Germ Cell Detachment Research Articles

Overexpression of PD-L1 causes germ cells to slough from mouse seminiferous tubules via the PD-L1/PD-L1 interaction.

Spermatogenesis is a cyclical process in which different generations of spermatids undergo a series of developmental steps at a fixed time and finally produce spermatids. Here, we report that overexpression of PD‐L1 (B7 homolog1) in the testis causes sperm developmental disorders and infertility in male mice, with severe malformation and sloughing during spermatid development, characterized by disorganized and collapsed seminiferous epithelium structure. PD‐L1 needs to be simultaneously expressed on Sertoli cells and spermatogonia to cause spermatogenesis failure. After that, we excluded the influence of factors such as the PD‐L1 receptor and humoral regulation, confirming that PD‐L1 has an intrinsic function to interact with PD‐L1. Studies have shown that PD‐L1 not only serves as a ligand but also plays a receptor‐like role in signal transduction. PD‐L1 interacts with PD‐L1 to affect the adhesive function of germ cells, causing malformation and spermatid sloughing. Taken together, these results indicate that PD‐L1 can interact with PD‐L1 to cause germ cell detachment and male infertility.

Aluminum exposure promotes histopathological and pro-oxidant damage to the prostate and gonads of male and female adult gerbils

Since the industrial revolution, all living beings have become susceptible to numerous sources of aluminum (Al) exposure. In addition to causing proven toxicity in many organs and systems, Al can also have estrogenic activity when absorbed by the body. The reproductive organs are commonly affected by environmental pollutants with estrogenic activity, but little is known about the effects of Al on the prostate and gonads. Therefore, the aim of this study was to evaluate the effects of subchronic Al exposure on the prostate and gonads of male and female adult gerbils. After 30 days of oral exposure to aluminum chloride (10 mg/kg/day), the animals were euthanized and the organs processed for cytochemical, ultrastructural, and biochemical assays. Ventral male prostates exposed to Al became hyperplastic and showed signs of cell aging. In addition, the male prostate showed decreased catalase (CAT) and superoxide dismutase (SOD) activity. The female prostate was structurally more affected than the ventral male prostate, since it presented hyperplasia and punctual foci of inflammation and prostatic intraepithelial neoplasia. However, CAT and SOD activities did not change in this gland. In the testis, Al promoted immature germ cell detachment and degeneration, as well as reduced CAT activity. In the ovaries, Al caused reduction in folliculogenesis and decreased SOD activity. Together, these results indicate that Al is toxic to the prostate and gonads of adult gerbils and that continuous exposure to this metal can impair the fertility of individuals of both sexes.

Subacute Testicular Toxicity to Cadmium Exposure Intraperitoneally and Orally.

The toxic effects of cadmium (Cd) on reproductive parameters are widely described in the literature. Experimental models often make use of the intraperitoneal route (i.p.), although human intoxication occurs preferentially by the oral route and can be continuous. However, little is known about the effect of Cd administration routes on the testicular structure. Thus, this study investigated the testicular impact of Cd exposure comparing both i.p. and oral routes, both single dose (SD), in addition to the oral route in fractional doses (FD). Swiss adult male mice received CdCl2 1.5 mg/kg i.p., 30 mg/kg oral SD, and 4.28 mg/kg oral FD for 7 consecutive days. The Cd bioaccumulation was observed in all routes, mainly in the oral FD route. The concentrations of testicular Ca and Cu decreased in all animals exposed to Cd, while Zn and Mn decreased only in the i.p. route. Testicular SOD activity was reduced in both routes of oral administration, while CAT increased in the i.p. route, and GST increased in all animals exposed to Cd. Changes in the tubular parameters and cell viability were observed in both routes of Cd administration but were more intense in the oral route, mainly in the FD. Serum testosterone concentration was reduced in both routes of oral administration. Tubular damage, such as the vacuolization of the seminiferous epithelium, germ cell detachment, and seminiferous tubule degeneration, occurred in all groups exposed to Cd. Therefore, the oral Cd administration presented greater potential to promote testicular damage, mainly when the metal was given in a fractionated way.

