In vivo and in vitro protective effects of the Wuzi Yanzong pill against experimental spermatogenesis disorder by promoting germ cell proliferation and suppressing apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuzi Yanzong pill (WZYZP) is a classical traditional Chinese medicine (TCM) formula originated from the Tang dynasty. WZYZP has a long history of use for reinforcing kidney and alleviating male infertility in China. AIM OF THE STUDY: The effect of WZYZP on male infertility and the mechanism underlying this effect was not clarified clearly. Therefore, this study aimed to investigate the protective effect of WZYZP in experimental spermatogenesis disorder via in vivo and in vitro studies, to promote the use of this formula for the treatment of spermatogenesis disorder. MATERIAL AND METHODS: Male SD rats were exposed to tripterygium glycosides to induce experimental spermatogenesis disorder, and WZYZP was subsequently administrated at different dosages for treatment. Sperm counts, sperm motility, and serum hormone levels were detected. HE staining and TUNEL staining were performed to evaluate the pathological lesions and apoptosis of testes, respectively. Next, germ cells were isolated from spermatogenesis disorder-model rats and treated with WZYZP- containing serum at different concentrations. CCK-8 assay and flow cytometry assay were performed to detect cell proliferation and apoptosis. Immunofluorescence assay, qRT-PCR and Western blotting analyses were performed to detect the expression of Beclin 1, LC3 and TGF-ß-PI3k/AKT-mTOR pathway - related factors, including TGF-ß, PI3K, AKT, mTOR, 4 EBP-1 and p70S6K. RESULTS: In vivo experiments showed that WZYZP protected against spermatogenesis disorder in model rats by improving sperm count and motility, as well as restoring serum hormone levels. HE and TUNEL staining demonstrated that the pathological injuries and cell apoptosis in testes of the model rats were alleviated by WZYZP treatment. Moreover, in vitro experiments of germ cells isolated from spermatogenesis disorder-model rats showed that WZYZP treatment increased the cell proliferation, inhibited cell apoptosis and autophagy. qRT-PCR and Western blotting assay results showed that this protective effect was associated with the regulation of the TGF-ß/PI3K/AKT/mTOR signaling pathway. The expression levels of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, 4 EBP-1 and p70S6K were increased, while TGF-ß was inhibited in the WZYZP treated groups. CONCLUSION: The results showed that WZYZP could protect against experimental spermatogenesis disorder by increasing the germ cell proliferation and inhibiting their apoptosis. Our support the clinical use of this formula for the management of spermatogenesis disorder.

Detection of Caenorhabditis elegans Germ Cell Apoptosis Following Exposure to Environmental Contaminant Mixtures: A Crude Oil-Dispersant Mixture Example.

Crude oil disasters, such as the Deepwater Horizon accident, have caused severe environmental contamination and damage, affecting the health of marine and terrestrial organisms. Some previous studies have demonstrated cleanup efforts using chemical dispersant induced more potent toxicities than oil alone due to an increase in bioavailability of crude oil components, such as PAHs. However, there still lacks a systematic procedure that provides methods to determine genotypic and phenotypic changes following exposure to environmental toxicants or toxicant mixture, such as dispersed crude oil. Here, we describe methods for identifying a mechanism of dispersed crude oil-induced reproductive toxicity in the model organisms, Caenorhabditis elegans (C. elegans). Due to the genetic malleability of C. elegans, two mutant strains outlined in this chapter were used to identify a pathway responsible for inducing apoptosis: MD701 bcIs39 [lim-7p::ced-1::GFP + lin-15(+)], a mutant strain that allows visualization of apoptotic bodies via a green fluorescent protein fused to CED-1; and TJ1 (cep-1(gk138) I.), a p53/CEP-1 defective strain that is unable to activate apoptosis via the p53/CEP-1 pathway. In addition, qRT-PCR was utilized to demonstrate the aberrant expression of apoptosis (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) and cytochrome P450 (cyp14a3, cyp35a1, cyp35a2, cyp35a5, and cyp35c1) protein-coding genes following exposure to dispersed crude oil. The procedure outlined here can be applicable to determine whether environmental contaminants, most of time contaminant mixture, cause reproductive toxicity by activation of the proapoptotic, p53/CEP-1 pathway.

