the controls. Overall, this study reported, for the first time, that DAT impairs energy metabolism, inhibits dimorphic spermatogenesis, and decreases sperm motility, while these abnormalities in sperm lead to adult-male infertility.

Low-moderate dietary phytoestrogens transiently disrupt spermatogenesis and the seminal plasma proteome in the ram.

In brief: Dietary phytoestrogens disrupt a specific stage of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa. This paper demonstrates for the first time that ram reproduction is compromised by oestrogenic pasture, whilst also providing a longitudinal model for the impact of phytoestrogens on male fertility. Abstract: Compounds with oestrogen-like actions are now common in both the Western diet. The long-term impacts and underlying mechanisms by which oestrogenic compounds alter male reproduction, however, are unclear. To investigate this, we used a longitudinal sheep model examining the impact of oestrogenic pasture consumption on semen quality and production, testicular size, sexual behaviour and the seminal plasma proteome of Merino rams (n = 20), over a full spermatogenic cycle and in the subsequent breeding season. Throughout the study period, sexual behaviour, sperm production and motility were similar between the exposed and non-exposed rams (P > 0.05). However, between 5 and 8 weeks of exposure to dietary phytoestrogens, rams produced a higher percentage of spermatozoa with a specific malformation of the sperm midpiece and reduced DNA integrity, compared to non-exposed rams (P < 0.001). Investigation into the seminal plasma proteome revealed 93 differentially expressed proteins between phytoestrogen-exposed and control rams (P < 0.05). Exposure to phytoestrogens increased the expression of proteins involved in cellular structure development, actin cytoskeleton reorganisation, regulation of cell function and decreased expression in those related to catabolic processes. The greatest fold changes were in proteins involved in the assembly of the sperm flagella, removal of cytoplasm, spermatid development and maintenance of DNA integrity. After returning to non-oestrogenic pasture, no differences in any measure were observed between treatment groups during the subsequent breeding season. We conclude that dietary phytoestrogens can transiently disrupt specific stages of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa.

N-acetylcysteine rescues meiotic arrest during spermatogenesis in mice exposed to BDE-209.

Deca-bromodiphenyl ethers (BDE-209) has been widely used in electronic devices and textiles as additives to flame retardants. Growing evidence showed that BDE-209 exposure leads to poorer sperm quality and male reproductive dysfunction. However, the underlying mechanisms of BDE-209 exposure caused a decline in sperm quality remains unclear. This study aimed to evaluate the protective effects of N-acetylcysteine (NAC) on meiotic arrest in spermatocytes and decreased sperm quality in BDE-209-exposed mice. In the study, mice were treated with NAC (150 mg/kg BW) 2 h before administrated with BDE-209 (80 mg/kg BW) for 2 weeks. For the in vitro studies, spermatocyte cell line GC-2spd cells were pretreated with NAC (5 mM) 2 h before treated with BDE-209 (50 µM) for 24 h. We found that pretreatment with NAC attenuated the oxidative stress status induced by BDE-209 in vivo and in vitro. Moreover, pretreatment with NAC rescued the testicular histology impairment and decreased the testicular organ coefficient in BDE-209-exposed mice. In addition, NAC supplement partially promoted meiotic prophase and improved sperm quality in BDE-209-exposed mice. Furthermore, NAC pretreatment effectively improved DNA damage repair by recovering DMC1, RAD51, and MLH1. In conclusion, BDE-209 caused spermatogenesis dysfunction related to the meiotic arrest medicated by oxidative stress, decreasing sperm quality.

The traditional Chinese medicine Qiangjing tablet prevents blood-testis barrier injury induced by CdCl2 through the PI3K/Akt/Rictor signaling pathway.