Protective effect of Zingerone against mouse testicular damage induced by zinc oxide nanoparticles.

The purpose of the present study was to evaluate the effect of Zingerone (Zing) on zinc oxide nanoparticle (ZNP)-induced spermatogenesis defects in mice. To this end, 50mg/kg of ZNP was prescribed to the mice as an intoxicated group for 35days. In protection groups, Zing (10, 20, and 40mg/kg) was given prior to ZNP treatment for seven days and then co-administration of ZNP for 35days. Epididymal sperm parameters, testicular histology, Johnsen's scoring, morphometric parameters, TUNEL staining, oxidative stress, and serum testosterone level were evaluated for determining ZNP and Zing effects on the mouse testicles. Effects of Zing and ZNP on the viability of mouse Leydig (TM3) and mouse Sertoli (TM4) cell lines were also done. Testicular weights, testosterone levels, sperm quality, morphometric parameters, Johnsen's score, and superoxide dismutase (SOD) and catalase (CAT) activities were significantly decreased in ZNP-intoxicated mice, while apoptotic index, Malondialdehyde (MDA) content, and histological features, including epithelial vacuolization, sloughing, and germ cell detachment, were improved significantly in ZNP-intoxicated mice. Pretreatment with 20 or 40mg/kg Zing significantly reduced the histological criteria, increased morphometric parameters, enhanced testosterone levels, attenuated apoptotic index, improved sperm quality, and reversed oxidative stress by reducing the level of MDA and incrementing the activity level of SOD and CAT enzymes. Zing dose-dependently enhanced the viability of ZNP-treated TM3 and TM4 cells in comparison with only ZNP-exposed cells. According to the results of our study, Zing effectively prevented the defects in spermatogenesis among mice treated by ZNP.

Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC1

Our previous studies showed that the prototypical testicular toxic phthalate monoester, mono-(2-ethylhexyl) phthalate (MEHP), suppresses Sertoli cell TIMP2 levels and allows for the activation of MMP2 in seminiferous epithelium. Activation of MMP2 is important for triggering germ cell apoptosis and instigating germ cell detachment from Sertoli cells. These novel findings led us to examine the transcriptional regulation of the Timp2 gene that accounts for the decrease in Sertoli cell TIMP2 levels following MEHP exposure. Sequential deletion of the Timp2 5'-upstream activating sequence (1200 bp) was used to survey transcriptional activation in the Timp2 promoter region in response to MEHP. Results indicate that under control conditions in rat Sertoli cells, CCAAT enhancer-binding protein alpha (CEBPA) acts as a transactivator to initiate Timp2 gene transcription, and its action is deactivated by exposure to MEHP. By contrast, MYC protein acts as an inhibitor of Timp2 gene transcription, and its activity is increased after MEHP treatment. Addition of follicle-stimulating hormone (FSH) to cells causes translocation of CEBPA into the Sertoli cell nucleus and rescues MEHP-suppressed TIMP2 levels. Down-regulation of TIMP2 expression by MEHP exposure is blocked by forskolin (a cAMP-elevating agent), suggesting that the decrease in Sertoli cell TIMP2 expression following MEHP exposure is cAMP-dependent. Taken together, these data indicate that MEHP both disrupts the FSH-stimulated cAMP signaling pathway and activates the inhibitory signaling mediated by MYC protein, to ultimately account for the cellular mechanism underlying the decreased expression of TIMP2 in Sertoli cells.

