ABSTRACT
Neonatal administration of diethylstilbestrol (DES) to rodents has adverse effects on spermatogenesis. However, not many studies have been conducted to determine which type of cell - germ or somatic - is the major target of DES. In order to clarify this, we tried reciprocal germ cell transplantation--transplantation of germ cells from DES-treated mice into intact mice and germ cells from normal mice into DES-treated mice. The donor germ cells were tagged with the green fluorescent protein (GFP) gene in order to distinguish the exogenous germ cells from the endogenous cells. Moreover, to obtain a large number of spermatogonia from the testes of adult mice, we performed fractionation by centrifugation with Percoll. Consequently, we found that the germ cells collected from DES-treated mice have differentiated into normal sperms in normal seminiferous tubules. However, in the case of the transplantation of normal germ cells into the seminiferous tubules of DES-treated mice, defective spermatogenesis was observed. In conclusion, DES has adverse effects on the somatic cells that are involved in spermatogenesis rather than the germ cells.
Subject(s)
Diethylstilbestrol/toxicity , Spermatogonia/drug effects , Testis/drug effects , Animals , Animals, Newborn , Antineoplastic Agents, Alkylating/toxicity , Busulfan/toxicity , Cell Proliferation/drug effects , Diethylstilbestrol/administration & dosage , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Injections, Subcutaneous , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence/methods , Seminiferous Tubules/cytology , Seminiferous Tubules/drug effects , Spermatogenesis/drug effects , Spermatogonia/cytology , Spermatogonia/transplantation , Stem Cell Transplantation/methods , Testis/cytology , Testis/transplantationABSTRACT
Summary In utero or neonatal exposure to high levels of exogenous steroid hormones, such as the potent synthetic diethylstilbestrol (DES), incurs an increased risk of malfunctional male reproduction. In this study, we investigated whether neonatal exposure to DES induces the alteration of mRNA expression in adult mouse testis. Using a cDNA subtraction method, we isolated seven gene clones whose expression was changed in neonatally DES-treated mouse testis. Northern blot analysis revealed that five up-regulated genes (AF326230, AF356521, AK004975, AK006136 and BM237156) and two down-regulated genes (AK017044, AK017130) were predominantly expressed in testes of 8-week-old mice. Moreover, we confirmed that the expression of these seven genes was altered by neonatal DES-exposure using Northern blot analysis. Our results suggest that neonatal exposure to DES leads to the alteration of gene expression in the testis in the long term. These genes might be useful as biological markers of foetal or neonatal exposure to exogenous steroid hormones, such as DES.
Subject(s)
Aging/metabolism , Animals, Newborn , Diethylstilbestrol/administration & dosage , Estrogens, Non-Steroidal/administration & dosage , Gene Expression/drug effects , Testis/metabolism , Animals , Blotting, Northern , DNA, Complementary , Down-Regulation , Drug Administration Schedule , Male , Mice , Mice, Inbred ICR , Molecular Sequence Data , RNA, Messenger/metabolism , Subtraction Technique , Up-RegulationABSTRACT
AIM: To isolate and transplant germ cells from adult mouse testes for transplantation. METHODS: In order to distinguish transplanted cells from endogenous cells of recipients, donor transgenic mice expressing green fluorescent protein (GFP) were used. Germ cells were collected from the donors at 10-12 weeks of age and spermatogonia were concentrated by percoll fractionation and transplanted into recipient seminiferous tubules that had been previously treated with busulfan at 5 weeks of age to remove the endogenous spermatogenic cells. RESULTS: Twenty weeks after the transplantation, a wide spread GFP signal was observed in the recipient seminiferous tubules. The presence of spermatogenesis and spermatozoa was confirmed in sections of 12 out of 14 testes transplanted (86 %). However, when germ cells were transplanted without concentration the success rate was zero (0/9). CONCLUSION: Germ cells from adult mouse testes can be successfully transplanted into recipient seminiferous tubules if the cell population is rich in spermatogonia and the percoll fractionation is useful in obtaining such a cell population.