In vitro reduction of Plasmodium falciparum gametocytes: Artemisia spp. tea infusions vs. artemisinin.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua has a long history of use in Southeast Asia where it was used to treat "fever", and A. afra has a similar history in southern Africa. Since their discovery, A. annua use, in particular, has expanded globally with millions of people using the plant in therapeutic tea infusions, mainly to treat malaria. AIM OF THE STUDY: In this study, we used in vitro studies to query if and how A. annua and A. afra tea infusions being used across the globe affect asexual Plasmodium falciparum parasites, and their sexual gametocytes. MATERIALS AND METHODS: P. falciparumstrain NF54 was grown in vitro, synchronized, and induced to form gametocytes using N-acetylglucosamine. Cultures during asexual, early, and late stage gametocytogenesis were treated with artemisinin, methylene blue, and A. annua and A. afra tea infusions (5 g DW/L) using cultivars that contained 0-283 µM artemisinin. Asexual parasitemia and gametocytemia were analyzed microscopically. Gametocyte morphology also was scored. Markers of early (PfGEXP5) and late stage (Pfs25) gametocyte gene expression also were measured using RT-qPCR. RESULTS: Both A. annua and A. afra tea infusions reduced gametocytemia in vitro, and the effect was mainly artemisinin dependent. Expression levels of both marker genes were reduced and also occurred with the effect mainly attributed to artemisinin content of four tested Artemisia cultivars. Tea infusions of both species also inhibited asexual parasitemia and although mainly artemisinin dependent, there was a weak antiparasitic effect from artemisinin-deficient A. afra. CONCLUSIONS: These results showed that A. annua and to a lesser extent, A. afra, inhibited parasitemia and gametocytemia in vitro.

Deleterious effects of Pfaffia glomerata (Spreng.) Pedersen hydroalcoholic extract on the seminiferous epithelium of adult Balb/c mice.

Several plant species such as Pfaffia glomerata are widely used in traditional Brazilian medicine as stimulants and aphrodisiacs. In this regard, the aim of our study was to explore the effects of the long-term intake of the hydro-alcoholic root extract of P glomerata on the germ and somatic cells within the seminiferous tubules in adult Balb/c mice. The experimental groups were placed as: controls (water and DMSO), and treated with 300 and 400 mg/kg of the root extract. The number of germ and somatic cells, the proportion of pathological seminiferous tubules, and the germ cell apoptotic levels were evaluated. The volume and proportion of the seminiferous epithelium was decreased after the extract intake due to the increased germ cell apoptotic levels. Vacuolization of Sertoli cell cytoplasm was observed widely in pathological tubules, along with fully disorganized epithelia, showing multinucleated cells, which lead to decreased daily sperm production. Taken together, our results indicate that long-term intake of the P glomerata caused deleterious effects on spermatogenesis by inducing apoptosis and altering the seminiferous tubule's epithelial dynamics.

Extract and the quassinoid ailanthone from Ailanthus altissima inhibit nematode reproduction by damaging germ cells and rachis in the model organism Caenorhabditis elegans.

Bark and leaves of Ailanthus altissima (Mill.) Swingle are widely used in European folk medicine to treat intestinal worm infections. The study aimed to rationalize a potential anthelmintic effect of A. altissima extract against the model organism Caenorhabditis elegans. A methanol-water (7:3, v/v) extract of the primary stem bark was tested on L4 larvae of C. elegans for induction of mortality and influence on reproduction. Bioactivity-guided fractionation was performed by chromatography on MCI-gel, preparative HPLC on RP18 stationary phase and fast-centrifugal-partition-chromatography. Structural elucidation of isolated quassinoids was performed by NMR and HR-ESI-MS. The sterilizing effect on C. elegans was investigated by light microscopy and atomic force microscopy of ultra-sections. Different GFP-tagged reporter strains were used to identify involved signaling pathways. A. altissima extract (1 mg/mL) irreversibly inhibited the reproduction of C. elegans L4 larvae. This effect was dependent on the larval stage since L3 larvae and adults were less affected. Bioactivity-guided fractionation revealed the quassinoid ailanthone 1 as the major active compound (IC50 2.47 µM). The extract caused severe damages to germ cells and rachis, which led to none or only poorly developed oocytes. These damages led to activation of the transcription factor DAF-16, which plays a major role in the nematode's response to stress. A regulation via the respective DAF-2/insulin-like signaling pathway was not observed. The current findings support the traditional use of A. altissma in phytotherapy to treat helminth infections and provide a base for standardization of the herbal material.

Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells.

BACKGROUND: As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown. METHODS: We generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR. RESULTS: We optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 µg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes. CONCLUSIONS: Taken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process.

Assessing effects of germline exposure to environmental toxicants by high-throughput screening in C. elegans.

Chemicals that are highly prevalent in our environment, such as phthalates and pesticides, have been linked to problems associated with reproductive health. However, rapid assessment of their impact on reproductive health and understanding how they cause such deleterious effects, remain challenging due to their fast-growing numbers and the limitations of various current toxicity assessment model systems. Here, we performed a high-throughput screen in C. elegans to identify chemicals inducing aneuploidy as a result of impaired germline function. We screened 46 chemicals that are widely present in our environment, but for which effects in the germline remain poorly understood. These included pesticides, phthalates, and chemicals used in hydraulic fracturing and crude oil processing. Of the 46 chemicals tested, 41% exhibited levels of aneuploidy higher than those detected for bisphenol A (BPA), an endocrine disruptor shown to affect meiosis, at concentrations correlating well with mammalian reproductive endpoints. We further examined three candidates eliciting aneuploidy: dibutyl phthalate (DBP), a likely endocrine disruptor and frequently used plasticizer, and the pesticides 2-(thiocyanomethylthio) benzothiazole (TCMTB) and permethrin. Exposure to these chemicals resulted in increased embryonic lethality, elevated DNA double-strand break (DSB) formation, activation of p53/CEP-1-dependent germ cell apoptosis, chromosomal abnormalities in oocytes at diakinesis, impaired chromosome segregation during early embryogenesis, and germline-specific alterations in gene expression. This study indicates that this high-throughput screening system is highly reliable for the identification of environmental chemicals inducing aneuploidy, and provides new insights into the impact of exposure to three widely used chemicals on meiosis and germline function.

Coffee mitigates cyclophosphamide-induced genotoxic damage in Drosophila melanogaster germ cells.

In the present study, coffee (CF) was evaluated for its protective effects against genotoxic damage and oxidative stress induced by the chemotherapeutic drug, cyclophosphamide (CPH). The sex-linked recessive lethal (SLRL) test was employed to study the induction of mutations in the larvae as well as in all the successive germ cell stages of treated males. Control and treated third instar larvae were used to monitor the biomarkers of oxidative stress response such as glutathione content (GSH), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (MDA content). Our results demonstrated that co-administration of CF (2%) with CPH (3 mM) has significantly reduced CPH-induced lethal mutations in the germ cells of larvae and adult flies. The reductions observed in mutation frequencies were: 75% in larvae and 62.4% in the adult. Significant enhancement in antioxidant enzymatic levels: CAT (46.6%) > SOD (43.0%) > GST (42.4%) > GSH (31.6%) and reduction in MDA levels (32.05%) in the pretreated third instar larvae demonstrated the antioxidant activity of CF against CPH-induced oxidative stress. The findings from the present study suggest that the Drosophila model is an ideal one for evaluating the antigenotoxic and antioxidant activity of complex mixtures like CF.

Effects of Moringa oleifera Lam Extract (MOLE) in the heat shock protein 70 expression and germ cell apoptosis on experimentally induced cryptorchid testes of rats.