Differential Expression of Connexin 43 in Adult Pig Testes During Normal Spermatogenic Cycle and After Flutamide Treatment

Evidence is mounting that the foetal and neonatal period of reproductive tract development is highly sensitive to hormonal disruption induced by various endocrine active compounds. Thus, we asked whether androgen withdrawal caused by prenatal (GD20, GD80) or neonatal (PD2) exposure to an anti-androgen flutamide alters Cx43 gene expression and may induce delayed effects on morphology and function of adult pig testes. Flutamide was given in five doses (50 mg/kg bw). Our histological analysis and TUNEL staining revealed varying degrees of seminiferous tubules abnormalities in all experimental pigs. Testes of pigs exposed to flutamide in utero exhibited moderate alterations of the spermatogenic process, whereas those of exposed neonatally were severely impaired. The most striking effects were spermatogenic arrest, germ cell detachment and a statistically significant increase in the frequency of germ cell apoptosis (p<0.01). Moreover, all pigs exposed to flutamide displayed Leydig cell hyperplasia. Because the network of cell-cell communication provided by gap junction channels plays an essential role in the regulation and maintenance of spermatogenesis, the physiological significance of Cx43-based gap junctions with regards to the gonadal impairment was evaluated by analysis of its expression using immunohistochemical, Western blot and qRT-PCR approaches. Significantly, lower Cx43 expression was found when flutamide was administered neonatally, which has coincided with severe disruption of spermatogenesis. Our data suggest that neonatal exposure to flutamide induces long-term effects on the spermatogenic capacity of the pig testis through alterations of Cx43-mediated intercellular communication and permanent alteration of both Sertoli and Leydig cell functions.

Mono-(2-Ethylhexyl) Phthalate-Induced Disruption of Junctional Complexes in the Seminiferous Epithelium of the Rodent Testis Is Mediated by MMP21

Tight junctions between Sertoli cells of the testicular seminiferous epithelium establishes the blood-testis barrier (BTB) and creates a specialized adluminal microenvironment above the BTB that is required for the development of the germ cells that reside there. Actin filament-based anchoring junctions between Sertoli cells and germ cells are important for maintaining close physical contact between these cells as well as regulating the release of mature spermatids into the lumen. Previously, we reported that Sertoli cell injury in rodents after mono-(2-ethylhexyl) phthalate (MEHP) exposure results in the activation of matrix metalloproteinase 2 (MMP2) and increases the sensitivity of germ cells to undergo apoptosis. A disruption in the physical association between Sertoli cells and germ cells and premature loss of germ cells from the seminiferous epithelium has been widely described after phthalate treatment. In this study, we investigate the functional participation of MMP2 in the mechanism underlying MEHP-induced disruption of junction complexes and the resultant loss of germ cells. Exposure of C57BL/6J mice to MEHP (1 g/kg, oral gavage) decreased the expression of occludin in the tight junctions between Sertoli cells and caused gaps between adjacent Sertoli cells as observed by transmission electron microscopy. A reduced expression of laminin-gamma3 and beta1-integrin in apical ectoplasmic specializations between Sertoli cells and germ cells in a time-dependent manner was also observed. Treatment with specific MMP2 inhibitors (TIMP2 and SB-3CT) both in vitro and in vivo significantly suppressed MEHP-induced germ cell sloughing and changes in the expression of these junctional proteins, indicating that MMP-2 plays a primary role in this process. Furthermore, the detachment of germ cells from Sertoli cells appears to be independent of the apoptotic signaling process since MEHP-induced germ cell detachment from Sertoli cells could not be prevented by the addition of a pan-caspase inhibitor (z-VAD-FMK).

Effect of argemone oil and argemone alkaloid, sanguinarine on Sertoli–germ cell coculture

