Inhibition of mitochondrial cyclophilin D, a downstream target of glycogen synthase kinase 3α, improves sperm motility.

BACKGROUND: Cyclophilin D (CypD) negatively regulates ATP production by opening of the mitochondrial permeability transition pore. This study aimed to understand the role of CypD in sperm motility regulation. METHODS: Changes in CypD during sperm capacitation and its interaction with glycogen synthase kinase 3α (GSK3α), a key kinase regulating sperm motility, were examined in mouse spermatozoa. The effects of CypD inhibitor cyclosporin A (CsA) and GSK3 inhibitor 6-bromo-indirubin-3'-oxime (BIO) on sperm motility, p-GSK3α(Ser21), mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), and ATP production were examined. The effect of proteasome inhibitor MG115 on the cellular levels of CypD was examined. RESULTS: In cauda epididymal spermatozoa, GSK3α was found in both cytosolic and mitochondrial fractions whereas CypD was primarily found in the mitochondrial fraction together with ATP synthase F1 subunit alpha (ATP5A), a mitochondrial marker. GSK3α and CypD were co-localized in the sperm midpiece. Interaction between GSK3α and CypD was identified in co-immunoprecipitation. CsA, a CypD inhibitor, significantly increased sperm motility, tyrosine phosphorylation, mPTP closing, MMP, and ATP levels in spermatozoa, suggesting that CypD acts as a negative regulator of sperm function. Under capacitation condition, both GSK3α and CypD were decreased in spermatozoa but ATP5A was not. The GSK3 inhibitor BIO markedly increased p-GSK3α(Ser21) and decreased CypD but significantly increased mPTP closing, MMP, ATP production, and motility of spermatozoa. This suggests that inhibitory phosphorylation of GSK3α is coupled with degradation of CypD, potentiating the mitochondrial function. Degradation of CypD was attenuated by MG115, indicative of involvement of the ubiquitin proteasome system. CONCLUSIONS: During sperm capacitation, CypD act as a downstream target of GSK3α can be degraded via the ubiquitin proteasome system, stimulating mitochondrial function and sperm motility.

Dibutyl phthalate disrupts glycogen synthase kinase 3α essential for sperm motility.

To unravel the toxic mechanism of phthalate ester plasticizer endocrine disruptor in spermatozoa, we examined the effect of dibutyl phthalate (DBP) on the stability and inhibitory phosphorylation of glycogen synthase kinase 3α (GSK3α), a protein kinase crucial for sperm motility in mice. In DBP-treated spermatozoa, reactive oxygen species (ROS) and lipid peroxide were significantly increased. In computer-assisted sperm analysis, DBP at concentrations of 10 - 100 µg/mL significantly decreased total motility and progressive motility of spermatozoa. On western blots, DBP decreased p-GSK3α(Ser21) and increased p-GSK3α(Tyr279) in spermatozoa. Similarly, hydrogen peroxide decreased p-GSK3α(Ser21) but not p-GSK3α(Tyr279) in spermatozoa. Immunofluorescent labeling demonstrated that DBP markedly decreased immunoreactivities of GSK3α and p-GSK3α(Ser21) but increased immunoreactivity of p-GSK3α(Tyr279) in spermatozoa. DBP at a concentration of 100 µg/mL significantly increased phosphatase activity in spermatozoa. Calyculin A, a protein phosphatase 1 and 2 A inhibitor, markedly increased p-GSK3α(Ser21) and sperm motility and attenuated a DBP-induced decrease of p-GSK3α(Ser21) and sperm motility. On western blot, 1-100 µg/mL DBP decreased GSK3α in spermatozoa. On immunoprecipitation western blot, DBP at 10 - 100 µg/mL increased polyubiquitinated sperm proteins including GSK3α. The MG115, proteasome inhibitor attenuated degradation of GSK3α in DBP-treated spermatozoa. Hydrogen peroxide at 10 µM increased polyubiquitinated sperm proteins, suggesting that DBP may increase ubiquitination of GSK3α via ROS induction. Together, DBP may decrease the cellular amount of GSK3α through the ubiquitin-proteasome pathway and p-GSK3α(Ser21) through ROS generation and activation of protein phosphatases, impairing sperm motility.