Subject(s)
Spermatogonia/physiology , Spermatogonia/transplantation , Animals , Cell Fractionation , Green Fluorescent Proteins , Luminescent Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Seminiferous Tubules/cytology , Seminiferous Tubules/physiology , Spermatogenesis/physiology , Testis/cytologyABSTRACT
In this study, we carried out toxicogenomic analysis using in-house cDNA microarray to ascertain the long-term effects of neonatal exposure to genistein, also known as phytoestrogen, on testicular gene expression in mice. Male ICR mice, 1 day after birth, were exposed for 5 days to genistein (1000 microg/mouse/day) or diethylstilbestrol (DES) (50 microg/mouse/day), used as an example of a potent estrogen, and their testes were used when they were 12 weeks old. Since exposure to DES was been reported to induce morphological changes and alteration of gene expression in reproductive organs, DES was used as a positive control. Genistein-treated mice did not show any histological abnormalities or increased apoptotic cells in testes, but these abnormalities and increases were found in DES-treated mice. On the other hand, mRNA expression analysis using in-house cDNA microarray revealed that 2 down-regulated genes (GeneBank accession No. W49392 and AI430907) were detected in genistein-treated mouse testes. Moreover, real-time PCR analysis revealed that mRNAs of the W49392 gene, estrogen receptor alpha (ERalpha) and androgen receptor (AR), were down-regulated in the testes of both genistein-treated and DES-treated mice. In our present study using toxicogenomic analysis, long-term alteration in testicular mRNA expression, without morphological change in testes, was detected after neonatal treatment with genistein, indicating that the W49392 gene, in addition to ERalpha and AR, might be useful as a biological marker for predicting the effects of neonatal exposure to DES and genistein.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Genistein/toxicity , Growth Inhibitors/toxicity , Testis/drug effects , Animals , Animals, Newborn , Apoptosis/drug effects , Body Weight/drug effects , Diethylstilbestrol/toxicity , Estrogen Receptor alpha , Estrogens, Non-Steroidal/toxicity , In Situ Nick-End Labeling , Male , Mice , Mice, Inbred ICR , Oligonucleotide Array Sequence Analysis , Organ Size/drug effects , RNA, Messenger/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Testis/metabolism , Testis/pathologyABSTRACT
In this study, we investigated the effects of neonatal exposure to exogenous estrogen (diethylstilbestrol: DES, 17beta-estradiol: E2) on testicular gene expressions. Male C57BL/6J mice, 1 day after birth, were subcutaneously injected with DES or E2 (3 micrograms/mouse/day) for 5 days, and then they were raised for 8 weeks. In morphological observation of 8-week-old mice testes, spermatozoa were absent from many seminiferous tubules in DES-treated mice testes, but there was no change in E2-treated mice testes. Analysis of in-house cDNA microarray (mouse cDNA 889 genes) revealed that 17 genes were altered in DES-treated mice testes at 8 weeks of age, compared to each control. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) analysis of these genes revealed that some genes, which were changed in E2-treated testis, were the same as in DES-treated testis, whereas in other cases there was a difference between DES-treated and E2-treated testis. The present results suggest that each exogenous estrogenic compound has both a common gene expression change pattern and its own testicular gene expression change pattern. Mol. Reprod. Dev. 67: 19-25, 2004.
