Dysregulation of lncRNA and circRNA Expression in Mouse Testes after Exposure to Triptolide.

BACKGROUND: Triptolide has been shown to exert various pharmacological effects on systemic autoimmune diseases and cancers. However, its severe toxicity, especially reproductive toxicity, prevents its widespread clinical use for people with fertility needs. Noncoding RNAs including lncRNAs and circRNAs are novel regulatory molecules that mediate a wide variety of physiological activities; they are crucial for spermatogenesis and their dysregulation might cause male infertility. However, whether they are involved in triptolide-induced reproductive toxicity is completely unknown. METHODS: After exposure of mice to triptolide, the total RNAs were used to investigate lncRNA/circRNA/mRNA expression profiles by strand-specific RNA sequencing at the transcriptome level to help uncover RNA-related mechanisms in triptolide-induced toxicity. RESULTS: Triptolide significantly decreased testicular weight, damaged testis and sperm morphology, and reduced sperm motility and density. Remarkable deformities in sperm head and tail were also found in triptolide-exposed mice. At the transcriptome level, the triptolide-treated mice exhibited aberrant expression profiles of lncRNAs/circRNAs/mRNAs. Gene Ontology and pathway analyses revealed that the functions of the differentially expressed lncRNA targets, circRNA cognate genes, and mRNAs were closely linked to many processes involved in spermatogenesis. In addition, some lncRNAs/circRNAs were greatly upregulated or inducibly expressed, implying their potential value as candidate markers for triptolide-induced male reproductive toxicity. CONCLUSION: This study provides a preliminary database of triptolide-induced transcriptome, promotes understanding of the reproductive toxicity of triptolide, and highlights the need for research on increasing the medical efficacy of triptolide and decreasing its toxicity.

[IMMUNOCYTOCHEMICAL ANALYSIS OF THE DISTURBANCES IN THE STRUCTURE OF SYNAPTONEMAL COMPLEXES IN SPERMATOCYTE NUCLEI IN MICE UNDER EXPOSURE TO ROCKET FUEL COMPONENT].

There was performed an assessment of genotoxic effects of rocket fuel component--unsymmetrical dimethylhydrazine (UDMH, heptyl)--on forming germ cells of male mice. Immunocytochemically there was studied the structure of meiotic nuclei at different times after the intraperitoneal administration of UDMH to male mice. There were revealed following types of disturbances of the structure of synaptonemal complexes (SCs) of meiotic chromosomes: single and multiple fragments of SCs associations of autosomes with a sex bivalent, atypical structure of the SCs with a frequency higher than the reference level. In addition, there were found the premature desinapsis of sex bivalents, the disorder offormation of the genital corpuscle and ring SCs. Established disorders in SCs of spermatocytes, analyzed at 38th day after the 10-days intoxication of animal by the component of rocket fuel, attest to the risk of permanent persistence of chromosomal abnormalities occurring in the pool of stem cells.

Histomorphological and biochemical changes induced by triptolide treatment in male lesser bandicoot rat, Bandicota bengalensis.

Mature and healthy male lesser bandicoot rats, Bandicota bengalensis (n = 40) were fed on bait (mixture of cracked wheat and powdered sugar in 98:2) containing different concentrations of triptolide (0, 0.15, 0.20 and 0.25% w/w) for 15 days in two-choice trials. Results revealed no significant effect of triptolide treatment on weights of vital organs after 30 and 60 days of treatment withdrawal. A significant (P ≤ 0.05) increase in plasma levels of TP, ALP, ACP, ALT and AST in response to stress induced in groups of rats treated with 0.20 and 0.25% triptolide was observed after 30 days of treatment withdrawal. No significant effect of treatment was observed on histomorphology of liver. A significant (P ≤ 0.05) effect of triptolide treatment was, however, observed on testicular function in the form of reduced diameter of seminiferous tubules and number of various spermatogenic cells indicating effect on spermatogenesis and spermiogenesis. The cell stages affected did not recover fully within 60 days period following treatment withdrawal. The present study suggests the potential of triptolide in the reproductive management of B. bengalensis by way of affecting testicular function.

Reproductive toxicity of triptolide in male house rat, Rattus rattus.