Moringa oleifera (Moringaceae) is a plant known for having high antioxidant potency, anticancer, hepatoprotective, cardioprotective etc. and many more activities. Besides these, Moringaceae has the potential for attenuating the male sexual dysfunction. Reactive oxygen species/ROS were increased in cryptorchidism and therefore cause infertility by damaging sperm DNA and germ cell apoptosis. There was an increase in heat shock proteins (HSP) in cells, which is affected by heat shock. In the present study, the antioxidant effects of two different doses of M. oleifera Lam Extract (MOLE) on experimentally induced cryptorchid testes of rats was investigated. Forty two male rats (16 days old) were divided into four groups: a normal control group, a cryptorchidism-induced control group and two cryptorchidism-induced groups treated orally with either 400 or 800 mg/kg MOLE for 2 weeks. Our study showed that there were ruptures from interstitial spaces, separation of the germ cells from basal membrane, falling of the germ cells into the lumen, perivascular fibrosis, oedema, increased level of HSP70, apoptosis, malondialdehyde (MDA) and decrease in the level of superoxide dismutase (SOD) after the cryptorchidism. We found that pathological damages, oxidative stress, expression of the HSP70 and germ cell apoptosis were decreased in treated groups with MOLE. In brief, we can say that aqueous extract of M. oleifera reduces the oxidative stress in a unilateral cryptorchidism induced rats, and it might attenuate histopathological damages, HSP expression and germ cell apoptosis.

[Protective effect of Wuzi Yanzong recipe on testicular germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress].

To study the protective effects of Wuzi Yanzong recipe on testis germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress (ERS), 16-month-old male SPF grade SD rats were randomly divided into three groups: ageing model group, and low and high-dose Wuzi Yanzong recipe groups (WZ, 1 and 4 g·kg⁻¹), with 10 rats in each group. In addition, 2-month-old SD male rats were used as adult control group. The ageing model group and the adult control group were fed with normal diet for 4 months. WZ groups were given the medicated feed for 4 months. After fasting for 12 hours, the rats were put to death. Then, the testes were immediately collected. The change of testicular tissue morphology was observed by HE staining. The expression levels of ER stress-related proteins GRP78, p-PERK, p-eif2α, ATF4, p-IRE1, XBP1, ATF6 and apoptosis-related proteins CHOP, caspase12 and p-JNK in testes were detected by Western blot. Compared with the ageing model group, Wuzi Yanzong recipe alleviated the morphological changes of testicular tissue. Western blot results showed that Wuzi Yanzong recipe significantly increased the expression levels of endoplasmic reticulum stress-related proteins GRP78, p-PERK, p-eif2α, ATF4, p-IRE1, XBP1, ATF6 and significantly decreased the expression levels of endoplasmic reticulum-induced apoptosis-related proteins CHOP, caspase 12 and p-JNK. In conclusion, Wuzi Yanzong recipe can alleviate the ageing-related apoptosis of testicular germ cells in natural ageing rats by regulating endoplasmic reticulum stress.

Melatonin attenuates radiofrequency radiation (900 MHz)-induced oxidative stress, DNA damage and cell cycle arrest in germ cells of male Swiss albino mice.

Increasing male infertility of unknown aetiology can be associated with environmental factors. Extensive use of mobile phones has exposed the general population to unprecedented levels of radiofrequency radiations (RFRs) that may adversely affect male reproductive health. Therefore, the present study investigated the effect of RFR Global System for Mobile communication (GSM) type, 900 MHz and melatonin supplementation on germ cell development during spermatogenesis. Swiss albino mice were divided into four groups. One group received RFR exposure for 3 h twice/day for 35 days and the other group received the same exposure but with melatonin ( N-acetyl-5-methoxytryptamine) (MEL; 5 mg/kg bw/day). Two other groups received only MEL or remain unexposed. Sperm head abnormality, total sperm count, biochemical assay for lipid peroxides, reduced glutathione, superoxide dismutase activity and testis histology were evaluated. Additionally, flow cytometric evaluation of germ cell subtypes and comet assay were performed in testis. Extensive DNA damage in germ cells of RFR-exposed animals along with arrest in pre-meiotic stages of spermatogenesis eventually leading to low sperm count and sperm head abnormalities were observed. Furthermore, biochemical assays revealed excess free radical generation resulting in histological and morphological changes in testis and germ cells morphology, respectively. However, these effects were either diminished or absent in RFR-exposed animals supplemented with melatonin. Hence, it can be concluded that melatonin inhibits pre-meiotic spermatogenesis arrest in male germ cells through its anti-oxidative potential and ability to improve DNA reparative pathways, leading to normal sperm count and sperm morphology in RFR-exposed animals.