Isosorbide, a versatile green chemical: Elucidating its ADME properties for safe use.

Isosorbide, an environmentally friendly and renewable substance, finds extensive application in diverse fields, such as a bisphenol A substitute, polymers, functional materials, organic solvents, fuels, and pharmaceuticals. Despite its increasing interest and widespread usage, there remains a notable absence of available reports regarding its absorption, distribution, metabolism, and excretion (ADME) properties. This study endeavors to investigate the ADME characteristics of isosorbide in rats. Isosorbide levels in biological samples were quantified based on the analytical method using gas chromatography-mass spectrometry (GC-MS). Following administration, isosorbide exhibited rapid absorption and elimination, with a bioavailability of 96.1%. The metabolic stability assay indicated that isosorbide remained stable during metabolism. The majority of absorbed isosorbide was promptly excreted, with urinary excretion as the primary route. This study furnishes valuable insights into the ADME of isosorbide, contributing to its safety assessment and fostering its continued application across various domains.

Increased testicular insulin-like growth factor 1 is associated with gonadal activation by recombinant growth hormone in immature rats.

BACKGROUND: In children, recombinant human growth hormone (rhGH) therapy for treatment of short stature has raised concerns of the early onset of puberty. Puberty is initiated by the activation of the hypothalamus-pituitary-gonad axis. Insulin-like growth factor-1 (IGF1) has been known to mediate physiologic effects of GH. To understand the mechanism of precocious sexual maturation following prepubertal GH therapy, the effects of rhGH on the hypothalamus-pituitary-gonad axis were examined in the immature male rats. METHODS: Immature male rats were given by daily injection of rhGH (1 or 2 IU/kg) from postnatal day (PND) 21 to PND 23 or 30. The effects of rhGH on kisspeptin-GnRH-LH system in the hypothalamus-pituitary axis, systemic and testicular IGF1, spermatogenesis, steroidogenesis, and circulating testosterone levels were examined. The effects of rhGH on the IGF1 expression and steroidogenesis were examined in progenitor LCs in vitro. RESULTS: Testicular steroidogenic pathway and spermatogenesis marker mRNA levels, number and size of 17ß-hydroxysteroid dehydrogenase (+) LCs, and blood testosterone levels of rhGH rats were significantly higher than those of controls on PNDs 24 and 31. Hypothalamic Kiss1 and Gnrh1 mRNA of rhGH rats were significantly higher than those of controls on PND 24, indicating early activation of hypothalamic kisspeptin-GnRH neurons by rhGH. Hypothalamic Igf1 mRNA levels of rhGH rats were significantly higher than those of controls on PND 24 but significantly lower than those of controls on PND 31. Testicular Igf1 mRNA levels were significantly higher in rhGH rats than in the controls on PNDs 24 and 31 whereas circulating IGF1 levels were not. In progenitor LCs, rhGH significantly increased Igf1 and steroidogenic pathway mRNA levels and testosterone production. CONCLUSIONS: Local increases in testicular IGF1 might be an important mediator of gonadal maturation via activation of LCs steroidogenesis in immature rats given rhGH.

Phthalate plasticizer decreases the prion-like protein doppel essential for structural integrity and function of spermatozoa.

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.

Assessment of endocrine-disrupting activities of alternative chemicals for bis(2-ethylhexyl)phthalate.

Plastic products are closely intertwined with modern life. Some plasticizers used in making plastics, such as phthalates, are reported to be endocrine-disrupting chemicals. Plasticizers can be released into the environment, and health risks related to plasticizer exposure have been reported. In addition, due to plastic waste that flows into the ocean, microplastics have been found in marine products, including non-biological seawater products such as sea salt. Plastics can affect the body via a variety of pathways, and therefore safer alternative chemicals are needed. Three chemicals were evaluated: acetyl tributyl citrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylacitrate (ATHC), replacing bis(2-ethylhexyl)phthalate (DEHP), a typical plasticizer. The endocrine-disrupting activities of each chemical, including estrogenic or anti-estrogenic activity (test guideline (TG) No. 455), androgenic or anti-androgenic activity (TG No. 458), steroidogenesis (TG No. 456), and estrogenic properties via a short-term screening test using the uterotrophic assay (TG No. 440), were assessed in accordance with the Organisation for Economic Co-operation and Development guidelines for chemical testing. Our results showed that DEHP, ATBC, ATEC, ATHC possess no estrogenic activity, whereas DEHP, ATBC and ATHC demonstrate anti-estrogenic activity and ATBC anti-androgenic activity. DEHP and ATHC exhibited a disruption in steroidogenesis activities. Additional tests are necessary, but our results suggest that ATEC is a good candidate plasticizer providing a suitable alternative to DEHP.

