Kwan Hee Kim

Transgenerational Effects of Di-(2-ethylhexyl) Phthalate on Testicular Germ Cell Associations and Spermatogonial Stem Cells in Mice

ABSTRACT Recent evidence has linked human phthalate exposure to abnormal reproductive and hormonal effects. Phthalates are plasticizers that confer flexibility and transparency to plastics, but they readily contaminate the body and the environment. In this study, timed pregnant CD1 outbred mice were treated with di-(2-ethylhexyl) phthalate (DEHP) from Embryonic Day 7 (E7) to E14. The subsequent generation (F1) offspring were then bred to produce the F2, F3, and F4 offspring, without any further DEHP treatment. This exposure scheme disrupted testicular germ cell association and decreased sperm count and motility in F1 to F4 offspring. By spermatogonial transplantation techniques, the exposure scheme also disrupted spermatogonial stem cell (SSC) function of F3 offspring. The W/WV recipient testes transplanted with F3 offspring germ cells from the DEHP-treated group had a dramatically lower percentage of donor germ cell-derived spermatogenic recovery in seminiferous tubules when compared to the recipient testes transplanted with CD1 control germ cells. Further characterization showed that the major block of donor germ cell-derived spermatogenesis was before the appearance of undifferentiated spermatogonia. Interestingly, the testes transplanted with the F3 offspring germ cells from the DEHP-treated group, when regenerated, replicated testis morphology similar to that observed in the testes from the F1 to F3 offspring of the DEHP-treated group, suggesting that the germ cell disorganization phenotype originates from the stem cells of F3 offspring. In conclusion, embryonic exposure to DEHP was found to disrupt testicular germ cell organization and SSC function in a transgenerational manner.

Differential effects of phthalates on the testis and the liver.

Abstract Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) α is equally abundant in the liver and the testis, whereas PPARγ and retinoic acid receptor (RAR) α are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RARα and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPARα and PPARγ in Sertoli cells, but they decreased the nuclear localization of RARα, as previously shown. Both PPARα and PPARγ were in the nucleus and cytoplasm of liver cells, but RARα was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RARα, PPARα, and PPARγ in these organs.

Expression of germ cell nuclear factor (GCNF/RTR) during spermatogenesis.

Germ cell nuclear factor (GCNF/RTR), a novel orphan receptor in the nuclear receptor superfamily of ligand‐activated transcription factors, is expressed predominantly in developing germ cells. In several mammalian species two GCNF/RTR mRNAs are present in the testis, with the smaller 2.3‐kb transcript generally expressed at higher levels than the larger 7.4‐ or 8.0‐kb transcript. In both the mouse and rat, the 2.3‐ and 7.4‐kb GCNF/RTR transcripts were detected in isolated spermatogenic cells, but not in Sertoli cells. Expression of these transcripts is differentially regulated, with the larger 7.4‐kb mRNA appearing earlier during testicular development. The major 2.3‐kb transcript is expressed predominantly in round spermatids in the mouse and rat. In situ hybridization studies in the rat demonstrated that GCNF/RTR transcripts reach maximal steady‐state levels in round spermatids at stages VII and VIII of the spermatogenic cycle, and then decline abruptly as spermatids begin to elongate. RNase protection assays were used to predict the 3′ termination site of the 2.3‐kb transcript. An alternative polyadenylation signal (AGUAAA) was identified just upstream of this termination site. These studies suggest that GCNF/RTR may regulate transcription during spermatogenesis, particularly in round spermatids just prior to the initiation of nuclear elongation and condensation. Mol. Reprod. Dev. 50:93–102, 1998.

Effects of Mono-(2-Ethylhexyl) Phthalate on Fetal and Neonatal Rat Testis Organ Cultures

Abstract Di-(2-ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to cause reproductive toxicity in both developing and adult animals. In this study, we used organ cultures of fetal and neonatal rat testes to assess the in vitro effect of MEHP on seminiferous cord formation in Embryonic Day 13 (E13) testes and on the development of E18 and Postnatal Day 3 (P3) testes. Interestingly, MEHP had no effect on cord formation in the organ cultures of E13 testes, indicating that it has no effect on sexual differentiation of the indifferent gonad to testis. Consistently, the expression of a Sertoli cell-specific protein, mullerian inhibiting substance (MIS), or the number of gonocytes did not change in E13 testes after MEHP treatment. In contrast, MEHP decreased the levels of MIS and GATA-4 proteins in Sertoli cells and impaired Sertoli cell proliferation in the organ cultures of E18 and P3 testes. These results suggest that MEHP negatively influences proliferation and differentiation of Sertoli cells in both fetal and neonatal testes. In addition, MEHP treatment did not alter the number of gonocytes in E18 testes, whereas the number of gonocytes in P3 testes decreased in a dose-dependent manner, apparently due to enhanced apoptosis. These results suggest that MEHP adversely affects the gonocytes, which are mitotically active and undergoing migration and differentiation in neonatal testes, but it has no effect on fetal gonocytes that are mitotically quiescent.