Protective effects of Tribulus terrestris hydroalcoholic extract against cisplatin-induced germ cell apoptosis in male mice / Efectos protectores del extracto hidroalcohólico Tribulus terrestris contra la apoptosis de células germinales inducida por cisplatino en ratones machos

SUMMARY: Toxic effects of anti-cancer and other drugs on the normal tissues could be reduced by the herbal plants and their fractions. This study investigated the protective effect of Tribulus terrestris (TT) on Cisplatin- induced cytotoxicity germ cell apoptosis in male mice. In this experimental study, thirty male Balb/c mice were divided randomly into 5 groups (n=6). A single dose of Cisplatin (5.5 mg/kg) and differ-ent concentrations of Tribulus terrestris were administrated for 14 consecutive days. Reverse transcription polymerase chain reaction (RT-PCR) of apoptosis-re-lated genes was performed with RNA extracted from testes of the mice. Statistical analysis was done using one-way ANOVA. In the Cisplatin group, there was a significant increase in mRNA expression of p53 (P=0.008), bax (P=0.004) and the ratio of bax/Bcl-2 (P=0.000), whereas there was an decrease in the expression of Bcl-2 (P=0.003), as compared to control group. In Cis+TT groups, the data showed that different concentrations of TT could improve the harmful effects caused by the Cisplatin. The best protective effects were achieved in Cis+TT (300 mg/kg). Tribulus terrestris protects testicular germ cell against Cisplatin induced apoptosis by affecting related genes regulation.

RESUMEN: Los efectos tóxicos en los tejidos normales, de los medicamentos contra el cáncer al igual que otras medicamentos podrían mejorar con el uso de plantas medicinales y hierbas. Este estudio investigó el efecto protector de Tribulus terrestris (TT) sobre la apoptosis de células germinales por citotoxicidad inducida por cisplatino en ratones machos. En este estudio se dividieron treinta ratones Balb/c macho aleatoriamente en 5 grupos (n = 6). Se administró una sola dosis de cisplatino (5,5 mg / kg) y diferentes concentraciones de Tribulus terrestris durante 14 días consecutivos. La reacción en cadena de la polimerasa de transcripción reversa de los genes relacionados con la apoptosis, se realizó con ARN extraído de los testículos de los ratones. El análisis estadístico se realizó usando ANOVA de una vía. En el grupo cisplatino, hubo un aumento significativo en la expresión de mRNA de p53 (P = 0,008), bax (P = 0,004) y la relación de bax / Bcl-2 (P = 0.000), mientras que hubo una disminución en la expresión de Bcl-2 (P = 0,003), en comparación con el grupo control. En los grupos Cis + TT, los datos mostraron que las diferentes concentraciones de TT podrían mejorar los efectos nocivos causados por el cisplatino. Los mejores efectos protectores se lograron en Cis + TT (300 mg / kg). Tribulus terrestris protege las células germinales testiculares contra la apoptosis inducida por cisplatino al afectar la regulación de los genes relacionados.

Male reprotoxicity associated with Sophora japonica treatment: evaluation of cellular and molecular events in vitro.

