Testis Function Research Articles

The use of deidentified organ donor testes for research.

Our knowledge of testis development and function mainly comes from research using mammalian model organisms, primarily the mouse. However, there are integral differences between men and other mammalian species regarding cellular composition and expression profiles during fetal and post-natal testis development and in the mature testis. Therefore, to specifically learn more about human testis development and function, there is a need to use human testis tissue for research. Human testicular tissues that have been donated for research have allowed extensive molecular and cytological assessments, as well as single-cell transcriptome and epigenome analyses. These tissues have also been used for the development of cell technologies and in vitro models that aim to improve infertility treatments and diagnostics. Biopsied material taken from patients and designated for research is usually very small in size and is unsuitable for comprehensive studies. On the other hand, research using whole testes obtained from deceased, deidentified donors has become a valuable resource to assess conservation between humans and other organisms and identify human-specific phenomena. This review discusses the acquisition of donated deidentified human testes and their use for basic science research.

Deletion of an evolutionarily conserved TAD boundary impacts spermatogenesis in mice

Abstract Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8. We then used CRISPR-Cas9 to generate a mouse line where 26 kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting mild increase in the DNA–DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, higher frequency of histological abnormalities, a drop of 47–52% in efficiency of meiosis, a 15–18% reduction in efficiency of spermatogenesis, and consistently, a 12–28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.

Appraisal the toxicity of CaO and Gold Nanoparticles in Wistar rats: A systematic review

Nanotechnology is a new science and these risks are not realistically defined or observed at present. Among these risks is that particles with a diameter of 300 nanometers or smaller can with no trouble penetrate the human body also penetrate cells, as well as that particles with a diameter of 70 nanometers or smaller can easily enter the nucleus of the cell. It is possible to imagine the potential danger to the body, as an interaction may occur between nanoparticles. Because of the small size of nanoparticles, masks and even protective masks of all types are not useful for protection against them, as these particles can penetrate them. The actual impact of nanoparticles on liver and kidney function otherwise male fertility and toxicity is assessed afterward a careful investigation of the obtainable literature. The chief role reviews animal patterns to comprehend the biomarkers related to the function of the liver and kidneys, also testis function of nanoparticles. Our principal outcomes put forward that nanoparticles are in general capable to reach the liver, kidneys, and the testicle in minor quantities where they persevere for more than a few months, nevertheless of the route of exposure. Notable, the majority of nanoparticles have little shortest toxicity to liver, kidneys, and the testis. Research conducted on mice and laboratory animals has shown that nanoparticles, after entering their bodies, tend to migrate and localize in the liver, kidney and that these particles can penetrate to reproductive system, and nanoparticles can also localize in other organs.

SARS-CoV-2-induced cytokine storm drives prolonged testicular injury and functional impairment in mice that are mitigated by dexamethasone.

Compromised male reproductive health, including reduced testosterone and sperm count, is one of the long COVID symptoms in individuals recovering from mild-severe disease. COVID-19 patients display testicular injury in the acute stage and altered serum fertility markers in the recovery phase, however, long-term implications on the testis remain unknown. This study characterized the consequences of SARS-CoV-2 on testis function. The K18-hACE2 mice that survived SARS-CoV-2 infection were followed for one month after infection and the testicular injury and function markers were assessed at different stages of infection and recovery. The long-term impact of infection on key testes function-related hormones and male fertility was measured. The efficacy of inflammation-suppressing drug in preventing testicular injury was also evaluated. The morphological defects like sloughing of spermatids into the lumen and increased apoptotic cells sustained for 2-4 weeks after infection and correlated with testicular inflammation and immune cell infiltration. Transcriptomic analysis revealed dysregulation of inflammatory, cell death, and steroidogenic pathways. Furthermore, reduced testosterone levels associated with a transient reduction in sperm count and male fertility. Most testicular impairments resolved within one month of infection. Importantly, dexamethasone treatment attenuated testicular damage, inflammation, and immune infiltration. Our results implicate virus-induced cytokine storm as the major driver of testicular injury and functional impairments, timely prevention of which limits testis damage. These findings serve as a model for evaluating therapeutics in long COVID patients and may guide clinical strategies to improve male reproductive health outcomes post-SARS-CoV-2 infection.