Effects of nonylphenols on embryonic development and metamorphosis of Xenopus laevis: FETAX and amphibian metamorphosis toxicity test (OECD TG231).

Nonylphenols (NPs) are a group of endocrine-disrupting surfactants that mimic estrogen. To determine the developmental toxicity and thyroid-disrupting effect of NPs, the effects of exposure to nonylphenol (NP), 4-nonylphenol (4-NP), and nonylphenol ethoxylate (NP-12) were examined according to the frog embryo teratogenesis assay-Xenopus (FETAX) and Organization for Economic Co-operation and Development test guidelines 231 (TG231). In FETAX, the LC50 values of NP, 4-NP, and NP-12 were 59.14 mg/L, 10.13 mg/L, and 14.60 mg/L, respectively. At 10.0 mg/L, NP, 4-NP, and NP-12 significantly decreased the total length of tadpoles, and NP and 4-NP increased gut malformation and bent tails. In surviving tadpoles, the EC50 values for malformation of NP, 4-NP, and NP-12 were 4.66, 6.51, and 13.08 mg/L, respectively. The teratogenic indices of NP, 4-NP, and NP-12 were 12.69, 1.56, and 1.08, respectively, suggesting the teratogenic potential of NP and 4-NP. In a range-finder assay for TG231, the 96-h LC50 values of NP, 4-NP, and NP-12 were 2.0, 2.0, and 10.57 mg/L, respectively. When NF stage 51 larvae were exposed for 21 days, larval growth was inhibited by NP, 4-NP, and NP-12 at 0.67, 0.07, and 0.37 mg/L, respectively. 4-NP at 0.07 mg/L accelerated the developmental stage and significantly increased hind limb length, while 0.67 mg/L 4-NP delayed the developmental stage and decreased hind limb length, suggesting a bimodal effect of 4-NP on metamorphosis. NP and NP-12 at test concentrations did not alter the larval stage, but NP-12 at 0.37 mg/L significantly decreased total length and tail length, suggesting growth inhibition in larvae. The total colloid area of thyroid follicles was significantly increased by 0.07 mg/L 4-NP but not by NP and NP-12, suggesting that 4-NP may interfere with thyroid function. Together, the developmental toxicity of NPs was in the following order: 4-NP, NP-12, and NP. 4-NP may alter metamorphosis driven by thyroid hormones in X. laevis.

Effects of citrate ester plasticizers and bis (2-ethylhexyl) phthalate in the OECD 28-day repeated-dose toxicity test (OECD TG 407).

Citrate esters are considered functional alternatives to phthalate plasticizers, but their toxicity remains poorly understood. The toxicity of citrate esters, including triethyl 2-acetylcitrate (ATEC) and trihexyl O-acetylcitrate (ATHC), were examined together with that of bis (2-ethylhexyl) phthalate (DEHP) using the Organization for Economic Co-operation and Development Test Guideline 407 (OECD TG407). Following 28-day oral administration, no significant differences in body weight or the weight of the brain, pituitary, heart, epididymis, seminal vesicles, or coagulating gland were found between the vehicle control and DEHP, ATEC or ATHC groups. In the 400 mg/kg day DEHP group, liver, adrenal, thymus, spleen, kidney, testis, and prostate weights were significantly increased. In the 400 mg/kg day ATHC group, kidney, adrenal, thymus, testis and prostate weights were significantly increased. In the 400 mg/kg day ATEC group, kidney, adrenal and testis weights were significantly increased. Hepatocyte size was significantly increased in the 400 mg/kg day DEHP group, suggestive of hepatotoxicity, but was not increased in the ATEC or ATHC groups. There were no significant differences in white blood cell, red blood cell or platelet counts, hemoglobin concentrations, hematocrit, mean corpuscular volume, fasting glucose, insulin, or testosterone concentrations between the vehicle control and DEHP, ATEC and ATHC groups. In the ATHC 400 mg/kg day group, T3 was decreased while T4 was increased, suggestive of disruption of thyroid function. The results of the OECD TG407 subacute repeated dosing toxicity test indicate ATEC is less toxic compared to ATHC or DEHP and could be recommended as an alternative to phthalate plasticizers.