Several incidences of reduction in the fertility (sperm count) have been reported in India and worldwide as well. Adulteration of food and consumption of adulterated mustard oil with argemone oil (AO) are presumed to be the factors for reduction in sperm count. In the present study we have studied the exfoliation of germ cells from Sertoli cell, its viability after detachment, cytotoxicity and execution of apoptosis via mitochondrial pathway for different concentration of AO, argemone alkaloid (AA) and its major constituent sanguinarine (SA). A dose dependent increase in germ cell detachment and decrease in viability of detached germ cells were observed (P<0.05). A significant inhibition was observed via 3-(4,5-dimethylthiazol-2-yl)-2,5-dipehyl tetrazolium bromide (MTT) assay in the proliferative activity of germ cell and leakage of cytosolic enzyme was observed via Lactate dehydrogenase(LDH) assay (P<0.05). A time and dose dependent inhibition of mitochondrial membrane potential was observed (P<0.05). Treatment of Sertoli-germ cells with the lowest concentration of AO/AA and SA for 24h resulted in 5.2- , 4.4- and 3.6-fold increase in the percentage of early apoptotic cells, respectively. This increase was enhanced to 8.3, 4.75 and 5.81-fold, respectively at 48 h in detached germ cells undergoing early apoptosis. These results suggest that alterations in germ cell apoptosis by a disruption in contact mediated communication between the Sertoli cells and germ cells, may subsequently lead to testicular impairment.

Hybrid GPCR/Cadherin (Celsr) Proteins in Rat Testis Are Expressed With Cell Type Specificity and Exhibit Differential Sertoli Cell—Germ Cell Adhesion Activity

Spermatogenesis requires Sertoli cell-germ cell adhesion for germ cell survival and maturation. Cadherins are a diverse superfamily of adhesion proteins; structurally unique members of this superfamily (celsr cadherins) are hybrid molecules containing extracellular cadherin repeats connected to a G protein-coupled receptor transmembrane motif. Here we demonstrate postnatal testicular mRNA expression of the 3 celsr paralogs (celsr1, celsr2, and celsr3), protein localization of celsr2 and celsr3, and functional analysis of celsr2 adhesion activity in primary Sertoli cell-germ cell co-cultures. Evaluation of celsr mRNA levels during a postnatal time course indicated that celsr1 and celsr2 were Sertoli cell and/or early-stage germ cell products, whereas celsr3 was expressed in later-stage germ cells. Cell type-specific expression was verified using the Sertoli cell line 93RS2, where celsr1 and celsr2 mRNA, but not celsr3, were detected. Immunostaining of testicular cryosections resulted in celsr2 protein localization to a spokelike pattern in the basal seminiferous epithelium and punctate figures in the apical epithelium, consistent with both Sertoli cell and germ cell expression. Celsr3 localized to punctate structures in the adluminal epithelium from postnatal day 40, consistent with elongate spermatid expression. The subcellular localization of celsr2 was examined further to define its localization in Sertoli cells and germ cells. Celsr2 localized to the Golgi complex in Sertoli cells and germ cells. In addition, germ cell celsr2 localized to a rab7-positive structure, which may be an endocytic compartment. Neither celsr2 nor celsr3 immunostaining was present at classic cadherin-based adhesion junctions. Nonetheless, the addition of a recombinant celsr2 protein fragment consisting of extracellular cadherin domains 4 through 8 to Sertoli cell-germ cell co-cultures resulted in germ cell detachment from Sertoli cells. Collectively, these data indicate that celsr cadherins have a cell type-specific expression pattern, and celsr2 may mediate Sertoli cell-germ cell adhesion outside of classic cadherin-based adhesion junctions.

Cytotoxic effect of endosulfan on rat Sertoli-germ cell coculture

Endosulfan induced testicular impairment has been reported in vivo in rats. The present study was conducted to evaluate the cytotoxic effects of endosulfan in vitro employing rat testicular cells in culture, that is Sertoli–germ cell coculture. Cytotoxic changes induced by endosulfan (0, 2, 20, 40, and 80 μM) in mixed cultures of Sertoli and germ cells were seen after 24 and 48 h of treatment. Endosulfan led to an increase in germ cell detachment from the Sertoli cell monolayer in a dose dependent manner. A loss in the viability of detached cells was observed in all treated groups. Substantial leakage of the cytosolic enzyme lactate dehydrogenase was observed in the medium after 24 and 48 h endosulfan treatment at concentrations of 20 to 80 μM. The extent of toxicity was greater after 48 h of treatment.