OBJECTIVE: Environmental contaminants such as cadmium (Cd) may have a deleterious impact on sperm and reduce male fertility by compromising the blood-testis barrier (BTB). Hence, the effects of the traditional Chinese medicine Qiangjing tablet (QJP) on sperm quality and BTB alterations induced by Cd in mouse testes were examined. METHODS: Adult KM mice challenged with Cd chloride were examined, QJP was administered to mice as an oral drug by gavage, and the experiments lasted 2 weeks. Testicular and epididymal weights, sperm quality, anti-sperm antibodies (AsAb), hormone levels, and histology were evaluated. Changes in the levels of N-cadherin, occludin, ZO-1, claudin-11, F-actin, and ß-tubulin and their mRNAs were evaluated. The effects of QJP on the PI3K/Akt/Rictor pathway were evaluated. RESULTS: CdCl2 decreased reproductive organ weight, sperm quality, and testosterone (T) levels; increased AsAb, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels; induced structural damage in testicles with BTB disruption; increased BTB permeability; and decreased N-cadherin, occludin, ZO-1, claudin-11, F-actin, and ß-tubulin expression. After treatment, QJP blocked the effects of Cd on reproductive organ weight, sperm quality, and T; mitigated germinal epithelium compartment alterations; decreased AsAb, FSH, and LH levels; and preserved BTB ultrastructure and function. In addition, QJP induced increases in N-cadherin, occludin, ZO-1, claudin-11, F-actin, and ß-tubulin levels and the expression of their mRNAs through the PI3K/Akt/Rictor pathway. After the application of JRAB2011, the levels of a specific mTORC2 suppressor, Rictor, and the BTB-protective effect of QJP were greatly reduced. CONCLUSIONS: We demonstrated the effect of QJP against Cd-induced damage to the BTB, and the results indicate that QJP may play a significant role in opposing the effects of Cd through the PI3K/Akt/Rictor pathway.

Wuzi-Yanzong-Wan prevents oligoasthenospermia due to TAp73 suppression by affecting cellular junction remodeling in testicular tissue in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuzi-Yanzong-Wan (WZYZW) is a classic Chinese herbal preparation, which has a significant clinical efficacy in tonifying the kidney and benefiting the sperm, and is widely used in the treatment of oligoasthenospermia with a long history. TAp73 inhibition results in the decrease of sperm quality, but the therapeutic mechanism of WZYZW on oligoasthenospermia caused by TAp73 gene inhibition remains elusive. AIMS OF STUDY: The purpose of this study is to investigate whether TAp73 suppression leads to oligoasthenospermia and the application of WZYZW treatment in condition of TAp73 suppression. METHODOLOGY: C57BL/6 male mice were injected with Pifithrin-α (2.5 mg/kg) intraperitoneally for 30 days to induce TAp73 suppression model, with WZYZW at 1.0, 2.0 and 4.0 g/kg were administrated in parallel. The blood, testis and epididymis were collected, with organ coefficient calculated. Makler sperm counter was used to analyze the density, motility, survival and malformation rate of sperm. Apoptosis of sperm was analyzed by flow cytometry. Serum hormone levels were determined using ELISA. HE staining and transmission electron microscopy (TEM) were used to observe histopathological changes of testis in blood-testis barrier (BTB), ectoplasmic specialization (ES) and other cell junctions. Expressions of cell adhesion factors including TAp73, Integrin-α6, N-cadherin, Nectin-2 and Occludin were determined by RT-PCR and western blotting. RESULTS: Compared to control mice, TAp73 inhibition dramatically decreased the epididymal coefficient, sperm quality, and serum testosterone (T) level, while increasing apoptosis in sperm in mice. HE staining and TEM showed that the tight junction (TJ) and apical ES structure were seriously abnormal in the testis in mice with TAp73 inhibition. Additionally, the expression of Occludin protein was elevated, while that of TAp73, Integrin-α6, N-cadherin, and Nectin-2 reduced in model mice. WZYZW treatment ameliorated testicular spermatogenic dysfunctions in TAp73 suppressed mice, restoring the decreased sperm quality, serum T level and testicular histopathological changes of TJ and ES, as well as decreasing sperm malformation rate and apoptosis. Moreover, WZYZW reversed the expressions of Occludin, TAp73, Integrin-α6, N-cadherin and Nectin-2 in TAp73 suppressed mice. CONCLUSIONS: By impairing spermatogenesis and maturation, TAp73 inhibition led to oligoasthenospermia in mice. WZYZW could rescue the oligoasthenospermia associated with TAp73 inhibition via affecting the dynamic remodeling of cellular junctions in testicular tissues in mice.