Modulating Role of Vernonia amygdalina Leaf Meal on Spermatogenic and Steroidogenic Functions of West African Dwarf Bucks Testis

Vernonia amygdalina has a unique nutritional and phytochemical property with numerous physiological, biochemical and morphological benefits due to its anti-oxidative and anti-helminthic properties. This study evaluated the effects of Vernonia amygdalina leaf meal (VALM) on follicle stimulating (FSH), luteinizing hormones (LH) and testosterone regulation of seminal characteristics of West African dwarf (WAD) bucks. Twenty-four (24) WAD bucks were randomly assigned to four treatments (A, B, C and D) in a completely randomized design for a nutritional trial of eight weeks. Animals were given diets containing 0, 20, 30 and 40g VALM/kg feed. Animals were weighed and blood samples were collected at the beginning of the experiment and subsequently forth-nightly before the end of the experiment for hormonal assay. Semen volume, semen color, sperm concentration, sperm motility and sperm morphology were determined from the semen samples obtained forth-nightly by electro-ejaculation. All data collected were analyzed using the General Linear Model procedure of SAS, (2002). Means were separated using Duncan’s Multiple Range Test of the same software. There were significant differences (p<0.05) in most of the hormones studied except testosterone which was not significantly affected by VALM. Sperm concentration, motility and morphology increased significantly (P<0.05) and was highest at 40 g of VALM/kg feed. The color of the semen from all the treatments was whitish grey. It was therefore concluded that VALM improved the FSH and LH production at 40 g of VALM/kg feed with improved sperm quality in WAD bucks, thereby enhancing the reproductive performance of the goats. Keywords: Vernonia amygdalina, semen quality, hormone, goats

Reprotoxicity of zinc oxide nanoparticles synthesized with Crataegus monogyna leaves extract: testis and sperm function

This work examines the effect of three doses of zinc oxide nanoparticles (ZnONPs), synthetized with Crataegus monogyna leaves using zinc acetate, on the sperm quality of Wistar rats. Animals were divided into 4 groups; the control group maintained without treatment, while ZNP1, ZNP2, and ZNP3 received respectively 10 mg ZNP/kgbw, 50 mg ZNP/kgbw, and 100 mg ZNP/kgbw by gavage for 15 days. Epididymis sperm was collected for sperm parameters: concentration, live sperm, motility, velocity (VCL, VSL, and VAP), linearity (LIN), amplitude lateral head (ALH), and beat cross frequency (BCF). DNA fragmentation was measured in three samples selected from control, ZNP1, and ZNP2. Testicular and epididymis malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GPx) were evaluated. Compared to the control, ZNP1 has a significant reduction of testicular and epididymis weights, sperm concentration, live sperm, motility, VCL, VSL, VAP, LIN, and BCF, with a significant increase of MDA and a significant decrease of GSH levels. The ZNP2 group demonstrated a significant increase in epididymis weight, a raise in sperm parameters (concentration, motility, VCL VSL, VAP, LIN, ALH, and BCF), and an augmentation in GSH and GPx levels. However, ZNP3 has a significant increase in VSL and ALH while ZNP1 and ZNP2 showed no effect on spermatozoa DNA. interestedly, we found that the lower dose of ZNP1 acted as toxic to testicular and epididymis parameters, while the higher ones may help to improve sperm quality and reduced oxidative stress.