Ligand-Dependent Regulation of Retinoic Acid Receptor α in Rat Testis: In Vivo Response to Depletion and Repletion of Vitamin A

Male animals are sterile due to testicular degeneration in the absence of retinoic acid (RA) or functional retinoic acid receptor-alpha (RAR alpha). This degeneration can be reversed by injecting retinol, a precursor of RA, into vitamin A-deficient (VAD) rats. To determine the relationship between this ligand-dependent testicular degeneration and regeneration and the expression levels of RAR alpha messenger RNA and protein, testes were depleted and then replenished with retinol in vivo. Results showed that RAR alpha messenger RNA and protein levels declined to VAD amounts after 7 weeks on a VAD diet. This decline was due to decreased RAR alpha levels in early meiotic spermatocytes and the loss of advanced germ cells. Interestingly, the advanced germ cells still contained RAR alpha, but the protein was primarily cytoplasmic instead of nuclear, indicating inactivity as a transcription factor. In VAD testis, RAR alpha levels were low and then increased primarily in Sertoli cells after retinol replenishment. TUNEL analyses showed that most germ cells at the basal aspect of seminiferous tubules were undergoing apoptosis during degeneration. These results indicate that RAR alpha is either down-regulated or inactivated in RA-deficient testis and coincident with that, testes degenerate by apoptosis or selective loss of germ cells.

Immunoprotective Properties of Primary Sertoli Cells in Mice: Potential Functional Pathways that Confer Immune Privilege

ABSTRACT Primary Sertoli cells isolated from mouse testes survive when transplanted across immunological barriers and protect cotransplanted allogeneic and xenogeneic cells from rejection in rodent models. In contrast, the mouse Sertoli cell line (MSC-1) lacks immunoprotective properties associated with primary Sertoli cells. In this study, enriched primary Sertoli cells or MSC-1 cells were transplanted as allografts into the renal subcapsular area of naive BALB/c mice, and their survival in graft sites was compared. While Sertoli cells were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells were rejected between 11 and 14 days, with 0% graft survival at 20 days posttransplantation. Nonetheless, the mechanism for primary Sertoli cell survival and immunoprotection remains unresolved. To identify immune factors or functional pathways potentially responsible for immune privilege, gene expression profiles of enriched primary Sertoli cells were compared with those of MSC-1 cells. Microarray analysis identified 2369 genes in enriched primary Sertoli cells that were differentially expressed at ±4-fold or higher levels than in MSC-1 cells. Ontological analyses identified multiple immune pathways, which were used to generate a list of 340 immune-related genes. Three functions were identified in primary Sertoli cells as potentially important for establishing immune privilege: suppression of inflammation by specific cytokines and prostanoid molecules, slowing of leukocyte migration by controlled cell junctions and actin polymerization, and inhibition of complement activation and membrane-associated cell lysis. These results increase our understanding of testicular immune privilege and, in the long-term, could lead to improvements in transplantation success.

Potential Functions of Retinoic Acid Receptor A in Sertoli Cells and Germ Cells During Spermatogenesis

Abstract: Elucidation of the retinoid signaling circuitry in the testis is critical to understanding how male germ cells develop to spermatozoa. Retinoic acid receptor A protein (RARA) is an essential mediator of retinoid signaling in the testis, as shown by a sterility phenotype observed for retinoic acid receptor A gene (Rara) knockout male mice. The seminiferous tubules of Rara knockout mice showed varying degrees of germ‐cell degeneration. A dramatic increase in apoptosis of early meiotic prophase spermatocytes was observed in these tubules compared to the wild‐type tubules. Germ‐cell loss was dependent on the stages of the spermatogenic cycle: germ‐cell loss was negligible in stages I–V, but severe after stages VIII and IX of the spermatogenic cycle. Using spermatogonial transplantation, the individual function of RARA in Sertoli cells or germ cells was determined. The wild‐type donor germ cells, transplanted into Rara knockout testes, colonized and proliferated in the RARA‐deficient microenvironment. The donor‐derived cells were mostly early meiotic prophase spermatocytes, with few more advanced germ cells detected. Conversely, when Rara‐deficient germ cells were injected into the microenvironment that express RARA, establishment of donor‐derived germ‐cell colonies was rare, but remarkably, once colonized, Rara‐deficient germ cells progressed normally through spermatogenesis. These results together suggest that RARA may function in Sertoli cells to promote the survival and development of early meiotic prophase spermatocytes, whereas RARA in germ cells functions to increase the proliferation and differentiation of spermatogonia, prior to meiotic prophase.