Dietary selenium excess affected spermatogenesis via DNA damage and telomere-related cell senescence and apoptosis in mice.

Selenium (Se) is a vital microelement for spermatogenesis and male fertility. The aim of this study was to investigate the effects of Se on the male reproductive function and possible mechanisms. Fourty male mice were randomly divided into 0, 0.1, 0.3 and 0.9 mg/kg Se supplementation groups and given with Se dietary intervention for 12 weeks. Our data showed that excessive Se intake damaged the tissue structure of testes and epididymides of the mice, resulting in decreased sperm quality and quantity. Moreover, excessive Se induced oxidative stress, causing DNA damage and activated DNA damage repair factors (Mre11/Rad50/Nbs1), and also disrupted telomere function by shortening telomere length and decreasing TERT expression. Se excess activated the senescence pathway p53/p21/p16, leading to germ cell senescence, and inhibited cell proliferation by suppressing the Sirt1/Foxo1/c-Myc pathway. All of this led to spermatogenic cell apoptosis, thereby causing a decrease of sperm quantity and quality. In conclusion, excessive Se caused reproductive toxicity via inducing telomere dysfunction due to DNA damage, leading to germ cellular senescence and apoptosis in the testes of male mice. Our research provide new proof to explain the underlying mechanism of male reproductive toxicity triggered by excessive Se intake.

Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function. AIM OF THE STUDY: This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery. METHODS: We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration. RESULTS: Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS). CONCLUSION: Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Histological Study of Alteration in Testes and Epididymis of Domestic Rabbits Caused by Tribulus Terrestris and Vitamin E.

Tribulus Terrestris (TT) is a common herbal plant with different categories that grows in many countries of the world. Traditional Chinese and Indian therapies have used TT for infertility treatment and also as a powerful antioxidant agent. Therefore, this study aimed to use this plant supplemented with vitamin E to study their combined effects on the histological condition of the testicle and epididymis of rabbits. This study was performed on 28 healthy male rabbits (445-950 g, 2.0-3.0 months old) that were randomly divided into four groups (n=7). All animals were subjected to clinical examination to ensure that they were free of external and internal parasites with the use of some preventive treatments. The animals were housed individually (cage size: 50 cm×50 cm×40 cm) over the 60-day study period starting from January 2022, with an adaptation period of two weeks. Tribulus Terrestris and vitamin E treatments were as follows: the first group (G1) was daily fed on a standard diet and kept as the control group, the second group (G2) was daily fed on the same ration plus 1 g of TT (animal/daily), the third group (G3) was daily fed on the same ration plus 1 g of TT supplemented with 60 mg of vitamin E (orally) (animal /daily), and the fourth group (G4) was daily fed on the same ration, with the addition of 60 mg vitamin E per animal (orally). The morphometric investigation, macroscopic variables (including body weight, testicular weight, and volume), and the microscopic parameters of the testicular seminal tubule were measured. The histological section showed the absence of negative effects after the oral administration of TT at a dose of 1 g per day and 60 IU vitamin E for each animal. However, there was a positive effect on spermatogonia and spermatocytes in all animals, while the spermatogonia in the experimental groups were more dense, especially in the second and third groups, compared to the control group. The seminiferous tubules were significantly lined with spermatogonia, spermatocytes, and round spermatids (P<0.5) in the experimental groups, compared to the control group. Nevertheless, the epididymis tissue did not show traces of histological changes, such as epididymal hyperplasia. Sperms were more frequent in the lumens of the epididymis as well as the lumens larger than those of the control. Based on the results of this study, it can be concluded that the pole plant and vitamin E have a positive effect on the epithelial lining of the seminiferous tubules and the epididymis with an increase in sperm formation and differentiation towards maturity.

Multi-omics analysis reveals that iron deficiency impairs spermatogenesis by gut-hormone synthesis axis.

