Association between environmental exposure to p, p'-DDE and lindane and semen quality.

Scientific concern exists about the toxic effect of dichlorodiphenyldichloroethylene (p, p'-DDE) and lindane on male infertility, and the mechanism underlying male reproductive toxicity of this pesticide remains unanswered. We investigated not only the possible association between the chlorinated pesticide levels and semen quality in nonoccupationally exposed men, but also the probable mode of action using mitochondrial membrane potential (MMP), reactive oxygen species (ROS), lipid peroxidation (LPO), and sperm chromatin structure assay (SCSA). A study in 278 men (21-40 years old) who visited Obstetrics and Gynecology Department, KGMU, Lucknow, for semen analysis was conducted. We performed semen analysis according to the WHO guidelines, while p, p'-DDE and lindane analysis was done by the GLC and LPO by the spectrophotometer, and the sperm mitochondrial status, ROS, and SCSA with the flow cytometer. The questionnaire data showed no significant difference in the demographic characteristics between the two groups, i.e., trying to conceive >1 year and proven fertility. However, a significant difference in the concentration of p, p'-DDE and lindane was observed between the groups. When the subjects were divided among four categories by quartile of exposure, the subjects in the highest quartile showed low sperm motility as compared to the subjects in the lowest quartile. Pearson's correlation showed a significant negative correlation between semen p, p'-DDE, lindane level, and sperm quality and positive association with the number of cells with depolarized mitochondria, elevation in ROS production and LPO, and DNA fragmentation index (DFI). The findings are suggestive that these toxicants might cause a decline in semen quality, and these effects might be ROS, LPO, and mitochondrial dysfunction mediated.

Endosulfan induced cell death in Sertoli-germ cells of male Wistar rat follows intrinsic mode of cell death.

Health of germ cells may affect production of quality gametes either due to endogenous or exogenous factors. Pesticides are among the exogenous factors that can enter the organisms through various routes of exposure and also can affect the reproductive system of an organism. Endosulfan is an organochlorine cyclodiene pesticide used widely for controlling agricultural pests. It has been shown to induce reproductive dysfunctions such as sperm abnormalities, reduced intracellular spermatid count in exposed organisms. Germ cells being the progenitor cells for male gametes and Sertoli cells as their nourishing cells, we examined whether endosulfan induces cell death in Sertoli-germ cells of male rats. Sertoli-germ cells, isolated from 28 d old male Wistar rats, were exposed to endosulfan (2.0, 20.0 and 40.0 µg mL(-1)) for 24-72 h. Cytotoxicity, endosulfan concentration, reactive oxygen species (ROS) generation, oxidative stress parameters were measured in these cells in the absence or presence of endosulfan for the above mentioned exposure periods and subsequently, cell death endpoints were measured. We detected endosulfan in the exposed cells and demonstrated increased cell death in exposed Sertoli-germ cells as evidenced by a significant increase in annexin-V staining, depolarization of mitochondrial membrane, caspase-9 and -3 activities and BAD and PARP cleavage activities and DNA ladder formation along with non-significant increase in autophagic cell death. The study suggests that endosulfan can cause cell death in exposed Sertoli-germ cells due to higher oxidative damage with the activation of intrinsic cell death pathway which may eventually affect the production of quality gametes.

Semen quality of environmentally exposed human population: the toxicological consequence.

Human data on the relationship of semen quality with pesticide and metals are mostly inconsistent. The purpose of the study is to confirm the toxicity of organochlorine pesticide ß- and γ-hexachlorocyclohexane (HCH), DDE and DDD, and metals lead or cadmium on sperm motility in epidemiological study among fertile and infertile men and to determine whether in vivo and in vitro results are in the same direction. Semen analysis and estimation of the toxicants were done in 60 fertile and 150 infertile men. In the in vitro studies, sperm were exposed to the highest levels of these toxicants found in vivo, as well as five and ten times higher, and to the mixture of all compounds. The study assesses sperm viability and motility for a period ranging between 30 min and 96 h. Epidemiological data showed an inverse correlation of toxicant with sperm motility. In vitro study showed that γ-HCH and lead after 12 h, cadmium after 8 h, and coexposure to toxicants after 6 h of exposure caused significant concentration- and duration-dependent decline in sperm motility. Data of in vitro study were concurrent with epidemiological finding that might be useful in establishing the possible association between exposure and effect of these selected pollutants on sperm motility.