The aim of study was to investigate the toxic effect of triptolide fed in bait on reproduction of male house rat, Rattus rattus. Feeding of cereal based bait containing 0.2% triptolide to male R. rattus for 5 days in no-choice feeding test, leading to mean daily ingestion of 20.45 mg/kg bw of triptolide, was found effective in significantly (P ≤ 0.05) reducing sperm motility and viability in cauda epididymal fluid by 80.65 and 75.14%, respectively, from that of untreated rats. Pregnancy rates were decreased by 100% in untreated cyclic female rats paired with male rats treated with 0.2% triptolide. Present studies suggest the potential of 0.2% triptolide bait in regulating reproductive output of R. rattus.

INSLF3-LGR8 ligand-receptor system in testes of mature rats after exposure to ethane dimethanesulphonate (short communication).

The cytotoxic agent ethane-1,2-dimethanesulphonate (EDS) specifically destroys the Leydig cells (LC) in the adult testis, followed by a complete regeneration. The process of LC renewal after exposure to EDS shows homology to the development of the adult-type LC population in prepubertal testis. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a peptide hormone, a novel member of the insulin/relaxin family, and seems to be localized predominantly in the gonadal tissues. INSL3 mRNA is expressed in the LC in a constitutive fashion and INSL3 thus seems to be a useful marker of LC differentiation status. The present study was aimed at establishing the chronology and dynamic of expression of INSL3 and its specific receptor LGR8 in the LC repopulation after exposure to mature rats to EDS. As material, testes of mature Wistar rats that received single intraperitoneal injection of EDS (75 mg/kg body weight) were used. The animals were killed 1, 7, 14, 21 and 35 days after the initial treatment. The pattern of INSL3-LGR8 expression in newly formed LC after EDS administration was established using a high sensitive immunohistochemical polymer detection kit. After treatment with EDS, the immunoreactivity for INSL3 and LGR8 disappeared from the testis and reappeared again at the time of regeneration of the first LC, 14 days after EDS. The INSL3-LGR8 positive cells grew in number concomitantly with the increase of the LC repopulation. Thirty-five days after EDS destruction a larger number of immunopositive LC were seen in form of clusters corresponding with the regeneration of adult type LC population. The present findings support the hypothesis that EDS-treated rats can serve as a model for studying the LC development in the prepubertal testis and indicate a specific role of hormonal factors like INSL3 in this process.

Lonidamine affects testicular steroid hormones in immature mice.

The effects on the hypothalamus-pituitary-testicular axis of the well-known antispermatogenic drug lonidamine (LND) has not been elucidated so far. In the present study, the possible changes of the testicular steroid hormones were evaluated in immature mice for a better characterization of the LND adverse effects both in its use as antitumoral agent and male contraceptive. Male CD1 mice were orally treated on postnatal day 28 (PND28) with LND single doses (0 or 100 mg/kg b.w.) and euthanized every 24 h from PND29 to PND32, on PND35 and on PND42 (1 and 2 weeks after the administration, respectively). Severe testicular effects were evidenced in the LND treated groups, including: a) significant testis weight increase, 24 h and 48 h after dosing; b) sperm head counts decrease (more than 50% of the control) on PND29-32; c) damage of the tubule morphology primarily on the Sertoli cell structure and germ cell exfoliation. All these reproductive endpoints were recovered on PND42. At the same time, a significant impairment of the testicular steroid balance was observed in the treated mice, as evidenced by the decrease of testosterone (T) and androstenedione (ADIONE) and the increase of 17OH-progesterone (17OH-P4) on the first days after dosing, while the testicular content of 17beta-estradiol (E2) was unchanged. The hormonal balance was not completely restored afterwards, as levels of T, ADIONE and 17OH-P4 tended to be higher in the treated mice than in the controls, on PND35 and PND42. These data showed for the first time that LND affects intratesticular steroids in experimental animals. However further data are needed both to elucidate the mechanism responsible for the impairment of these metabolic pathways and to understand if the androgens decrease observed after LND administration could be partially involved in the testicular damage.

2,5-hexanedione and carbendazim coexposure synergistically disrupts rat spermatogenesis despite opposing molecular effects on microtubules.