Toxicological studies are urgently needed to confirm the safety of Sophora japonica extracts for clinical use. In particular, in addition to pharmacy and pharmacology, phytotherapy, herbal medication, Sophora japonica extracts are widely used as biologically active supplements. Scientfic data suggests that some of Sophora japonica extract components have very comprehensive biological effects. In the present study, our hypothesis assumed the potential reprotoxicity of Sophora japonica extract in respect to mouse germ cells (GC-1 spg, GC-2 spd) in vitro. Specifically, we were interested in the high-performance liquid chromatography (HPLC) identification of extract components and its stress-related effects on cellular and biochemical features, such as mitochondrial metabolism, cell cycle progression, oxidative stress balance and micronuclei formation. The results indicate that Sophora japonica extract induces oxidative/nitrosative stress-mediated impairment of the mechanism for free radicals scavenging, which may provoke genotoxic events in germ cells, by cell cycle arrest and micronuclei formation. Therefore, the interplay between reactive oxygen species (ROS)/reactive nitrogen species (RNS) and antioxidant system is critical for normal testicular function maintenance in the their environment. The specific pathways and mechanisms involved in the reprotoxicity of Sophora japonica need to be further investigated.

Differentiation of mesenchymal stem cells to germ-like cells under induction of Sertoli cell-conditioned medium and retinoic acid.

The aim of this research was to find a way to differentiate germ cells from umbilical cord Wharton's jelly mesenchymal stem cells (MSCs) to support in vitro spermatogenesis. A small piece of Wharton's jelly was cultured in high-glucose Dulbecco's modified Eagle's medium in present of 10% foetal calf serum. After the fourth passage, the cells were isolated and cultured in Sertoli cell-conditioned medium under induction of two different doses of retinoic acid (10-5 , 10-6  m). The differentiation of MSC to germ-like cells was evaluated by expression of Oct4, Nanog, Plzf, Stra8 and Prm1 genes during different days of culture through qPCR. The results showed that there were downregulation of Oct4 and Nanog and upregulation of pre-meiotic germ cell marker (stra8) and haploid cell marker (Prm1) when MSCs are differentiated over time. The expression of Bax gene (an apoptotic marker) was significantly observed in high dosage of retinoic acid (RA). As a result, RA has positive effects on proliferation and differentiation of MSCs, but its effects are related to dosage. The success of this method can introduce umbilical cord MSC as a source of germ cells for treatment of infertility in future.

A Phytochemical Approach to Promotion of Self-renewal in Murine Spermatogonial Stem Cell by Using Sedum Sarmentosum Extract.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, which is dependent on the ability to self-renew and differentiation. Controlling self-renewal and differentiation of SSCs could apply to treatment of disease such as male infertility. Recently, in the field of stem cell research, it was demonstrated that effective increase in stem cell activity can be achieved by using growth factors derived from plant extracts. In this study, our aim is to investigate components from natural plant to improve the self-renewal of SSCs. To find the components, germ cells were cultured with comprehensive natural plant extracts, and then the more pure fraction, and finally single compound at different concentrations. As a result, we found 5H-purin-6-amine at 1 µg/mL, originated from Sedum sarmentosum, was a very effective compound induced SSCs proliferation. Our data showed that germ cells cultured with 5H-purin-6-amine could maintain their stable characteristics. Furthermore, transplantation results demonstrated that 5H-purin-6-amine at 1 µg/mL increased the activity of SSCs, indicating the compound could increase true SSC concentration within germ cells to 1.96-fold. These findings would be contributed to improve further reproductive research and treat male infertility by using natural plant extracts.

Methotrexate-induced germ cell toxicity and the important role of zinc and SOD1: Investigation of molecular mechanisms.

Zinc (Zn) was proved to be a germ cell protectant against various disease conditions and toxic insults. Besides other mechanisms, here we have explored the important role of Zn and Zn-dependent SOD1in methotrexate (MTX)-induced germ cell damage. MTX was given 5 mg/kg i.p. once a week for four consecutive weeks, while Zn was supplemented daily at the doses of 3 and 6 mg/kg i.p. for four consecutive weeks. After four weeks of treatment the animals were sacrificed and observed for various end points. There were several histopahtological alterations in the testes like desquamation and altered tubular structures. DNA damage was also increased by MTX as evident by TUNEL assay. Sperm head abnormalities were increased in case of MTX treated animals. Protein expressions of PCNA, BCl-2/Bax, SOD, catalase and GPX5 were found to be altered by the MTX treatment. To further investigate the role of Zn and Zn-dependent SOD1, rats were injected intratesticularly with diethyldithiocarbamate (DEDTC) for three days after MTX 20 mg/kg i.p. was given on the first day. DEDTC in combination with MTX was found to significantly decrease the protein expressions of SOD1, catalase, Nrf2 and GPX4, along with deranged histology. This study adds to the point that Zn might be a better germ cell protectant and deserve further investigation.