Effects of co-exposure of benzene, toluene and xylene to Drosophila melanogaster: alteration in hsp70, hsp60, hsp83, hsp26, ROS generation and oxidative stress markers.

Benzene, toluene and xylene are monocyclic aromatic hydrocarbon compounds, used both as individual compound and as mixtures, in industry as well as household. Previous studies involving exposures to these compounds, individually, have shown that benzene was more toxic compared to toluene or xylene. Here, we tested a working hypothesis that toluene and/or xylene in a mixture containing benzene affect benzene induced toxicity in a non-target organism, Drosophila melanogaster. We exposed D. melanogaster larvae transgenic for hsp70, hsp83 or hsp26 and wild type (Oregon R strain) larvae to 25.0-100.0mM benzene, 25.0-100.0mM toluene and 25.0-100mM xylene, individually or in mixtures. Subsequently, we examined the expression of stress genes (encoding heat shock proteins, hsps), generation of reactive oxygen species (ROS), induction of anti-oxidant stress markers and emergence of flies under treatment as well as control conditions. We observed that all these endpoints were significantly altered in all the treatment groups compared to their respective controls. However, the magnitude of toxicity of a benzene-toluene (BT) or benzene-xylene (BX) or benzene-toluene-xylene (BTX) mixture was significantly lower in the organism than that of individual chemical. Our results also show the modulation of toluene toxicity by xylene. Present study suggests antagonistic effect of xylene and toluene on benzene toxicity and additive/synergistic effect of xylene on toluene induced toxicity. Thus, expression of stress genes may be used as an assay for detection of early cellular toxicity. Further, our study supports the use of Drosophila as an alternative animal model for first tier screening of adverse effects of chemical mixtures.

Induction of hsp70, hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene and xylene exposed Drosophila melanogaster: role of ROS generation.

Exposure to benzene, toluene and xylene in the human population may pose a health risk. We tested a working hypothesis that these test chemicals cause cellular toxicity to a non-target organism, Drosophila melanogaster. Third instar larvae of D. melanogaster transgenic for hsp70, hsp83 and hsp26 and Oregon R(+) strain were exposed to 1.0-100.0 mM benzene, toluene and xylene for 2-48 h to examine the heat shock proteins (hsps), ROS generation, anti-oxidant stress markers and developmental end points. The test chemicals elicited a concentration- and time-dependent significant (p<0.01) induction of the hsps in the exposed organism in the order of hsp70>hsp83>or=hsp26 as evident by beta-galactosidase activity after 24 h. RT-PCR amplification studies in Oregon R(+) larvae revealed a similar induction pattern of these genes along with hsp60 in the order of hsp70>hsp60>hsp26>or=hsp83. Under similar experimental conditions, a significant induction of ROS generation and oxidative stress markers viz. superoxide dismutase, catalase, glutathione S-transferase, thioredoxin reductase, glutathione, malondialdehyde and protein carbonyl content was observed. Sub-organismal response was propagated towards organismal response i.e., a delay in the emergence of flies and their reproductive performance. While hsp70 was predominantly induced in the organism till 24 h of treatment with the test chemicals, a significant or insignificant regression of Hsp70 after 48 h was concurrent with a significant induction (p<0.01) of hsp60>hsp83>or=hsp26 in comparison to the former. A significant positive correlation was observed between ROS generation and these hsps in the exposed organism till 24 h and a negative correlation between ROS generation and hsp70 in them after 48 h indicating a modulatory role of ROS in the induction of hsps. The study suggests that among the tested hsps, hsp70 may be used as an early bioindicator of cellular toxicity against benzene, toluene and xylene and D. melanogaster as an alternative animal model for screening the risk posed by environmental chemicals.

Biomonitoring of metal deposition by using moss transplant method through Hypnum cupressiforme (Hedw.) in Mussoorie.

