A comparative study of the effects of 3 testicular toxicants in cultures of seminiferous tubules of rats or of domestic cats (veterinary waste): An alternative method for reprotoxicology.

In response to the EU cosmetics directive regulation and REACH legislation which encourage cell culture methods in order to reduce or replace the use of animals in toxicology studies, we settled the culture of prepubertal domestic cat seminiferous tubules in our validated BioAlter® model, usually used with prepubertal rat, called here BioAlter®-rat, by opposition to BioAlter®-cat settled here. We carried out a comparative study on the effects of 3 testicular toxicants, 1,3-dinitrobenzene at 60 μM, 2-methoxyacetic acid at 2.5 mM and carbendazim at 50 nM or 500 nM in both BioAlter®-cat and BioAlter®-rat over a 3-week culture period.

Low concentrations of glyphosate alone affect the pubertal male rat meiotic step: An in vitro study.

Glyphosate is the most used herbicide in the world. Controversial studies exist on its effect on the male reproductive system. We used the validated BioAlter® model to test the effects of low concentrations of Glyphosate.

An in vitro bioassay to assess the potential global toxicity of waters on spermatogenesis: a pilot study.

Many toxicants are present in water as a mixture. Male infertility is one of the environmental impacts in developed countries. Using our rat seminiferous tubule culture model, we evaluated the effects of waters of different origins, on several parameters of the seminiferous epithelium.

Effects of a mixture of low doses of atrazine and benzo[a]pyrene on the rat seminiferous epithelium either during or after the establishment of the blood-testis barrier in the rat seminiferous tubule culture model.

Atrazine (ATZ), a widely used agricultural pesticide and benzo[a]pyrene (BaP), a ubiquitous environmental human carcinogen can induce alterations of spermatogenesis. In the present study, we showed first that our seminiferous tubule culture model, in bicameral chambers, allowed the settlement of the blood-testis barrier (BTB) in 8-day-old male rat cultures and the differentiation of spermatogonia into round spermatids.The effect of a mixture of 1 μg/L of ATZ and 1 μg/L of BaP was then investigated either during or after the establishment of the BTB by using 8- or 20-22-day-old rats.

Dynamic Structuration of Physical Chitosan Hydrogels.

We studied the microstructure of physical chitosan hydrogels formed by the neutralization of chitosan aqueous solutions highlighting the structural gradients within thick gels (up to a thickness of 16 mm). We explored a high polymer concentrations range (C ≥ 1.0% w/w) with different molar masses of chitosan and different concentrations of the coagulation agent.

Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study.

It has been shown that non-cytotoxic doses of Carbendazim (CBZ), a broad-spectrum benzimidazole fungicide, possess endocrine-disrupting (androgen-like) actions, ex vivo, on the pubertal rat seminiferous epithelium. Iprodione (IPR), a dicarboximide fungicide, is also known to be an endocrine-disrupter (anti-androgen). The effect of a mixture of these two pesticides was investigated in the validated rat seminiferous tubule culture model.

Ex vivo assessment of testicular toxicity induced by carbendazim and iprodione, alone or in a mixture.

To measure the testicular toxicity of two fungicides (carbendazim and iprodione), alone or in a mixture, we used a rat ex vivo model of seminiferous tubules, greatly reducing the number of rodents used, in accordance with the 3R rule (Replacement, Reduction, and Refinement). This model allows the representation of puberty, a critical life period with regard to endocrine disruptors. The cellular modifications were followed for three weeks through transcriptomic and proteomic profiling analysis.

Complete Human and Rat Ex Vivo Spermatogenesis from Fresh or Frozen Testicular Tissue.

Until now, complete ex vivo spermatogenesis has been reported only in the mouse. In this species, the duration of spermatogenesis is 35 days, whereas it is 54 days in the rat and 74 days in humans. We performed long-term (until 60 days) cultures of fresh or frozen rat or human seminiferous tubule segments in a bioreactor made of a hollow cylinder of chitosan hydrogel.

Use of a rat ex-vivo testis culture method to assess toxicity of select known male reproductive toxicants.

Due to the complex physiology of the testes, in vitro models have been largely unsuccessful at modeling testicular toxicity in vivo. We conducted a pilot study to evaluate the utility of the Durand ex vivo rat seminiferous tubule culture model [1-3] that supports spermatogenesis through meiosis II, including the formation of round spermatids. We used this system to evaluate the toxicity of four known testicular toxicants: 1,3-dinitrobenzene (DNB), 2-methoxyacetic acid (MAA), bisphenol A (BPA), and lindane over 21 days of culture.

Exposure to low-dose bisphenol A impairs meiosis in the rat seminiferous tubule culture model: a physiotoxicogenomic approach.

Background: Bisphenol A (BPA) is one of the most widespread chemicals in the world and is suspected of being responsible for male reproductive impairments. Nevertheless, its molecular mode of action on spermatogenesis is unclear. This work combines physiology and toxicogenomics to identify mechanisms by which BPA affects the timing of meiosis and induces germ-cell abnormalities.

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway.

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells.

Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model.

Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities.

Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells.

Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2-week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture.