Apoptosis precedes detachment of germ cells from the seminiferous epithelium after hormone suppression by short-term oestradiol treatment of rats.

Spermatogenesis is a highly synchronized process in which FSH and testosterone are considered the major regulators. Nevertheless, the mechanism by which these hormones act on germ cells is unclear. Cell adhesion has been proved to play an essential role in the regulation of programmed cell death in epithelial cells and it is now known that FSH and testosterone withdrawal results in the triggering of apoptosis as well as germ cell detachment from the seminiferous epithelium. Therefore, it seemed important to investigate whether the triggering of apoptosis in germ cells by experimental hormone suppression occurred as a result of their previous detachment from the epithelium. To achieve this goal, adult male rats were injected with 50 micrograms oestradiol benzoate for 1, 2, 3, 4, 5 or 10 days to suppress gonadotrophin secretion and thus intratesticular levels of testosterone. Germ cell apoptosis was assessed in testes from these animals by in situ 3' end-labelling of DNA fragments and quantified in seminiferous tubule sections at stages VII-VIII. Serial sections throughout the epididymides from these animals were analysed to search for immature germ cells detached from the epithelium. These cells were scored and quantified in non-consecutive randomly selected epididymal sections. Our data indicate that the triggering of apoptosis in germ cells precedes germ cell detachment, suggesting that detachment of germ cells from the epithelium, occurring after hormone suppression, is not necessary for germ cell apoptosis.

The modulating effects of antioxidants in rat embryos and Sertoli cells in culture.

The male and female reproductive systems are targets for the toxicity of a wide range of compounds. There is a paucity of information regarding the modulating effects of antioxidants in such systems. Enzymically generated oxygen radicals have been shown to be toxic and/or mutagenic in a variety of in vitro test systems. It is known that vitamins C and E can modify responses in such systems. Malformations and growth reductions have been observed in whole rat embryo cultures in this laboratory after treatment with the oxygen radical generating system of xanthine/xanthine oxidase. Groups of 9.5-day-old rat embryos were treated with this system with or without vitamin C or E. Vitamin C at the doses given totally abolished neural suture defects while vitamin E only partially did so. Vitamins C and E administered alone had no effect on the embryos. Germ cell detachment has been shown to occur in mixed cultures of Sertoli and germ cells in response to some known in vivo testicular toxins. Such cultures were also treated with the oxygen radical generating system of xanthine/xanthine oxidase. There was an increase in germ cell detachment with this treatment which was reduced by vitamin C but not by vitamin E at the doses administered. These findings would suggest that vitamin supplementation could protect somatic cells of reproductive systems against toxins that act through oxygen radical mechanisms.

Effects of thallium on primary cultures of testicular cells.

The objective of this in vitro study was to examine the response of mixed cultures of Sertoli and germ cells to treatment with thallium (Tl) at the range of concentrations that, in previous studies, was shown in vivo to affect reproduction. Cultures were prepared from the testis of Sprague-Dawley rats. Cultures containing approximately 3.75 x 10(6) cells/ml were treated with Tl concentrations corresponding to 35, 7, and 1.4 micrograms Tl/g testis, estimated from protein content of cultures. Observations at 24, 48, and 72 h after treatment showed a significant release of germ cells into the culture medium that was both concentration and time dependent. Cultures treated with 35 micrograms Tl/g testis showed a threefold increase in germ-cell detachment compared with controls after only 24 h of exposure. As the treatment time increased to 48 h of exposure, even cultures exposed at the lowest Tl concentration (1.4 micrograms Tl/g testis) showed significant loss of germ cells. After 48 h, cultures exposed to 7 micrograms Tl/g testis exhibited a 2.5-fold increase in germ-cell detachment, and those exposed to 35 micrograms Tl/g testis exhibited a 10-fold increase over controls. Morphological investigations of cell cultures showed evident loss of germ cells with significant reduction in prepachytene and pachytene spermatocytes and changes in the shape of Sertoli cells. These results are in agreement with in vivo studies, in which thallium treatment at comparable exposure levels manifested its earliest toxic testicular effects in Sertoli and germ cells. They also demonstrate the usefulness of this in vitro culture technique to assess toxic testicular damage rapidly.