Metals Cu, Zn, Cd and Pb were surveyed at 14 sampling sites by using moss Hypnum cupressiforme through active monitoring technique. Samples were transplanted in all four directions of Mussoorie city and were harvested after exposure of four months (representing each season) to analyze metal precipitation and its trend at different sites during 2005. Bioaccumulation ability for metals was evaluated seasonally exhibiting maximum in summer followed by winter and minimum in rainy season. However, at some places Cu shows highly significant values in rainy season in comparison to winter. In case of Zn and Pb significantly different (p < or = 0.05) values were observed between summerand rainy season. Baseline concentration of Cu, Pb and Zn was significantly different at 5% in comparison to other transplant sites. Result indicates Dhanaulti as most polluted location might be due to higher tourist activity and vehicular load, whereas, same was found low at Chamba might be due to place was free from pollution sources or away from in proximity to road and have low human interference. The present study allows us to determine the extent of the area affected by metal precipitation load in different rural and urban areas and abundance of metals in order of Zn > Pb > Cu > Cd.

Chlorinated pesticide concentration in semen of fertile and infertile men and correlation with sperm quality.

In recent years controversy has evolved regarding the role of environmental pollutants especially chlorinated pesticides and heavy metals on male infertility. Previous data generated on the correlation of chlorinated pesticides with human semen are scarce and controversial. The objective is to explore the possibility of correlation if any between the chlorinated pesticides and sperm count and motility. Semen analysis were performed in 50 samples collected each from fertile and infertile men and pesticides estimation for dichlorodiphenyltrichloroethane (DDT) and its metabolites dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE); 1,1,1-trichloro-2,2-bis(p-chlorophenyl ethane) (pp'DDT); 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (pp'DDD), 1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (op'DDT), aldrin, hexachlorocyclohexane (HCH) isomers alpha (α), beta (ß), gamma (γ), delta (δ) were done by gas liquid chromatography. The higher concentrations of pesticide, viz. α-, ß-, γ-, δ-HCH, DDT and its metabolites (pp'DDE and pp'DDD) were detected in semen samples of infertile males. The data showed significant association between ß-HCH, γ-HCH, pp'DDE, pp'DDD with semen quality parameters.

Comparative toxic potential of market formulation of two organophosphate pesticides in transgenic Drosophila melanogaster (hsp70-lacZ).

This study investigated the working hypothesis that two widely used organophosphate pesticides; Nuvan and Dimecron, exert toxic effects in Drosophila. Transgenic D. melanogaster (hsp70-lacZ) was used as a model for assaying stress gene expression and AchE activity as an endpoint for toxicity and also to evaluate whether stress gene expression is sufficient to protect against toxic insult of the chemicals and to prevent tissue damage. The study was extended to investigate the effect of the pesticides on the life cycle and reproduction of the organism. The study showed that Nuvan affected emergence of the exposed flies more drastically than Dimecron and the effect was lethal at the highest tested concentration (0.075 ppm). While Nuvan at 0.0075 and 0.015 ppm concentrations affected reproduction of the flies significantly, the effect of Dimecron was significant only at 0.015 and 0.075 ppm. Nuvan-exposed third-instar larvae exhibited a 1.2-fold to 1.5-fold greater hsp70 expression compared to Dimecron at concentrations ranging from 0.0075 to 0.075 ppm following 12 and 18 h exposure. While maximum expression of hsp70 was observed in Nuvan-exposed third-instar larval tissues following 18 h exposure at 0.075 ppm, Dimecron at the same dietary concentration induced a maximum expression of hsp70 following 24 h exposure. Further, concomitant with a significant induction of hsp70, significant inhibition of AchE was observed following chemical exposure and temperature shock. Concurrent with a significant decline in hsp70 expression in Nuvan-exposed larvae after 48 h at 0.075 ppm, tissue damage was evident. Dimecron-exposed larvae exhibited a plateau in hsp70 induction even after 48 h exposure and moderate tissue damage was observed in these larvae. The present study suggests that Nuvan is more cytotoxic than Dimecron in transgenic Drosophila melanogaster.

Genotoxicity of industrial solid waste leachates in Drosophila melanogaster.