Melatonin protects spermatogenic cells against DNA damage and necroptosis induced by atrazine

Atrazine (ATZ), a widely used triazine herbicide, has been shown to disrupt reproductive development in organisms. Melatonin (MLT) is a natural hormone and has been shown to have strong antioxidant properties. Due to its lipophilicity, it can cross biological barriers freely and act on germ cells directly. However, the mechanism through which melatonin affects atrazine-induced damage to male sperm cells remains unclear. This study aimed to investigate the effects of ATZ on spermatocyte development and to elucidate MLT's role in preventing ATZ-induced spermatogenesis failure. Pubertal mice were randomly divided into four groups: blank control group (Con), 5 mg/kg melatonin group (MLT), 170 mg/kg atrazine group (ATZ), and ATZ + MLT group. GC-1 cell culture was employed to access the in vitro effects of MLT and ATZ on spermatogonia. The results indicate that atrazine affected protein and metabolite composition, and reduced sperm viability, sperm motility (VAP, VSL and VCL) and levels of proteins related to spermatogenesis function in the mice testis. Melatonin alleviated the development of cellular DNA damage and necroptosis caused by atrazine both in vivo and in vitro. Moreover, we proposed that it was GC-1 cells developing necroptosis, but not other cell types in the testis. In conclusion, this study suggests that atrazine disrupts the development process, causing DNA damage in spermatozoa during spermatogenesis. Additionally, ATZ-induced necroptosis specifically targets spermatogenic cells. Notably, melatonin alleviates atrazine-induced necroptosis and DNA damage in spermatogenic cells. This study provides new insights into potential therapeutic strategies for atrazine-induced male infertility.

Non-alkylating agents-induced gonadotoxicity in pre-pubertal males: Insights on the clinical and pre-clinical front.

Whilst chemotherapy regimens have proven to be more successful for pediatric cancer patients over the years, their influence on long-term side effects is relatively poorly understood. One of the possible targets is the gonads, with gonadotoxic agents representing those that threaten the patient's ability to have children post surviving the primary disease treatment. Many risk stratification guidelines have categorized these agents based on the severity of their effect on the pre-pubertal testis. While the consensus is that those agents factored with a cyclophosphamide equivalent dosage pose the highest threat to fertility (e.g. alkylating agents), other agents might still contribute to a reduced testis function; especially in the case of combination therapies. Besides, it is important to note that studies deciphering the effect of other non-alkylating agents on the pre-pubertal testis lack standardized conclusions for clinically relevant outcomes. This makes it imperative to ensure the knowledge gap is addressed between the clinic and pre-clinic to understand potential gonadotoxic effects, ultimately leading to improved patient care. Therefore, this review will summarize the key findings in understanding the gonadotoxic effects of the most commonly researched non-alkylating agents: vincristine, etoposide, doxorubicin, and imatinib on the pre-pubertal testis.

Poly(A)-Specific Ribonuclease Deficiency Leads to Deregulated Expression of Genes Involved in Sex Determination and Gonadal Maturation in Zebrafish

Abstract Background Deadenylation, the process of removal of poly (A) tail of messenger ribonucleic acids (mRNAs), is a rate-limiting step in mRNA stability, and poly(A)-specific ribonuclease (PARN) is the most important exonuclease involved in this process. Besides mRNA stability, PARN is also involved in several other processes including telomere maintenance, noncoding RNA maturation, ribosome biogenesis, and TP53 function. Previously, we have shown that zebrafish PARN null mutants are viable and fertile but turn out to only develop into males, indicating a role in oogenesis. The present study was focused on analyzing the expression of genes involved in sex determination and gonadal development in PARN mutant zebrafish. Materials and Methods Total RNA was extracted and quantitative real-time polymerase chain reaction was performed to determine the expression level of genes involved in gonad development in PARN mutant embryos (4 days postfertilization [dpf]) and adults (120 dpf) in comparison to their wild-type siblings. The expression levels were estimated by the ΔΔCT relative quantification method. Results At 4 dpf, the expression of germ cell-specific genes did not show any significant difference in the null mutants compared to the heterozygous and their wild-type siblings, suggesting no effect on germ cell differentiation due to the loss of PARN. However, the majority of the ovary-associated genes analyzed showed an increased expression in PARN null and heterozygous mutants compared to the wild-type siblings. Intriguingly, the expression of testis-associated genes showed decreased expression in the mutants compared to their wild-type siblings at 4 dpf. In adult stages, as expected, the expression of genes that jointly regulate the proper formation and function of ovaries and testes showed decreased expression in PARN null mutants. Interestingly, the expression of genes involved in the differentiation of testes, despite showing a decreased expression in the mutants, was comparable between the null and heterozygous mutants. Conclusion Taken together, these results suggest that the loss of PARN does not affect germ cell differentiation but affects the sexual differentiation that happens at later stages of development, particularly the process of oogenesis, in zebrafish.