2,5-Hexanedione (2,5-HD), a taxol-like promoter of microtubule assembly, and carbendazim (CBZ), a colchicine-like inhibitor of microtubule assembly, are two environmental testicular toxicants that target and disrupt microtubule function in Sertoli cells. At the molecular level, these two toxicants have opposite effects on microtubule assembly, yet they share the common physiologic effect of inhibiting microtubule-dependent functions of Sertoli cells. By studying a combined exposure to 2,5-HD and CBZ, we sought to determine whether CBZ would antagonize or exacerbate the effects of an initial 2,5-HD exposure. In vitro, 2,5-HD-treated tubulin had a decreased lag time and an increased maximal velocity of microtubule assembly. These 2,5-HD-induced in vitro alterations in microtubule assembly were normalized by CBZ exposure. In vivo, adult male rats were exposed to a 1% solution of 2,5-HD in the drinking water for 2.5 weeks. CBZ was administered by gavage (200 mg/kg body weight) at the same time as unilateral surgical ligation of the efferent ducts, 24 h before evaluation of the testis. Measures of testicular effect (testis weight, histopathologic changes [sloughing and vacuolization], and seminiferous tubule diameters) were all significantly altered with combined exposure. The testicular effects in the combined exposure group were either different (seminiferous tubule diameters), additive (% vacuolization), or greater than additive (% sloughing) compared to the effects of the individual toxicant exposure groups referenced to the controls. Therefore, CBZ coexposure does not antagonize the effects of an initial 2,5-HD exposure, as might be expected if their molecular effects on microtubule assembly were solely responsible for their combined toxicity; instead, 2,5-HD and CBZ act together to exacerbate the testicular injury.

Gestational exposure to ethane dimethanesulfonate permanently alters reproductive competence in the CD-1 mouse.

Although the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant CD-1 mice were treated with 160 mg/kg on Gestation Days 11-17, and reproductive development in male offspring was evaluated. Prenatal administration of EDS compromised fetal testosterone (T) levels, compared with controls. EDS-exposed pups recovered their steroidogenic capacities after birth because T production by hCG-stimulated testis parenchyma from prepubertal male offspring was unchanged. However, prepubertal testes from prenatally exposed males contained seminiferous tubules (STs) devoid of germ cells, indicating a delay in spermatogenesis. In adults, some STs in exposed males still contained incomplete germ cell associations corroborating observed reductions in epididymal sperm reserves, fertility ratios, and litter size. Morphometry revealed an EDS-induced increase in interstitial area and a concomitant decrease in ST area, but stereology revealed an unexpected decrease in the number and size of the LCs per testis in exposed males. Paradoxically, there was an increase in both serum LH and T production by adult testis parenchyma, indicating that the LCs were hyperstimulated. These data demonstrate permanent lesions in LC development and spermatogenesis caused by prenatal exposure in mice. Thus, although adult mouse LCs are insensitive to EDS, EDS appears to have direct action on fetal LCs, resulting in abnormal testis development.

Early testicular response to intraperitoneal administration of ethane dimethanesulphonate (EDS) in the goat (Capra hircus).

Early morphological changes in the goat testis after a single intraperitoneal injection of ethane dimethanesulphonate (EDS) were investigated using both light and electron microscopy. The compound was administered at two dose levels: 75 mg/kg and 25 mg/kg. While the former resulted in some deaths due to toxicity, the latter had no noticeable toxic effects on the animals. The testicular effects at both dose levels were similar. Six (6) days post-treatment, Leydig cells were refractory to EDS challenge but there was a marked disruption of spermatogenesis. These Leydig cells exhibited ovoid or irregularly round nuclei, abundant cytoplasm containing spherical, ovoid or elongate mitochondria and a preponderance of smooth endoplasmic reticulum typical of the normal cells. Lipid droplets were rare. In the seminiferous tubules germ and Sertoli cell degeneration was observed. Changes in the germ cells included: spermatogonial degeneration, condensed chromatin in leptotene spermatocytes and failure of chromatin re-organization resulting in the formation of clumps in the cells at the telophase stage of cell division (stage 4 of the seminiferous cycle). The nuclear envelope of primary spermatocytes showed marked irregularity and there was an overall reduction in cell size. There was peripheral re-distribution of chromatin in developing spermatids of stages 1, 2 and 5, often resulting in thick margination along the nucleolemma and leaving a pale nucleoplasm. An accompanying retention of maturation phase spermatids in stage 2 tubules was also observed. Sertoli cells exhibited extensive accumulation of intracytoplasmic vesicles, obscuring the rest of the organelles. Intercellular vacuoles also occurred within the epithelium. The results suggest that while EDS does not have any effect on goat Leydig cells, it causes severe disruption of the spermatogenic process. Furthermore, it is concluded from the results that the optimum dose in this species is 25 mg/kg.