The potential toxicity of industrial solid wastes is a major environmental concern. The present study evaluated the genotoxicity of industrial waste leachates on the gut cells of Drosophila melanogaster (Oregon R+), using a modified alkaline comet assay. Leachates were prepared from control soil and solid wastes generated by a flashlight battery factory, a pigment plant, and a tannery, using different pHs (7.0, 4.93, and 2.88). Newly emerged first instar Drosophila larvae (22 +/- 2 hr) were transferred to standard Drosophila diet containing 0.05%, 0.1%, 0.5%, 1.0%, and 2.0% of the leachates, and allowed to grow. At 96 +/- 2 hr, the anterior midgut of control and treated larvae was dissected out; single cell suspensions were prepared; and the comet assay was performed on the cells. All the leachates produced significant (P < 0.05), dose-dependent increases in DNA damage, in the gut cells. Leachates prepared at pH 7.0 were significantly less genotoxic than leachates prepared at pH 4.93 or 2.88. A comparison of the comet parameters among the exposed groups indicated that leachates of the pigment plant solid waste produced the least DNA damage, while leachates prepared from the flashlight battery factory solid waste were the most genotoxic. The present study indicates that leachates of solid wastes from flashlight battery factories, pigment plants, and tanneries possess genotoxic activity and that D. melanogaster is a useful in vivo model for assessing the genotoxicity of these potential environmental contaminants.

Validation of Drosophila melanogaster as an in vivo model for genotoxicity assessment using modified alkaline Comet assay.

The single cell gel electrophoresis or Comet assay is one of the most popular techniques for genotoxicity assessment. The present study was undertaken to validate our previously modified version of the Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with four well-known mutagenic and carcinogenic alkylating agents, i.e. ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and cyclophosphamide (CP). Third instar larvae (74 +/- 2 h) of D.melanogaster were fed different concentrations of EMS, MMS, ENU and CP (0.05, 0.5 and 1.0 mM) mixed standard Drosophila food for 24 h. At 98 +/- 2 h, the anterior midgut from control and treated larvae were dissected out, single-cell suspensions were prepared and Comet assay was performed. Our results show a dose-dependent increase in DNA damage with all the four alkylating agents, in comparison to control. The lower concentration (0.05 mM) of the test chemicals, except MMS, did not induce any DNA damage in the gut cells of the exposed larvae. When comparison of Comet parameters was made among the chemicals, MMS was found to be the most potent genotoxicant and ENU the least. The present study validated our previous observation and shows that D.melanogaster is a sensitive and suitable model for the in vivo assessment of genotoxicity using our modified alkaline Comet assay.

Correlation of chlorinated pesticides concentration in semen with seminal vesicle and prostatic markers.

Semen samples of fertile and infertile men were analysed by gas liquid chromatography (GLC) for the presence of dichlorodiphenyltrichloroethane (DDT) and its metabolites (1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene) (pp'-DDE); (1,1,1-trichloro-2,2-bis(p-chlorophenyl ethane) (pp'-DDT); (1,1-dichloro-2,2 bis(p-chlorophenyl) ethane) (pp'-DDD); (1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane) (op'-DDT), hexachlorocyclohexane (HCH) and its isomers alpha (alpha), beta (beta), gamma (gamma), delta (delta) and aldrin. The biochemical analysis of seminal vesicle and prostatic marker was done by spectrophotometer. The concentrations of alpha-, beta-, gamma-HCH, pp'-DDE and pp'-DDD were higher in the semen of infertile than in that of fertile men. An elevation in the fructose level along with decrease in the gamma-glutamyl transpeptidase and acid phosphatase activity was noticed in infertile men as compared to fertile subjects. The data showed correlation between alpha-, beta-, gamma-HCH, pp'-DDE, pp'-DDD and seminal vesicle marker fructose and prostatic marker gamma-gluatamyl transpeptidase and acid phosphatase in infertile men. The study suggests that the chlorinated pesticides may influence the semen quality by affecting the seminal and prostatic functions in infertile men.

Heat shock response: hsp70 in environmental monitoring.

Heat shock proteins (Hsps) are a ubiquitous feature of cells in which these proteins cope with stress-induced denaturation of other proteins. Among the different families of Hsps, the 70 kDa family (hsp70) is the most highly conserved and has been most extensively studied. Apart from their primary role in cellular defense under stress condition, a number of studies in recent years have shown the immense potential of hsp70 in pollution monitoring using even transgenic approach both in vivo and in vitro. This article reviews the recent developments in the widespread application of hsp70 in environmental risk assessment.

Argemone oil induced cellular damage in the reproductive tissues of transgenic Drosophila melanogaster: protective role of 70 kDa heat shock protein.