Decline of semen quality among 10 932 males consulting for couple infertility over a 20-year period in Marseille, France.

Semen from 10 932 male partners of infertile couples was analysed and sperm parameter trends were evaluated at the Reproduction Biology Laboratory of the University Hospital of Marseille (France) between 1988 and 2007. After 3-6 days of abstinence, semen samples were collected. Measurements of seminal fluid volume, pH, sperm concentration, total sperm count, motility and detailed morphology of spermatozoa were performed.

Validation of a rat seminiferous tubule culture model as a suitable system for studying toxicant impact on meiosis effect of hexavalent chromium.

There is evidence that exposure to environmental factors is at least partly responsible for changes in semen quality observed over the past decades. The detection of reproductive toxicants under Registration, Evaluation and Authorisation of Chemicals (REACH) will impact animal use for regulatory safety testing. We first validated a model of culture of rat seminiferous tubules for toxicological studies on spermatogenesis.

[European regulation REACH and the assessment of testicular toxicity].

Several studies suggest that exposure to environmental pollutants is partly responsible for testicular pathologies that have considerably increased over the last decades (cryptorchidism, hypospadias, cancer, decrease in the number of ejaculated spermatozoa). However, the cellular and molecular mechanisms involved in this reprotoxicity remain mostly unknown. One of the challenges of the european regulation REACH is to improve the knowledge on the chemical, toxic and ecotoxic properties of substances used in everyday life.

Role of miR-34c microRNA in the late steps of spermatogenesis.

Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into haploid spermatozoa. This process is highly regulated, notably at the post-transcriptional level. MicroRNAs (miRNAs), single-stranded noncoding RNA molecules of about 20-25 nucleotides, are implicated in the regulation of many important biological pathways such as proliferation, apoptosis, and differentiation.

Analysis of the intratesticular control of spermatogenesis by ex-vivo approaching.

Spermatogenesis involves the realization of a particular genetic program which requires a specific environment ("niche"). Multiplication, differentiation and apoptosis of male germ cells are finely regulated by pituitary hormones (mainly LH and FSH), and by a complex network of factors originating from both the somatic cells and the germ cells of the testis. It is becoming clear that hormones and intra-testicular regulatory factors can compensate, at least in part, for the absence of some hormones or factors including FSH and LH or androgen receptors.

Cytostatic factor proteins are present in male meiotic cells and beta-nerve growth factor increases mos levels in rat late spermatocytes.

Background: In co-cultures of pachytene spermatocytes with Sertoli cells, beta-NGF regulates the second meiotic division by blocking secondary spermatocytes in metaphase (metaphase II), and thereby lowers round spermatid formation. In vertebrates, mature oocytes are arrested at metaphase II until fertilization, because of the presence of cytostatic factor (CSF) in their cytoplasm. By analogy, we hypothesized the presence of CSF in male germ cells.

Redundancy of the effect of TGFbeta1 and beta-NGF on the second meiotic division of rat spermatocytes.

We have previously shown that in cocultures of late pachytene/diplotene spermatocytes (PS/DS) with Sertoli cells, beta-nerve growth factor (beta-NGF) or transforming growth factor (TGFbeta1) regulates the second meiotic division by blocking secondary spermatocytes in metaphase II, and thereby lowers round spermatid formation. In this study, we raised the question if beta-NGF and TGFbeta1 have additional or redundant effects on this step. Hence, we addressed the effect of beta-NGF in combination with TGFbeta1, as compared to those of beta-NGF or TGFbeta1 separately, on the completion of meiosis by rat late PS/DS.

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously.

Quantification of stem cell factor mRNA levels in the rat testis: usefulness of clusterin mRNA as a marker of the amount of mRNA of Sertoli cell origin in post pubertal rats.

Spermatogenesis is a complex cellular process regulated by gonadotrophins and local cell-cell interactions. Stem cell factor (SCF) is one of the paracrine factors, produced by the Sertoli cells, involved in the local regulation of spermatogenesis. Measurement of its testicular level is important for addressing its role in testis physiopathology.

Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes in vitro.

Background: TGF beta and its receptors are present in both germ cells and somatic cells of the male gonad. However, knock-out strategies for studying spermatogenesis regulation by TGF beta have been disappointing since TGF beta-or TGF beta receptor-null mice do not survive longer than a few weeks.

Methods: In the present study, we addressed the role of TGF beta-1 on the completion of meiosis by rat pachytene spermatocytes (PS) cocultured with Sertoli cells.

Development of the meiotic step in testes of pubertal rats: comparison between the in vivo situation and under in vitro conditions.

The present work aimed to compare some features of the meiotic process which develops in the testis of pubertal rats, in vivo and in vitro, paying special attention to the time-course of the phenomenon. The differentiation of spermatocytes was assessed in testes of 20- to 46-day-old rats and in tubule segments of 20- or 28-day-old rats cultured over a 4-week period. Very similar results were obtained in vivo and in vitro, during the first week of culture, when considering the changes in the cell populations of different ploidy, the gene expression of germ cells, the kinetics of differentiation of BrdU-labeled early or middle pachytene spermatocytes and the levels of apoptosis in the different cell populations.