Toxicological assessment of phthalates and their alternatives using human keratinocytes.

Phthalates are mainly used as binders and plasticizers in various industrial products including detergents, surfactants, waxes, paints, pharmaceuticals, food products, and cosmetics. However, they have been reported to be endocrine disruptors, which are chemicals that can mimic or disturb endocrines, causing interference to the endocrine system. Recently, there have been numerous reports showing that phthalates have negative health impacts such as asthma, breast cancer, obesity, type II diabetes, and male infertility. Due to these effects, there is an urgent need for phthalate alternatives. In this study, the potential cytotoxicity of phthalates and their substitutes were screened in HaCaT cells, a human keratinocyte cell line, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) thiazolyl blue assay, immunocytochemistry, flow cytometric analysis, and western blotting. We confirmed that common phthalates such as butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP) have genotoxic effects, leading to cell death. Among the known phthalate substitutes, tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) were tested for cytotoxicity. As a result, ATEC showed similar levels of cytotoxicity with the phthalates whereas ATBC and ATHC did not show significant cytotoxicity even in high doses (5 mg/ml).

Titanium dioxide nanoparticles induce apoptosis by interfering with EGFR signaling in human breast cancer cells.

Titanium dioxide nanoparticles, due to their smaller size and increased surface area comparted to the bulk form, are known to be bioreactive and have unexpected toxicological outcomes. Previous studies have shown that nanoscale titanium dioxide induces reactive oxygen species (ROS)-mediated cytotoxicity and genotoxicity. Although many reports have discussed the ROS-mediated cytotoxic effects of titanium dioxide nanoparticles (TiO2-NPs), their effects on the receptor-ligand association are unknown. In this study, the possibility that TiO2-NPs can interfere with the receptor-ligand binding was assessed by monitoring alterations in the phosphorylation status of proteins downstream of the epidermal growth factor receptor (EGFR) signaling cascade. TiO2-NPs blocked ligand-induced EGFR autophosphorylation, leading to the deactivation of EGFR downstream effectors such as Akt and extracellular signal-regulated kinase signaling, inducing cell death.

Cytotoxicity measurement of Bisphenol A (BPA) and its substitutes using human keratinocytes.

Bisphenol-A (BPA) was first synthesized in the 1890s and has been used in many plastic products. However, BPA is known to act as an endocrine disruptor and has been found to be toxic to human health. Many alternative substances have been developed to replace BPA, but it is still widely used worldwide. In this study, we identified the potential cytotoxicity of BPA by evaluating toxicity using human keratinocytes. Also, we evaluated cytotoxicity of BPA substitutes to determine their suitability as an alternative to BPA. The proliferation assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and western blot analysis showed that BPA significantly affect cell viability, induction of apoptotic fraction and increased activation of DNA-damage marker protein. In addition, through the same experiments, the substitutes of BPA were shown to be significantly less toxic than BPA, and the least toxicity was observed with 1,4-cyclohexanedimethanol (CHDM) and terephthalic acid (TPA). In conclusion, this study suggests that cytotoxicity of BPA induces apoptosis of human keratinocytes, and that CHDM and TPA are the most suitable substitutes for BPA.

Comparative toxicological evaluation of nonylphenol and nonylphenol polyethoxylates using human keratinocytes.