We explored the reproductive toxicity of argemone oil and its principal alkaloid fraction in transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9). The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Freshly eclosed first instar larvae of transgenic Drosophila melanogaster were transferred to different concentrations of argemone oil and its alkaloid fraction contaminated food. Virgin flies that eclosed from the contaminated food were pair-mated to look into the effect on reproduction. The study was further extended by investigating hsp70 expression and tissue damage in larval gonads, genital discs, and reproductive organs of adult fly. Our results showed that argemone oil was more cytotoxic than its principal alkaloid fraction. Moreover, it was the male fly that was more affected compared to its opposite number. The accessory glands of male reproductive system of the fly, which did not express hsp70, exhibited severe damage as evidenced by Trypan blue staining. This prompted us to explore the ultrastructural morphology of the gland, which showed acute signs of necrosis in both the cell types as evident by necrotic nuclei, higher vacuolization, and disorganized endoplasmic reticulum, decrease in the number of Golgi vesicles and disorganized, loosely packed filamentous structures in the lumen of the accessory gland, at the higher concentrations of the adulterant. The study showed the reproductive toxicity of argemone oil and its alkaloid fraction in transgenic Drosophila melanogaster and further confirmed the cytoprotective role of hsp70.

Lead and cadmium concentration in the seminal plasma of men in the general population: correlation with sperm quality.

The concentration of lead and cadmium in the seminal plasma of men in the general population, including fertile and infertile subjects, was measured. Semen samples were categorised as demonstrating oligospermia (sperm concentration < 20 x 10(6)/ml), asthenospermia (<50% motilesperm), oligoasthenospermia (a combination of the two criteria), and azoospermia (no sperm). An increase in lead and cadmium levels was observed in infertile men and there was a significant negative correlation of cadmium and lead semen concentration with sperm motility and sperm concentration in oligoasthenospermic men.

Correlation of lead and cadmium in human seminal plasma with seminal vesicle and prostatic markers.

The objective of this study was to investigate the correlation between lead and cadmium with seminal vesicle and prostatic markers. Semen samples categorized into fertile and infertile were evaluated for the presence of lead and cadmium and biochemical markers in the seminal plasma. Associations between lead and fructose, acid phosphatase and gamma-glutamyl transpeptidase (gamma-GT) were observed. However, no such relationships were noticed for cadmium. It is concluded that lead may be one of the pollutants indirectly affecting semen quality by altering the functions of accessory sex glands.

Evaluation of toxic potential of captan: Induction of hsp70 and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9.

The study investigated the working hypothesis that a widely used fungicide captan exerts toxic effects on nontarget organisms. Transgenic Drosophila melanogaster (hsp70-lacZ) was used as a model by assaying stress gene expression as an endpoint for cytotoxicity and also to evaluate whether stress gene expression is sufficient enough to protect and to prevent tissue damage against toxic insult of the chemical. The study was further extended to understand the effect of the pesticide on development, life cycle, and reproduction of the organism and finally to evaluate a concentration of the chemical to be nontoxic to the organism. The study showed that (i) captan causes cytotoxicity at and above 0.015 ppm; (ii) at 0.0015 ppm captan, absence of hsp70 expression in the exposed organism was evaluated as the concentration referred to as no observed adverse effect level (NOAEL) for Drosophila; (iii) emergence pattern of flies was affected only at the highest concentration of captan by 4 days, while hatching and survivorship were unaffected even at this concentration; (iv) reproductive performance was significantly affected only at 125.0 and 1250.0 ppm captan, while in the lower dietary concentrations no such deleterious effects were observed; (v) at 1250.0 ppm, hsp70 failed to protect the cells from toxicant assault after 48 h exposure, thus leading to tissue damage as revealed by Trypan Blue staining. The present study shows the cytotoxic potential of captan and further reveals the application of stress genes in determining NOAEL and its expression as bioindicator of exposure to environmental contaminants.

Evaluation of the No Observed Adverse Effect Level of Solvent Dimethyl Sulfoxide in Drosophila melanogaster.