Subject(s)
Diethylstilbestrol/pharmacology , Estradiol/pharmacology , Estrogens, Non-Steroidal/pharmacology , Gene Expression Profiling , Testis/drug effects , Testis/physiology , Toxicogenetics , Animals , Animals, Newborn , Gene Expression Regulation, Developmental , Male , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Spermatozoa/cytology , Spermatozoa/metabolism , Testis/cytologyABSTRACT
We investigated whether neonatal exposure to diethylstilbestrol (DES) induces the alteration of mRNA expression in adult mouse epididymis, which plays an important role in sperm maturation. Using a cDNA subtraction method, we isolated 15 changed gene clones in neonatally DES-treated mouse epididymides, and we found a clone homologous with a disintegrin and metalloprotease (ADAM) 7 in the epididymis, as a suppressed gene, by means of neonatal DES treatment in 8-week-old mice. Indeed, it was confirmed by Northern blot analysis that the ADAM7 mRNA expression in the epididymis was at a lower level in neonatally DES-treated mice than in non-treated mice. Moreover, in situ hybridization analysis and real-time reverse transcription and polymerase chain reaction (real-time RT-PCR) revealed that ADAM7 expression was markedly reduced in the corpus region of the epididymis of DES-treated mice as compared with non-treated mice. Our results suggest that neonatal exposure to DES leads to the suppression of ADAM7 expression in the epididymis in the long term. ADAM7 gene expression might be a biological marker of fetal or neonatal exposure to estrogenic compounds, including endocrine disruptors.
Subject(s)
Diethylstilbestrol/pharmacology , Disintegrins/metabolism , Epididymis/metabolism , Estrogens, Non-Steroidal/pharmacology , Metalloendopeptidases/metabolism , Suppression, Genetic/genetics , Animals , Blotting, Northern , Disintegrins/genetics , Epididymis/drug effects , Male , Metalloendopeptidases/genetics , Mice , Mice, Inbred ICRABSTRACT
The ever-increasing presence of environmental toxicants and their disruptive effect on the reproductive systems of wildlife raises the question of possible damage to the human reproductive system. Using medicolegal data from over 20,000 Japanese men subjected to necropsy from 1948 to 1998, we investigated temporal changes in testis weight to find possible evidence of male reproductive disorders. We also carried out a histological examination of 747 testes collected from 1978 to 1998. Our detailed analyses of the development of testis weight over the past 50 years have revealed four clear phenomena: (i) the age at which testis weight reaches its maximum has decreased; (ii) peak weight showed a general increase until it started to decline in boys born after 1960; (iii) the decline-rate at which testis weight decreases after its peak has greatly accelerated; and (iv) the onset of increasing testis weight of boys has occurred at a progressively younger age. Our quantitative analyses of testis weight indicate the possibility of a subtle reproductive disorder in Japanese men, especially in those born after 1960. Together with the accelerated development and decline in testis weight during the past 50 years, the decline in peak weight might be indicative of a subtle interference of environmental toxicants with male reproductive organogenesis.
Subject(s)
Infertility, Male/pathology , Testis/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Humans , Infant , Infertility, Male/etiology , Male , Middle Aged , Organ Size , Reproduction/physiology , Spermatogenesis , Testis/physiology , Time FactorsABSTRACT
We examined the effect of neonatal exposure to diethylstilbestrol (DES) on mouse testicular gene expression, using in-house mouse fetus (day 14.5) cDNA microarrays. Newborn male ICR mice were exposed to DES (50 microg/mouse/day) from neonatal day 1 to 5. Differential expression was detected in 14 genes in 4-week-old (day 28) mouse testes by cDNA microarray analysis; 11 genes (AI035263, AU080565, AU080361, AU080678, AI131681, AU080631, AA986882, AI037066, AA986537, AI156816, and AI596237) were up-regulated and three genes (AI131656, AI118968, and AI117606) were down-regulated in DES-treated mouse testes. Higher expression levels of the former eight genes, out of the up-regulated genes picked-up by the microarray, were also confirmed by reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) analysis. However, the differential expression of other genes could not be confirmed. Real-time RT-PCR analysis also revealed that expression levels of the eight genes were still higher in DES-treated testes at 8 and 12 weeks of age. Our results suggest that cDNA microarray analysis is a useful method by which a large number of gene expressions are simultaneously detected and changes in gene expression are screened. In addition, our results suggest that these genes, whose expressions are changed in the testes of adult mice by fetal or neonatal exposure to exogenous chemicals, might be candidates for predictive biological markers.