Deletion of an evolutionarily conserved TAD boundary compromises spermatogenesis in mice.

Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8 . We then used CRISPR-Cas9 to generate a mouse line where 26kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting increase in the DNA-DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, abnormal testis histology, a 35% drop in the estimated efficiency of spermatogenesis and a 28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences

BackgroundExposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h.ResultsLead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria.ConclusionsPb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.Graphical

Trace elements alone or in mixtures associated with unconventional natural gas exploitation affect rat fetal steroidogenesis and testicular development in vitro

Biomonitoring studies have shown that pregnant women living in regions of unconventional natural gas (UNG) exploitation have higher levels of trace elements. Whether developmental endocrine disruption can be expected at these exposure levels during pregnancy is unclear. In this study, we aimed to test the impact of five trace elements alone or in mixtures using in vitro cell- and tissue-based assays relevant to endocrine disruption and development. Manganese, aluminum, strontium, barium, and cobalt were tested at concentrations including those representatives of human fetal exposure. Using transactivation assays, none of the tested elements nor their mixture altered the human estrogen receptor 1 or androgen receptor genomic signalling. In the rat fetal testis assay, an organ culture system, cobalt (5 μg/l), barium (500 μg/l) and strontium (500 μg/l) significantly increased testosterone secretion. Cobalt and strontium were associated with hyperplasia and/or hypertrophy of fetal Leydig cells. Mixing the five elements at concentrations where none had an effect individually stimulated testosterone secretion by the rat fetal testis paralleled by the significant increase of 3β-hydroxysteroid dehydrogenase protein level in comparison to the vehicle control. The mechanisms involved may be specific to the fetal testis as no effect was observed in the steroidogenic H295R cells. Our data suggest that some trace elements in mixture at concentrations representative of human fetal exposure can impact testis development and function. This study highlights the potential risk posed by UNG operations, especially for the most vulnerable populations, pregnant individuals, and their fetus.

Adropin promotes testicular functions by modulating redox homeostasis in adult mouse.

Adropin is an emerging metabolic hormone that has a role in regulating energy homeostasis. The present study aimed to explore the impact of adropin on redox homeostasis and its possible role in testicular functions in adult mouse testis. Western blot, flow-cytometry, and TUNEL assay were performed to explore the impact of intra-testicular treatment of adropin (0.5 μg/testis) on testicular functions of adult mice. Hormonal assay was done by ELISA. Further, antioxidant enzyme activities were measured. Adropin treatment significantly increased the sperm count and testicular testosterone by increasing the expression of GPR19 and steroidogenic proteins. Also, adropin treatment reduced the oxidative/nitrosative stress by facilitating the translocation of NRF2 and inhibiting NF-κB into the nucleus of germ cells. Enhanced nuclear translocation of NRF2 leads to elevated biosynthesis of antioxidant enzymes, evident by increased HO-1, SOD, and catalase activity that ultimately resulted into declined LPO levels in adropin-treated mice testes. Furthermore, adropin decreased nuclear translocation of NF-κB in germ cells, that resulted into decreased NO production leading to decreased nitrosative stress. Adropin/GPR19 signaling significantly increased its differentiation, proliferation, and survival of germ cells by elevating the expression of PCNA and declining caspase 3, cleaved caspase 3 expression, Bax/Bcl2 ratio, and TUNEL-positive cells. FACS analysis revealed that adropin treatment enhances overall turnover of testicular cells leading to rise in production of advanced germ cells, notably spermatids. The present study indicated that adropin improves testicular steroidogenesis, spermatogenesis via modulating redox potential and could be a promising target for treating testicular dysfunctions.