The in vitro effects of four isomers of dinitrotoluene on rat Sertoli and Sertoli-germ cell cocultures: Germ cell detachment and lactate and pyruvate production

The present study was undertaken to evaluate the in vitro effects of four isomers of a known testicular toxicant, dinitroluene (DNT). Rat Sertoli or Sertoli-germ cell cocultures were treated, after 3 days in culture, with DNT isomers (0.01 to 100 μ m) or 1,3-dinitrobenzene (1,3-DNB) for 24 hr. Cellular morphology, germ cell detachment (GCD) and lactate pyruvate production were used as sensitive effect markers of in vitro toxicity. Morphologically the Sertoli cell monolayer remained intact 24 hr after exposure to DMSO, 1,3-DNB, or DNT isomers. Some apparent cytotoxicity was observed at 100 μ m 3,4-DNT: the monolayer was disrupted with extensive vacuolation of the Sertoli cells. Cocultures treated with concentrations of 50 μ m DNT isomers closely resembled cells treated with 100 μ m 1,3-DNB. GCD increased in a dose-dependent manner (0.01 and 10 μ m DNT isomers) increasing between 2- and 10-fold over control. Both lactate and pyruvate production increased with rising concentrations of DNT isomers. The most sensitive effect was seen with 3,4-DNT (10 to 25 μ m). In the case of 2,6-DNT, despite increases in GCD and lactate production, only a minimal increase in pyruvate was demonstrated. Overall, the ratio of lactate to pyruvate production declined with increasing doses of DNT. These results indicate that the four isomers of DNT directly affected Sertoli cell morphology and function, effects comparable to those seen with the Sertoli cell toxicant 1,3-DNB. Further, the data support the hypothesis that DNT may be a Sertoli cell toxicant.

Modulation of m-dinitrobenzene and m-nitrosonitrobenzene toxicity in rat Sertoli-germ cell cocultures

Previous work has shown that m-dinitrobenzene is a testicular toxicant in rats in vivo, and in vitro produces comparable morphological changes in rat testicular Sertoli-germ cell cocultures. m-dinitrobenzene is metabolized both in vivo and in the in vitro system to m-nitroaniline and m-nitroacetanilide. These metabolites do not provoke testicular toxicity in vivo or in vitro. We have therefore proposed a pathway for the metabolism of m-dinitrobenzene to m-nitroaniline and m-nitroacetanilide, which involved the intermediate m-nitrosonitrobenzene(1-nitroso-3-nitrobenzene, NNB). When tested, m-nitrosonitrobenzene, at equimolar doses to m-dinitrobenzene, produced similar morphological changes in the culture system to those exhibited by m-dinitrobenzene. However, m-nitrosonitrobenzene produced a greater toxicity than did m-dinitrobenzene (as measured by germ cell detachment). When the intracellular thiol levels were reduced in the cocultures pretreated with diethyl maleate, the toxicity of both m-dinitrobenzene and m-nitrosonitrobenzene was enhanced. In contrast, pretreatment of cocultures with agents known to increase cellular thiol (cysteamine) or scavenge reactive intermediates (cysteamine or ascorbate) reduced the toxicity of m-dinitrobenzene and m-nitrosonitrobenzene. We propose that m-dinitrobenzene requires metabolic activation before it can exert its toxicity to Sertoli cells, and it appears that the toxic species is m-nitrosonitrobenzene or a further metabolite of m-nitrosonitrobenzene.