Nonylphenol polyethoxylates (NPEOs) are a major group of nonionic surfactants widely used in various detergents, cleaners, plastics, papers, and agro-chemical products. Nonylphenol (NP), which is a final degraded metabolite derived from NPEOs, has been reported as an endocrine disrupter, known to mimic or disturb reproductive hormone functions. Concern about the hazards of NP and NPEOs has generated legal restrictions and action plans worldwide. Considering the fact that NP and NPEOs are majorly used in the production of products such as detergents, shampoos, and cosmetics which frequently come into contact with the skin, we investigated the effects of NP and NPEOs on a human keratinocyte cell line (HaCaT). In this study, the toxicity of NP and NPEOs was screened in HaCaT cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue assay and Western blotting. The potential cytotoxicity of substitutes was assessed by dose-response assays, relative cell viability, and genotoxicity caused by specific alterations in DNA damage response proteins (including ataxia-telangiectasia mutated, p53, Chk1, Chk2, and Histone H2A.X). We demonstrated that NP and NPEOs are toxic to HaCaT cells, as revealed by the decreased cell viability after 24 h treatment. NPs and NPEOs also induced apoptosis and DNA damage as shown by the activation of Poly(ADP-ribose) polymerase, Caspase-3, and Histone H2A.X.

Effect of particulate matter 2.5 on gene expression profile and cell signaling in JEG-3 human placenta cells.

Particulate matter the environmental toxicant, with a diameter less than or equal to 2.5 µm (PM2.5 ) is a common cause of several respiratory diseases. In recent years, several studies have suggested that PM2.5 can influence diverse diseases, such as respiratory diseases, cardiovascular diseases, metabolic diseases, dementia, and female reproductive disorders, and unhealthy birth outcomes. In addition, several epidemiological studies have reported that adverse health effects of PM2.5 can differ depending on regional variations. In the present study, to evaluate specific adverse health effects of PM2.5 , we collected two different PM2.5 samples from an underground parking lot and ambient air, and we evaluated cytotoxicity with eight different cell lines originating from human organs. Then, we selected JEG-3 human placenta cells, which show high cytotoxicity to both PM samples. Through RNA sequencing, gene expression profiling, and a gene ontology (GO) analysis of JEG-3 after exposure to two different PM2.5 samples, we identified 1021 commonly expressed genes involved in immune responses, the regulation of apoptosis, and so forth, which are known to induce several adverse health effects. In addition, we identified genes related to the calcium-signaling pathway, steroid hormone biosynthesis, and the cytokine-cytokine receptor interaction through a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Then, we confirmed these gene expressions using qRT-PCR, and the protein levels of mitogen-activated protein kinases and COX-2 with progesterone decreased using western blotting and enzyme-linked immunosorbent assay. In conclusion, this study suggests the possible toxic mechanism of human placenta that might be associated with PM2.5 -induced female reproductive disorders.

Detection of Matrix Metalloproteinase Activity by Bioluminescence via Intein-Mediated Biotinylation of Luciferase.

We report bioluminescence analysis of matrix metalloproteinase (MMP) activity in biological substances using a surface-bound luciferase probe. Intein-fused luciferase protein enables site-specific biotinylation of luciferase in the presence of N-terminus cysteine-biotin via intein-mediated splicing process, resulting in a strong association with high bioluminescence signal onto a NeutrAvidin-coated surface. When the peptide substrate for MMP-7 was inserted into a region between luciferase and intein, the biotinylated probe detected MMP-7 activity by cleaving the peptide, and surface-induced bioluminescence signal was strongly reduced in the MMP-secreted media or mouse tissue extracts, compared with that in MMP-deficient control set. Our approach is anticipated to be useful for generating biotinylated proteins and for their applications in diagnosing MMP activity in human diseases.