Effects of lonidamine on testicular and epididymal proteins in the rat.

The mechanism responsible for the antispermatogenic activity of lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a drug with low systemic toxicity and lack of significant hormonal effects, is still unclear but may be related to alterations of Sertoli cell proteins. Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis. When the testes and the epididymides of animals treated with or without LND were homogenized and analyzed by the Bradford assay, a significant increase of total protein content was observed after 24 and 48 h. When these homogenates were analyzed by concanavalin blotting, specific changes of the major rat macroglobulins, i.e. alpha(1)-inhibitor-3, alpha(2)-macroglobulin, and alpha(1)-macroglobulin, were noted. In particular, LND caused a decrease of testicular alpha(1)-inhibitor-3, but not an increase of testicular alpha(2)-macroglobulin, indicating a mild local inflammatory response to the drug.

In vitro fertilization after in vivo treatment of rats with three reproductive toxicants.

One objective of these experiments was to establish a sensitive assay to evaluate fertilizing potential of rat gametes in vitro. A second objective was to evaluate this in vitro fertilization (IVF) assay as a method to detect in vivo effects of reproductive toxicants on male and female gametes using three known reproductive toxicants as model systems. The IVF assay with zona-free oocytes was more precise than the assay with cumulus-intact oocytes in these studies (coefficients of variation of 8.7 and 14.4%, respectively). Sperm fertilizing potential for zona-free oocytes was reduced by treatment of rats with m-dinitrobenzene (10-10 000 microg/kg) and ethylene glycol monomethyl ether (50-100 mg/kg) that had no effect on sperm motility. Molinate (60 mg/kg for 5 days) reduced sperm fertilizing potential concurrently with reductions in sperm motility. Neither molinate (60 mg/kg for 5 days) nor dinitrobenzene (0.002% in the drinking water for 14 days) administered to females seemed to affect the fertilizability of their oocytes. Ethylene glycol monomethyl ether treatment (0.15-0.25% in the drinking water for 14 days) reduced the number of ovulated oocytes. IVF is a means to evaluate toxicant effects on female gametes and demonstrates sperm's ability to interact with the oocyte plasma membrane.

Arginine acts as a protective and reversal agent against glycolytic inhibitors in spermatozoa.

It is known that the amino acid arginine stimulates sperm motility and glycolytic activity. We have earlier studied its efficacy as a stimulator of glycolysis in goat spermatozoa under anaerobic conditions. Here, we have assessed the influence of arginine in reversing the impairment caused by glycolytic inhibitors, iodoacetamide and iodoacetic acid. Glycolysis has been monitored by measuring the consumption of 13C labeled glucose and the amount of 13C labeled lactate produced under different experimental conditions, using 13C NMR. It is observed that both L- and D-arginine are able to prevent and reverse the inhibitory action of glycolytic inhibitors. The reversal effect of arginine gives rise to about eight times higher metabolic activity as compared to the inhibited cells while structurally related amino acids such as nitro-arginine, homo-arginine, lysine and ornithine are ineffective. The energetics of spermatozoa as measured by 31P NMR show a reduction in ATP level in cells incubated with iodoacetamide. Treatment of these cells with both L- and D-arginine restores the ATP level. The results may have significance in the treatment of male infertility.

The reproductive toxicity of molinate and metabolites to the male rat: effects on testosterone and sperm morphology.

Molinate causes an impairment in reproductive capability in the male rat. Administration of molinate to rats (40 mg/kg/day for 7 days) caused a distinctive sperm lesion. At higher doses of molinate (140 mg/kg for 7 days) this lesion was accompanied by morphological changes to the testis that were consistent with a delayed release of the late spermatids to the seminiferous tubular lumen, a process controlled by the release of testosterone. In accordance with this, molinate (>/=40 mg/kg) caused a marked decrease in the concentration of circulating and testicular testosterone. The Leydig cells of the testis appear to be the primary target site in that radiolabel from [3H]molinate specifically localized within this cell type. In addition, esterase activity in the Leydig cells was inhibited following molinate administration. In vitro, molinate is a poor inhibitor of esterase activity, whereas molinate sulfoxide, a major metabolite of molinate in rats, and molinate sulfone were shown to be potent inhibitors of this process, suggesting that metabolic activation of molinate is required in vivo. Molinate sulfoxide (>/=10 mg/kg) caused an identical sperm lesion to that of molinate and markedly decreased plasma and testicular testosterone concentration. These effects were not seen with the molinate metabolites 4-hydroxymolinate (10 mg/kg), molinate sulfone (10 mg/kg), and hexamethyleneimine (10 mg/kg). Since the sperm lesion is a secondary event caused by a disruption of spermatogenesis, this would imply that the testis lesion and the reproductive impairment are also a consequence of molinate sulfur oxidation.