α-Lipoic acid attenuates transplacental nicotine-induced germ cell and oxidative DNA damage in adult mice.

BACKGROUND: Smoking during pregnancy is associated with numerous fetal and developmental complications and reproductive dysfunctions in the offspring. Nicotine is one of the key chemicals of tobacco responsible for addiction. The present study was aimed to investigate the protective role of α-lipoic acid (ALA) during the transplacental nicotine-induced germ cell and DNA damage in the offspring of Swiss mice. METHODS: Pregnant mice were treated with nicotine (20 mg/kg/day) in drinking water from 10 to 20 days of gestation period, and ALA (120 mg/kg/day) was administered orally for the same period. Endpoint of evaluation includes general observations at delivery and throughout the study, litter weight and size, sperm count and sperm head morphology, while structural damages and protein expression were assessed by histology and immunohistochemistry, respectively. RESULTS: Maternal nicotine exposure led to decreased growth rate, litter and testicular weight, testosterone level, 3ß-HSD expression and sperm count as well as increased sperm head abnormalities, micronucleus frequency and 8-oxo-dG positive cells, and the effects have been restored by ALA supplementation. CONCLUSIONS: The present study clearly demonstrated that ALA ameliorates nicotine-associated oxidative stress, DNA damage and testicular toxicity in the offspring by improving steroidogenesis, spermatogenesis and sperm count.

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression.

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that ß-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit the expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of ß-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases.

Improvement of gamete quality by stimulating and feeding the endogenous antioxidant system: mechanisms, clinical results, insights on gene-environment interactions and the role of diet.

Oxidative damage triggers extensive repair in gametes and thereafter in the zygote but it results in clinically relevant damage when affecting the maturation of the gametes chromatin, i.e. padlocking and epigenetic marking. It associates with defective DNA methylation and/or with oxidation of the methyl marks leading to derangement of gamete epigenetics, defects of chromatin condensation and aneuploidy. A proper feed to the one carbon cycle has the potential to stimulate the endogenous antioxidant defences, i.e. gluthatione synthesis, and to activate compensative homeostatic mechanisms restoring both the oxy-redox balance and DNA methylation, which are indeed strictly cross-regulated. This has been shown to produce measurable clinical improvements of male reproductive potential in pilot studies herein summarised. However, the effects of dietary habits and of supplementations are variable according to the individual genetic substrate, as genetic variants of several of the concerned enzymes occur with high frequency. Individual risk assessments and personalised interventions are still difficult to implement, in the meantime, a very varied diet may facilitate metabolic compensation in the majority of the cases. This review aims to report on the mechanisms of damage, on the opportunities to modulate the physiologic oxy-redox homeostasis by means of a varied diet or dietary supplements and on the open issues related to the genetic variability of the population.

Toxicological assessment of multi-walled carbon nanotubes in vitro: potential mitochondria effects on male reproductive cells.

Multi-walled carbon nanotubes (MWCNTs) have been widely used in many fields and were reported to cause reversible testis damage in mice at high-dose. However the reproductive effects of low dose MWCNTs remained elusive. Herein, we used the mice spermatocyte cell line (GC-2spd) to assess the reproductive effects of MWCNTs. Size distribution, zeta potential, and intensity of MWCNTs were characterized. A maximal concentration of 0.5 µg/mL MWCNTs was found to be nonlethal to GC-2spd. At this dose, cell cycles and the ROS levels were in normal status. We also found MWCNTs accumulated in mitochondria, which caused potential mitochondrial DNA damage in spermatocyte. Furthermore, the expression level of mitochondria-related genes, the oxygen consumption rate, and cellular ATP content were declined compared to controls, even at the nonlethal dose. Our results suggested for the first time that, in germ cells, mitochondrion was a cellular organelle that accumulated MWCNTs.