Dimethyl sulfoxide, a solvent commonly used in toxicological studies, was investigated for its cytotoxic potential and its effect on development and reproductive performance in transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9. Various concentrations (0.0, 0.1, 0.3, 0.5, 1.0, 2.0, and 3.0%) of the solvent were mixed with food and fed to the flies and larvae. The toxic effects were studied by examining hatchability, emergence, fecundity, reproductive performance, and hsp70 expression by means of in situ beta-galactosidase staining in the tissues of third-instar larvae and in the reproductive organs of male and female flies of the strain. Our results showed that dimethyl sulfoxide at and above 0.5% of dietary concentration evoked cytotoxicity, as evidenced by hsp70 expression in the larval tissues, and also impaired the development and reproductive performance of the flies. This study suggests that the no observed adverse effect level of dimethyl sulfoxide is 0.3% of dietary concentration for developmental toxicity, reproductive toxicity, and cytotoxicity studies in Drosophila melanogaster.

Toxicity of argemone oil: effect on hsp70 expression and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9.

The effect of argemone oil on hsp70 expression and tissue damage was investigated by studying beta-galactosidase activity, Western blotting and hybridization, and trypan blue staining in the larval tissues of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. Different concentrations of argemone oil were mixed with food and third-instar larvae were allowed to feed on them for different time intervals (2, 4, 24, and 48 h). Argemone oil was found to induce hsp70 even in the lowest concentration of the adulterant while maximum tissue damage was observed in the higher two treatment groups. Malpighian tubules and midgut tissue reflected maximum damage as evidenced by both high beta-galactosidase activity and trypan blue staining in these tissues. A prior temperature shock treatment to the larvae was enough to protect the larvae from argemone oil-induced tissue damage as evidenced by little or no trypan blue staining. The present study suggests the cytotoxic potential of argemone oil and further strengthens the evidence for the use of hsp70 as a biomarker in risk assessment.

Behaviour of a sperm surface transmembrane glycoprotein basigin during epididymal maturation and its role in fertilization in mice.

Basigin (bsg) is a transmembrane glycoprotein belonging to an immunoglobulin superfamily and is localized on the surface of the sperm tail. The behaviour of bsg during epididymal maturation and its role in fertilization were examined using an anti-bsg antibody. Spermatozoa from caput, corpus and cauda epididymides were immunostained by indirect immunofluorescence (IIF). Immunostaining revealed that bsg is localized on the principal piece of caput spermatozoa and the molecule was found on the middle piece during transit in the corpus and cauda epididymides. Concomitantly, the molecular mass of bsg was reduced from 37 kDa (testis) to 26 kDa (cauda epididymidis). IVF experiments were designed to assess the effect of anti-bsg antibody on the fertilization events. Anti-bsg antibody significantly inhibited primary binding to the cumulus-invested oocytes with intact zonae pellucidae in a dose-dependent manner. Consequently, the fertilization rate of cumulus-invested oocytes with intact zonae pellucidae was also inhibited. The bsg molecule was also detected on the head of live capacitated spermatozoa by IIF under IVF conditions. These findings indicate that testicular bsg is a glycosylated protein that undergoes molecular processing and deglycosylation during its transit in the epididymis. The bsg molecule that was detected on the sperm head after capacitation may facilitate the primary binding or might be involved in distinct events required for primary binding of spermatozoa to the zona pellucida during capacitation and sperm-cumulus interaction.

Are germ cell factors essential in the testicular enlargement after neonatal hypothyroidism recovery? A study using W/Wv mutant mice model.

We examined the issue of whether germ cell factors are required for testicular enlargement that occurs after recovery from neonatal hypothyroidism. Experiments were performed using W/Wv mutant mice (lacking germ cells) and normal mice (ICR). The pups in experimental group (neonatal hypothyroid) received 6 propyl 2-thio-uracil (PTU) treatment, administered by adding 0.1% (w/v) to the water provided to the mother from day 1 of birth through day 25 postpartum, while the pups of control group received drinking water only. Mice were sacrificed at the age of day 25, 50 and 90, in the case of ICR mice, or at day 25 and 90 in the case of W/Wv mutant mice. In both groups, early hypothyroidism caused a partial recoverable decrease in body growth and testicular development. Both ICR and W/Wv mutant mice, those recovered from neonatal hypothyroidism showed an increase in testis weights, the number of Sertoli cells, and the diameter of the semniferous tubules. This study demonstrates that neonatal hypothyroidism led recovery caused testicular enlargement not only in ICR mice but also in germ cell depleted W/Wv mutant mice. Hence these findings deny direct involvement of the germ cell factors in the process of testicular enlargement in recovered mice even in vivo, and reaffirm the notion that thyroid hormone directly regulates the dynamics of Sertoli cell maturation.