Considering that research has mainly focussed on how excessive iron supplementation leads to reproductive cytotoxicity, there is a lack of in-depth research on reproductive system disorders caused by iron deficiency. To gain a better understanding of the effects of iron deficiency on the reproductive system, especially spermatogenesis, we first constructed a mouse model of iron deficiency. We employed multi-omic analysis, including transcriptomics, metabolomics, and microbiomics, to comprehensively dissect the impact of iron deficiency on spermatogenesis. Moreover, we verified our findings in detail using western blot, immunofluorescence, immunohistochemistry, qRT-PCR and other techniques. Microbiomic analysis revealed altered gut microbiota in iron-deficient mice, and functional predictive analysis showed that gut microbiota can regulate spermatogenesis. The transcriptomic data indicated that iron deficiency directly alters expression of meiosis-related genes. Transcriptome data also revealed that iron deficiency indirectly regulates spermatogenesis by affecting hormone synthesis, findings confirmed by metabolomic data, western blot and immunofluorescence. Interestingly, competing endogenous RNA networks also play a vital role in regulating spermatogenesis after iron deficiency. Taken together, the data elucidate that iron deficiency impairs spermatogenesis and increases the risk of male infertility by affecting hormone synthesis and promoting gut microbiota imbalance.

Exposure to automobile exhaust-derived PM2.5 induces spermatogenesis dysfunction by damaging UPRmt of prepubertal rats.

Automobile exhaust-derived particulate matter 2.5 (PM2.5) can cause spermatogenic cell damage, potentially resulting in male infertility. This study uses male prepubertal Sprague Dawley (SD) rats to explore the molecular mechanisms by which automobile exhaust-derived PM2.5 causes spermatogenic cell damage and induces spermatogenesis dysfunction during sexual maturity by disrupting the mitochondrial unfolded protein response (UPRmt) in spermatogenic cells. Male prepubertal SD rats were randomly divided into four groups: control (intratracheal instillation of normal saline), low-dose PM2.5 (5 mg/kg), high-dose PM2.5 (10 mg/kg), and PM2.5 10 mg/kg +Vit (100 mg/kg of vitamin C and 50 mg/kg of vitamin E). The rats were treated for four weeks, with five consecutive treatment days and two non-treatment days, followed by cohabitation. Testicular and epididymal tissues were harvested for analysis. The mitochondria in spermatogenic cells were observed under an electron microscope. UPRmt-, oxidative stress-, and apoptosis-related markers in spermatogenic cells were examined. Spermatogenic cell numbers and conception rate declined significantly with increasing PM2.5 dose, with their mitochondria becoming vacuolated, swollen, and degenerated to varying degrees. The apoptosis of spermatogenic cells was abnormally enhanced in PM2.5 exposed groups compared to the control group. Spermatogenic cell numbers of conception rate gradually recovered, mitochondrial damage in spermatogenic cells was alleviated, and spermatogenic cell apoptosis was significantly reduced after vitamin intervention. In addition, protein levels of superoxide dismutase 1 (Sod1), nuclear factor erythroid 2-related factor 2 (Nrf2), and B-cell lymphoma 2 (Bcl-2) were significantly lower, while those of Bcl2-associated X apoptosis regulator (Bax), cleaved caspase 3 (Casp3), and cytochrome c (Cyt-c) and malondialdehyde (MDA) levels were significantly higher in the high-dose PM2.5 group than in the control group. The levels of UPRmt-related proteins C/EBP homologous protein (Chop), heat shock protein 60 (Hsp60), and activating transcription factors 4 (Atf4) and 5 (Atf5) were higher in the low-dose PM2.5 group, lower in the high-dose PM2.5 group, and gradually recovered in PM2.5 10 mg/kg +Vit group. Our results show that exposure to automobile exhaust-derived PM2.5 induces oxidative stress responses, leads to post-sexual maturation UPRmt dysfunction and mitochondrial impairment, and abnormally enhances spermatogenic cell apoptosis in prepubertal rats, resulting in male infertility.