Bisphenol A induces COX-2 through the mitogen-activated protein kinase pathway and is associated with levels of inflammation-related markers in elderly populations.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical, and it is one of the highest volume chemicals produced worldwide. Even though several in vivo and in vitro studies showed positive associations of BPA exposure with pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-6, the mechanism by which BPA induces inflammation is unclear. We investigated the mechanism by which BPA induces inflammation (expression of inflammation-related genes, changes in oxidative stress, and cell proliferation and migration) and evaluated the effect of BPA exposure on inflammation-related markers in epidemiologic studies using repeat urine and serum samples from elderly subjects. BPA induced COX-2 expression via nuclear translocation of NF-κB and activation of mitogen-activated protein kinase (MAPK) by phosphorylation of ERK1/2 and enhanced the migration of lung cancer A549 and breast cancer MDAMB-231 cells. In two epidemiologic studies, we detected associations of BPA with six inflammation-related markers (WBC, CRP, IL-10, ALT, AST, and γ-GTP levels). Our findings probably suggest that BPA exposure induces inflammation and exacerbates tumorigenesis.

Coxsackievirus and Adenovirus Receptor, a Tight Junction Protein, in Peri-Implantation Mouse Embryos.

To understand the role of Coxsackievirus and adenovirus receptor (CAR), a tight junction (TJ) protein, in peri-implantation embryos, developmental expression of CAR and its role in paracellular permeability were examined in mouse embryos. Splice variants for transmembrane CAR, Car1, Car2, and Car3 mRNA, were expressed from 2-cell, morula, and blastocyst stages onward, respectively, whereas mRNA for soluble CAR was expressed in MII oocytes and 4-cell stage onward. On Western blot, ∼46 kDa CAR proteins were detected in blastocysts. During the 4-cell embryos to morula stage, CAR was gradually concentrated at the contacts between blastomeres. In blastocysts, CAR was expressed at the cell contacts within the inner cell mass as well as in the trophectoderm (TE) where CAR was found together with ZO1 at the apical contacts, suggesting that CAR builds up apical TJs in TE and mediates cell adhesion in TE and inner cell mass. In blastocysts, CAR-blocking antibodies under Ca(2+) switching increased the dextran permeability and decreased the volume of blastocoel and H19 and Cdx2 mRNA, suggesting the pivotal role of CAR in the blastocyst development and paracellular permeability barrier in TE. CAR was expressed in TE of implanting embryos as well as endometrial epithelium, suggesting the involvement of CAR in the interaction between implanting embryos and endometrium. At 5-6 days postcoitum, CAR was expressed together with ZO1 in the primitive endoderm, visceral endoderm, and epiblasts facing the pro-amniotic cavity, suggesting that CAR TJs contribute to the separation of epiblast from the blastocoel and development of the pro-amniotic cavity within epiblasts.

Changes in Inflammatory Cytokines Accompany Deregulation of Claudin-11, Resulting in Inter-Sertoli Tight Junctions in Varicocele Rat Testes.

PURPOSE: To elucidate the changes that occur in the blood-testis barrier during varicocele we examined changes in Cldn11 (claudin-11), an element of the blood-testis barrier, as well as steroidogenesis and proinflammatory cytokines in a model of varicocele rat testes. MATERIALS AND METHODS: Male rats with experimentally induced varicocele were sacrificed 4 weeks after operation. Testicular histology and blood testosterone concentrations were examined. The expression of tight junctions, steroidogenic enzymes, apoptosis and immune cell markers, and proinflammatory cytokines in the testes were evaluated by reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry. RESULTS: Weight and Johnsen scores of varicocele testes were lower than those of normal testes. mRNA expression of Bad and Bax increased whereas Bcl-xl and Bcl2 mRNA decreased in varicocele testes compared to controls. Although blood testosterone did not change, Leydig cell 3ßHsd immunoreactivity, testicular 3ßHsd6 and 17ßHsd3 mRNA were significantly decreased in varicocele testes. Cldn11 mRNA and protein levels in varicocele testes were higher than in normal testes together with altered expression of Ocln, Zo1 and N-cadherin mRNA. Cldn11 immunoreactivity appeared as wavy strands at the periphery of the seminiferous epithelium in normal testes but was frequently found in the Sertoli cell cytoplasm in varicocele testes. In varicocele testes Tnfα, Il1α, Il6, Cd45, Cd3g and Cd3d mRNA was increased. CONCLUSIONS: An increase in proinflammatory cytokines might be responsible for deregulation of Cldn11 in the Sertoli cells in varicocele testes, leading to alterations in the permeability of the blood-testis barrier and immunological barriers to normal spermatogenesis.