Perfluorooctanoic acid triggers premature ovarian insufficiency by impairing NAD+ synthesis and mitochondrial function in adult zebrafish.

High-dose perfluorooctanoic acid (PFOA) impairs oocyte maturation and offspring quality. However, the physiological concentrations of PFOA in follicular fluids of patients with premature ovarian insufficiency (POI) were detected at lower levels, thus the relationship between physiological PFOA and reproductive disorders remains elusive. Here, we investigated whether physiological PFOA exposure affects gonad function in adult zebrafish. Physiological PFOA exposure resulted in POI-like phenotypes in adult females, which exhibited decreased spawning frequency, reduced number of ovulated eggs, abnormal gonadal index, and aberrant embryonic mortality. Meanwhile, oocytes from PFOA-exposed zebrafish showed mitochondrial disintegration and diminished mitochondrial membrane potential. Unlike the high-dose treated oocytes exhibiting high reactive oxygen species (ROS) levels and excessive apoptosis, physiological PFOA reduced the ROS levels and did not trigger apoptosis. Interestingly, physiological PFOA exposure would not affect testis function, indicating specific toxicity in females. Mechanistically, PFOA suppressed the NAD+ biosynthesis and impaired mitochondrial function in oocytes, thus disrupting oocyte maturation and ovarian fertility. Nicotinamide mononucleotide (NMN), a precursor for NAD+ biosynthesis, alleviated the PFOA-induced toxic effects in oocytes and improved the oocyte maturation and fertility upon PFOA exposure. Our findings discover new insights into PFOA-induced reproductive toxicity and provide NMN as a potential drug for POI therapy.

Kiwifruit (Actinidia deliciosa) aqueous extract improves hyperglycemia, testicular inflammation, apoptosis, and tissue structure induced by Streptozotocin via oxidative stress inhibition

Diabetes mellitus (DM) is a well-known hyperglycemic metabolic condition identified by oxidative stress and biological function disruption. Kiwifruit is a valuable source of polyphenols and vitamin C with great antioxidant, nutritional, and health-promoting effects. Therefore, this study was initiated to explore the antioxidant and anti-hyperglycemic effects of kiwifruit aqueous extract (KFE) against oxidative injury and testis dysfunction in rats with diabetes. Twenty-four male Wistar Albino rats (160–170 g) were divided into four groups: Group 1 served as the control, Group 2 supplemented orally with kiwifruit extract (KFE; 1 g/kg/day) for one month, Group 3 was treated with a single streptozotocin dose (STZ; 50 mg/kg ip), and Group 4 where the diabetic rats were administered with KFE, respectively. According to the results, the GC-MS analysis of KFE revealed several main components with strong antioxidant properties. In diabetic rats, lipid peroxidation and hyperglycemia were accompanied by perturbations in hormone levels and sperm characteristics. Antioxidant enzymes, glutathione content, aminotransferase, phosphatase activities, and protein content were decreased. Furthermore, histology, immunohistochemical PCNA expression, and histochemical analysis of collagen, DNA, RNA, and total protein. were altered in rat testis sections, supporting the changes in biochemistry. Furthermore, diabetic rats supplemented with KFE manifested considerable amendment in all the tested parameters besides improved tissue structure and gene expressions (NF-kB, p53, IL-1β, Bax, IL-10, and Bcl2) relative to the diabetic group. In conclusion, KFE has beneficial effects as it can improve glucose levels and testis function, so it might be used as a complementary therapy in DM.

Aphrodisiac and androgenic effects of the aqueous extract of the roots of Vepris afzelii on cyproterone acetate-induced hypogonadism in rat.