The detection of thyrotropin-releasing hormone (TRH) and TRH receptor gene expression in Siberian hamster testes.

Thyrotropin-releasing hormone (TRH) from the hypothalamus is the major regulator of TSH synthesis and secretion. Most recently, TRH and TRH receptors (TRH-R), as well as their mRNAs, have been identified in rat testis. To expand our knowledge on the testicular TRH and TRH receptor gene expression in different species, in the present study the mRNA levels of testicular TRH and TRH-R were investigated in Siberian hamsters. To further localize the cellular sites of the gene expression, the animal model was treated with a single injection of ethylene dimethane sulfonate (EDS) (i.p., 80 mg/kg body weight), a compound known as to specifically eliminate testicular Leydig cells. The elimination of Leydig cells induced by EDS treatment was confirmed by histological studies of the testis sections and by serum hormonal analyses, which showed a dramatic reduction of serum testosterone (T) levels and significantly elevated serum LH concentrations. Messenger RNA levels of TRH and TRH-R in the testes were determined by Northern blot analyses quantitated with densitometry scanning. The results showed that specific TRH-R mRNA, 3.8 kb in size, was identified in Siberian hamster testes and the mRNA levels were significantly elevated in the EDS-treated testes compared to the controls (p < 0.01). Testicular TRH mRNA was also detected; however, no significant differences in TRH mRNA levels were found between EDS-treated and control groups. The size of TRH mRNA was characterized as about 1.2 kb in hamster testes, which was smaller than that observed in the rat hypothalamus (1.6 kb) and in the rat testis (2.0 kb). Further studies by RNase H digestion revealed the presence of smaller TRH transcripts in the hamster testes than those in the rat testis. No hybridization signal for TRH mRNA was detected by RNase protection assay, when a rat TRH riboprobe was applied to hamster testis RNA, suggesting the limited homology of TRH gene sequences between these two species. Our results demonstrate that both TRH and TRH-R genes are expressed in Siberian hamster testes, and a significant increase of TRH-R mRNA levels occurs in the Leydig cell eliminated hamster testes. Unlike the rat testicular TRH mRNA mainly detected in Leydig cells, in hamster TRH mRNA could also be detected in other testicular compartment.

[Study of the reproductive function in albino rats after inhalation of 4,4'-diaminodiphenyl ether of resorcin]. / Issledovanie reproduktivnoi funktsii belykh krys pri ingaliatsionnom vozdeistvii 4,4'-diaminodifenilovogo éfira resortsina.

The article contains experimental data on the morphofunctional state of gonads in male rats after chronic inhalation of diamine P, and on the state of embryogenesis after inhalant introduction of diamine P into pregnant rats in threshold-level concentrations. Results of the study revealed that, in chronic inhalant introduction at 29.3 and 4.2 mg/m3, diamine P demonstrated gonadotoxic properties, which was proved by the higher percentage of dead spermatozoa and spermatogonium diseases. The changes in the gonads' state reflected the pathological processes caused by diamine P in the animals due to the general toxicity properties of the substance. The embryotoxic effect of diamine P in inhalations at 5 mg/m3 (close to the threshold levels of total toxicity) was not statistically relevant.

The embryotoxicity of a new class of antispermatogenic agents: the 3-indazole-carboxylic acids.

The effects of two antispermatogenic 3-indazole-carboxylic acids on pregnancy in the rat have been studied. AF 1312/TS (1-(4-chlorobenzyl)-1H-indazole-3-carboxylic acid) when administered from day 6 to day 15 of pregnancy produced embryolethality with an LD50 of 145 mg/kg. The more potent antispermatogenic lonidamine was 7.25 times more effective, with an LD50 of 20 mg/kg. No significant teratogenicity was exerted by AF 1312/TS, whilst lonidamine was slightly effective only at embryolethal doses. The latter was also effective after a single administration on day 9, with an embryonal LD50 of 25 mg/kg, whereas the teratogenic effect was greatest on day 10. The similarity of the embryonal LD50 after single or repeated administrations suggests an interference with a specific epigenetic crisis.