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

Claudin 11 inter-sertoli tight junctions in the testis of the korean soft-shelled turtle (Pelodiscus maackii).

Expression of claudin 11 (CLDN11), a tight junction (TJ) protein, was examined in the Korean soft-shelled turtle (Pelodiscus maackii) testis. Spermatogenesis began during the breeding season and peaked at the end of the breeding season. Spermiation started in summer and peaked in autumn. The deduced amino acid sequence of P. maackii CLDN11 was similar to those of avian and mammalian species. During the nonbreeding season when spermatogenesis and testosterone production were active, testicular Cldn11 levels were high. In the seminiferous epithelium, strong, wavy CLDN11 strands parallel to the basement membrane delaminate the spermatogonia, and early spermatocytes are in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm. Punctate zonula occludens 1 (ZO-1) immunoreactivity was found within the CLDN11 strands parallel to the basement membrane or at the outermost periphery of the seminiferous epithelium close to the basal lamina. During the breeding season, when circulating testosterone levels and spermatogenic activity was low, testicular CLDN11 level was lower than those during the nonbreeding season. CLDN11 was found at apicolateral contact sites between adjacent Sertoli cells devoid of the postmeiotic germ cells. At this time, lanthanum tracer diffused to the adluminal compartment of seminiferous epithelium. In cultured testis tissues, testosterone propionate significantly increased the level of Cldn11 mRNA. In P. maackii testis, CLDN11 participates in the development of the blood-testis barrier (BTB), where the CLDN11 expression was coupled with spermatogenic activity and circulating androgen levels, indicating the conserved nature of TJs expressing CLDN11 at the BTB in amniotes.

Role of p57KIP2 in Stem and Progenitor Leydig Cells of Mouse Testes.

PURPOSE: Precise control of proliferation and differentiation of Leydig cells is important for gonadal androgenesis and spermatogenesis. Though cyclin-dependent kinase inhibitors are crucial for cell proliferation and differentiation, their role in the development of early adult Leydig cells (ALCs) remained unanswered. To understand mechanism for ALC development, functional expression of p57KIP2 (cdkn1c) was investigated in the stem Leydig cells (SLCs) and progenitor Leydig cells (PLCs) in mice. MATERIALS AND METHODS: The roles of p57KIP2 in the proliferation, differentiation, apoptosis, and steroidogenesis in SLCs and PLCs were investigated by antibodies and bromodeoxyuridine (BrdU) labeling in the early neonatal testes and p57kip2 siRNA in the isolated SLCs and PLCs. Steroidogenic differentiation of PLCs was examined by progesterone and testosterone production in cell culture. RESULTS: From postnatal day (PND) 1 to 14, p57KIP2(+) spindle-shaped cells in the testis interstitium were α-smooth muscle actin (αSMA)(-), a peritubular myoid cells marker, suggesting that they are SLCs and PLCs. Besides, p57KIP2 was also expressed in HSD3ß(+) fetal Leydig cells. From PND1 to 14, BrdU(+)/αSMA(-), Ki67(+)/p57KIP2(+), and BrdU(+)/p57KIP2(+) spindle-shaped cells were gradually decreased. From PND1 to 14, p57KIP in the αSMA(-)/p57KIP2(+) cells was peaked at PND7 and decreased thereafter. In THY1(+) isolated SLCs, p57kip2 siRNA significantly increased ki67 and pcna mRNA and pdgfrα mRNA, a differentiation marker and decreased nestin mRNA, a SLC marker. No significant difference in apoptosis related genes mRNA was found after p57kip2 siRNA treatment. In HSD3ß(+) PLCs, p57kip2 siRNA increased proapoptotic genes mRNA, annexin V(+) early-apoptotic cells. Importantly, p57kip2 siRNA significantly decreased hsd3ß6 and cyp17a1 mRNA and progesterone production. CONCLUSIONS: p57KIP2 may suppress proliferation and support stemness of SLCs. In PLCs, p57KIP2 may suppress apoptosis and potentiate the steroidogenic differentiation.