This work aimed to evaluate the effects of the aqueous extract of Vepris afzelii roots on a rat model of hypogonadism. Phytochemical screening and acute toxicity of the extract were performed using different procedures. Hypogonadism was induced orally in adult Wistar rats using cyproterone acetate (30 mg/kg) for ten days. Besides six normal rats (10 ml/kg of distilled water, normal control), 30 hypogonadal rats were subdivided into five groups of six animals each, receiving for 14 days: distilled water (10 ml/kg, hypogonadal control), testosterone (4 mg/kg/3days) and the extract of V. afzelii (100, 200 and 400 mg/kg). Sexual behavior, sperm parameters, testes function and structure were assessed. Compared to the normal controls, significant (p = 0.0000) increases in mount (24 ± 0.94 seconds vs. 1200 ± 00 seconds) and intromission (49.16 ± 10.85 seconds vs. 1200 ± 00 seconds) latencies, and post-ejaculatory interval (381.72 ± 37.55 seconds vs. 1200 ± 00 seconds) were observed in all groups receiving cyproterone acetate on day 0. Total inhibitions of mounts (63.50 ± 8.91 vs. 00 ± 00), intromissions (36.66 ± 3.51 vs. 00 ± 00) (p = 0.0000), ejaculations (2.83 ± 00 vs. 00 ± 00, p = 0.0002) frequencies and mean copulatory interval (627.30 ± 81.80 vs. 00 ± 00, p = 0.0000) were also observed in these groups. Moreover, decreases in daily sperm production (2.65 ± 0.19 vs. 1.17 ± 0.08, p = 0.0498), percentage of sperm mobility (78.64 ± 8.41 vs. 10.12 ± 2.32), serum testosterone level (8.39 ± 0.63 ng/dl vs. 1.68 ± 0.19 ng/dl), diameter of seminiferous tubules (111.97 ± 0.51 µm vs. 94.51 ± 0.57 µm) and height of germinal epithelium (46.58 ± 0.34 µm vs. 33.74 ± 0.66 µm) (p = 0.0000) associated with increases in sperm transit (3.13 ± 0.45 vs. 11.07 ± 1.45, p = 0.0000) were also observed in these groups. Interestingly, compared to hypogonadal control and day 0, the administration of V. afzelii extract induced significant (p = 0.0000) improvements in all these altered parameters with 400 mg/kg being the most active dose. These results, attributed to saponins, flavonoids, polyphenols and triterpenes detected in this plant's extract confirm its traditional usage and could be useful for the management of patients suffering from hypogonadism.

Age-dependent changes in the expression and localization of LYZL4, LYZL6 and PCNA during testicular development in the Ashidan yak

Lysozyme like 4 (LYZL4), lysozyme like 6 (LYZL6) and proliferating cell nuclear antigen (PCNA) are implicated in the regulation of testicular function, but there was no research reported available on the expression patterns of LYZL4, LYZL6 and PCNA genes at different developmental stages of yak testes. In this study, we used the qRT-PCR, western blotting and immunohistochemistry estimated the LYZL4, LYZL6 and PCNA gene expression and protein lo-calization at different developmental stages of yak testes. The qPCR results showed that the mRNA expression of LYZL4, LYZL6 and PCNA genes significantly increased with age in the testes of yaks. Western blot results showed that the protein abundance of LYZL4, LYZL6 and PCNA in yak testes was significantly higher after puberty than before puberty. Furthermore, the results of immunohistochemistry indicated that LYZL4, LYZL6 and PCNA may be involved in the regulation of spermatogonia proliferation and Leydig cell function in immature testis. In adult yak testes, LYZL4, LYZL6 and PCNA may involve in the development of round spermatids and primary spermatocytes during testicular development. Our results indicated that LYZL4, LYZL6 and PCNA may be involved in the development of Sertoli cells, Leydig cells and gonocytes in yak testes.

Stem Leydig cells support macrophage immunological homeostasis through mitochondrial transfer in mice

As testicular mesenchymal stromal cells, stem Leydig cells (SLCs) show great promise in the treatment of male hypogonadism. The therapeutic functions of mesenchymal stromal cells are largely determined by their reciprocal regulation by immune responses. However, the immunoregulatory properties of SLCs remain unclear. Here, we observe that SLCs transplantation restore male fertility and testosterone production in an ischemia‒reperfusion injury mouse model. SLCs prevent inflammatory cascades through mitochondrial transfer to macrophages. Reactive oxygen species (ROS) released from activated macrophages inducing mitochondrial transfer from SLCs to macrophages in a transient receptor potential cation channel subfamily member 7 (TRPM7)-mediated manner. Notably, knockdown of TRPM7 in transplanted SLCs compromised therapeutic outcomes in both testicular ischemia‒reperfusion and testicular aging mouse models. These findings reveal a new mechanism of SLCs transplantation that may contribute to preserve testis function in male patients with hypogonadism related to immune disorders.