Expression of Receptors during the Cycle of the Seminiferous Epitheliuma

The data presented in this manuscript are all based on some inferences drawn from past experimental observations. The first is that the synchronized testis model is representative of the normal testis. Support for this premise comes from the studies on SGP-2, SGP-1 and transferrin where results using in situ hybridization and Northern blots are similar for normal and for synchronized testes. The second inference is that normalizing all of the data to the relative levels of SGP-1 mRNA adjusts for the possible differential loading of mRNA samples. The logic of this practice is based on the observation obtained using in situ hybridization that SGP-1 mRNA levels did not change across the cycle. The third assumption was that total mRNA levels do not change greatly across the cycle. Wholesale changes in testicular mRNA such as the doubling of all of the mRNA transcripts per testis would not be accounted for by these studies. We feel that this is an unlikely complication because of the strong correlation between much of the data and the known biology. In addition, there is a strong correlation between our data on the FSHR mRNA and the binding data for FSH obtained by another laboratory and different techniques. The available data in the literature reveals that most of the Sertoli cell products which change in relative concentrations during the cycle of the seminiferous epithelium appear to have a maximum in either stages VII or IX or in stages XIII to III. Thus, the Sertoli cells in the cycle could be described has having two different functional modes. In mode A maximal levels of mRNA for a specific Sertoli cell product are roughly found in stages VII-IX and in mode B the maximal levels are found in stages XIII to III (Fig. 5). The distribution of the receptor mRNA and ABP mRNA can also be described in terms of these two modes of Sertoli cell function. Transferrin receptors, retinoic acid receptors, and androgen-binding protein appear to fall into mode A while FSH and androgen receptors fall into mode B (Table 1). Products which have antithetical functions such as FSH and inhibin or cystatin and cathepsin L are found in different modes. We propose that most of the actions of the Sertoli cell during the cycle can be specified by the dual modes described above rather than by an infinite number of operational modes.(ABSTRACT TRUNCATED AT 400 WORDS)

Retinoic Acid Inhibits Rat XY Gonad Development by Blocking Mesonephric Cell Migration and Decreasing the Number of Gonocytes

Abstract Vitamin A (also called retinol) and its derivatives, retinoic acids (RAs), are required for postnatal testicular function. Abnormal spermatogenesis is observed in rodents on vitamin A-deficient diets and in retinoic acid receptor α (RARα) knockout mice. In contrast, RA has an inhibitory effect on the XY gonad development in embryos. To characterize this inhibitory effect of RA, we investigated the cellular events that are required for the XY gonad development, including cell migration from the adjacent mesonephros into the gonad, fetal Sertoli cell differentiation, and survival of gonocytes. In organ cultures of Embryonic Day 13 (E13) XY gonads from rats, all-trans-retinoic acid (tRA) inhibited mesonephric cell migration into the gonad. Moreover, treatment with tRA decreased the expression of Müllerian-inhibiting substance in Sertoli cells and dramatically reduced the number of gonocytes. Increased apoptosis was detected in the XY gonads cultured with tRA, suggesting that the loss of gonocytes could be due to increased apoptosis. In addition, Am580, a synthetic compound that exhibits RARα-specific agonistic properties, mimicked the inhibitory effects of tRA on the XY gonad development including mesonephric cell migration and gonocyte survival. Conversely, a RARα-selective antagonist, Ro 41-5253, suppressed the inhibitory ability of tRA on the XY gonad development. These results suggest that retinoic acid acting through RARα negatively affects fetal Sertoli cell differentiation and gonocyte survival and blocks the migration of mesonephric cells, thereby leading to inhibition of the XY gonad development.

Small Ubiquitin-Like Modifier-2 Modification of Retinoic Acid Receptor-α Regulates Its Subcellular Localization and Transcriptional Activity

The retinoic acid receptor-alpha (Rara) gene is critical for germ cell development in the testis, as demonstrated by infertile Rara knockout male mice. The encoded protein for Rara (RARA) is expressed in both Sertoli cells and germ cells, but it is not always in the nucleus. Previously, all-trans retinoic acid (ATRA) was shown to increase the nuclear localization and transcriptional activity of RARA in Sertoli cells. Here, we identified a small ubiquitin-like modifier-2 (SUMO-2) modification as a novel posttranslational regulatory mechanism controlling the ATRA-dependent RARA subcellular localization and transcription. ATRA increased the SUMO-2 modification of RARA. In the presence of ATRA, lysine 166 (K166) and K171 of RARA were modified at a physiological concentration of SUMO-2, whereas in the absence of ATRA, K399 was the only site that was modified, but at a higher SUMO-2 concentration. However, K399 was critical for ATRA-controlled nuclear trafficking of RARA. In the presence of ATRA, a K399 mutation to arginine resulted in the cytoplasmic localization of K399R mutant, indicating that K166 and K171 sumoylations were inhibitory to nuclear localization. This may be due to SUMO/sentrin-specific peptidase 6 (SENP6) not being able to bind K399R mutant to desumoylate K166 and K171 in Sertoli cells, whereas it can bind RARA with intact K399. On the other hand, functional K166 and K171 sites for sumoylation were required for a full transcriptional activity, when K399 was intact. These results together suggest that both K166 and K171 sumoylation and desumoylation are critical for optimal RARA function.