Integrated analysis of lncRNA, miRNA and mRNA expression profiles reveals regulatory pathways associated with pig testis function

Long noncoding RNA (lncRNA) and microRNA (miRNA) are known to play pivotal roles in mammalian testicular function and spermatogenesis. However, their impact on porcine male reproduction has yet to be well unraveled. Here, we sequenced and identified lncRNA and miRNA expressed in the testes of Chinese indigenous Banna mini-pig inbred line (BMI) and introduced Western Duroc (DU) and Large White (LW) pigs. By pairwise comparison (BMI vs DU, BMI vs LW, and DU vs LW), we found the gene expression differences in the testes between Chinese local pigs and introduced Western commercial breeds were more striking than those between introduced commercial breeds. Furthermore, we found 1622 co-differentially expressed genes (co-DEGs), 122 co-differentially expressed lncRNAs (co-DELs), 39 co-differentially expressed miRNAs (co-DEMs) in BMI vs introduced commercial breeds (DU and LW). Functional analysis revealed that these co-DEGs and co-DELs/co-DEMs target genes were enriched in male sexual function pathways, including MAPK, AMPK, TGF-β/Smad, Hippo, NF-kappa B, and PI3K/Akt signaling pathways. Additionally, we established 10,536 lncRNA-mRNA, 11,248 miRNA-mRNA pairs, and 62 ceRNA (lncRNA-miRNA-mRNA) networks. The ssc-miR-1343 had the most interactive factors in the ceRNA network, including 20 mRNAs and 3 lncRNAs, consisting of 56 ceRNA pairs. These factors played extremely important roles in the regulation of testis function as key nodes in the interactive regulatory network. Our results provide insight into the functional roles of lncRNAs and miRNAs in porcine testis and offer a valuable resource for understanding the differences between Chinese indigenous and introduced Western pigs.

Exercise and insulin glargine administration in mothers with diabetes during pregnancy ameliorate function of testis in offspring: Consequences on apelin-13 and its receptor

AimsDespite the evidence exhibited that diabetes during gestation (DDG) is linked with reproductive dysfunction in offspring, the underlying cellular mechanisms involved are not precisely defined. This study was designed to assess the impact of voluntary exercise and insulin glargine on DDG-induced metabolic and reproductive disorders in male offspring. Main methodsFifty female Wistar rats (three weeks old) received a control diet (n = 10) or high-fat-high-sucrose diet (to induce DDG; n = 40) for six weeks before breeding. From the 7th day of pregnancy onwards, blood glucose over 140 mg/dL was characterized as DDG. Then, the DDG animals were randomly divided into four subgroups with/without voluntary exercise and/or insulin glargine. To evaluate insulin resistance, a glucose tolerance test was performed on the 15th day of pregnancy. After three weeks, male offspring were weaned, and fed a control diet until 12 weeks old. At the end of the experiment, the lipid profile, sex hormones, and apelin-13 in the serum, mRNA expression of apelin receptors (APJ) in the testis and sperm analysis were assessed. Key findingsOur results indicated that voluntary exercise and/or insulin glargine administration in mothers with DDG ameliorated lipid profile, and sex hormones alterations, reduced the serum level of apelin-13, as well as increased APJ expression in testis, and quality of sperm in offspring. SignificanceCombined administration of voluntary exercise and insulin glargine during pregnancy by regulating of apelinergic system and inhibiting the metabolic and reproductive complications induced by DDG, can be considered as a suitable therapeutic strategy for improving sub-or in-fertility in the male offspring.