Toxicity and metabolism of 1,3-dinitrobenzene in rat testicular cell cultures

The testicular toxicity of 1,3-dinitrobenzene (DNB) has been modelled in primary cultures of rat testis. The morphological response obtained in vitro following direct addition of the compound to the cultures was analogous to that seen in vivo. This was characterized by Sertoli cell vacuolation and germ cell detachment. The effect could be quantified using germ cell exfoliation into the culture medium, with a significant response occurring at toxicologically relevant concentrations of DNB (5 × 10 −6 m and above). Both Sertoli-germ cell co-cultures and Sertoli cell cultures were shown to be capable of xenobiotic metabolism, with nitroreduction of DNB being the predominant route. It is postulated that DNB or a Sertoli cell metabolite (probably an intermediate of nitroreduction produced in situ) is responsible for the testicular damage observed following administration of the compound in vivo.

Testicular toxicity of 2-methoxyacetaldehyde, a possible metabolite of ethylene glycol monomethyl ether, in the rat

2-Methoxyacetaldehyde (MALD) was shown to produce specific cellular toxicity to pachytene spermatocytes in mixed testicular cell cultures as evidenced by morphological changes to these cells, an increase in germ-cell detachment and leakage of the pachytene spermatocyte marker enzyme lactate dehydrogenase-X. These effects occurred at concentrations where the known testicular toxicant, 2-methoxyacetic acid (MAA) was without effect (0.2 and 0.5 mM). In vivo, MALD also produced the characteristic testicular lesion reported previously for MAA and its parent compound ethylene glycol monomethyl ether (EGME). It is likely that MALD plays an important role in EGME-induced testicular toxicity.

Aspects of the testicular toxicity of phthalate esters.

Di(2-ethylhexyl) phthalate (DEHP) produced seminiferous tubular atrophy and reductions in seminal vesicle and prostate weight in 4-week-old, but not in 15-week-old rats. Di-n-pentyl phthalate (DPP) did produce atrophy in the older rats but this developed more slowly than in young animals. Coadministration of testosterone or gonadotrophins did not protect against phthalate-induced testicular toxicity but did partly reverse the depression of seminal vesicle and prostate weight. Secretion of seminiferous tubule fluid and androgen binding protein by the Sertoli cells was markedly suppressed within 1 hr of a dose of DPP or mono-2-ethylhexyl phthalate (MEHP) in immature rats. This occurred less rapidly in mature rats. [14C]Mono-n-pentyl phthalate and [14C]MEHP penetrated the blood testis barrier only to a very limited extent. These findings and the early morphological changes in the Sertoli cells produced by DPP suggest that phthalate esters may act initially to cause Sertoli cell injury, the subsequent loss of germ cells occurring as a consequence of this. Some features of the testicular lesion could be reproduced in primary cocultures of rat Sertoli and germ cells. Structure activity studies with a range of phthalate monoesters showed good agreement between the induction of germ cell detachment in culture and testicular toxicity in vivo. Three metabolites of MEHP (metabolites V, VI, and IX) were much less toxic in culture than MEHP itself, suggesting that the latter may be the active testicular toxin from DEHP.

Effects of di-(2-ethylhexyl) phthalate and five of its metabolites on rat testis in vivo and in in vitro.

In an attempt to establish which compound or compounds are responsible for the testicular damage observed after administration of di-(2-ethylhexyl) phthalate (DEHP) in rats, the effects of the parent compound and five of its major metabolites (mono-(2-ethylhexyl) phthalate (MEHP), 2-ethylhexanol (2-EH), mono-(5-carboxy-2-ethylpentyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate and mono-(2-ethyl-5-hydroxyhexyl) phthalate) were investigated in vivo and in vitro. The concentrations of MEHP and the three MEHP-derived metabolites in plasma were determined after single and multiple oral doses of DEHP. The plasma concentrations and areas under the plasma concentration-time curves (AUC's) of each of the MEHP-derived metabolites were considerably lower than those of MEHP both after single and after repeated administration of 2.7 mmol of DEHP/kg body weight. The mean elimination half-life of MEHP was significantly shorter in animals given repetitive doses than in those given a single dose, but there was no statistically significant difference between the mean AUC values. No testicular damage was observed in young rats given oral doses of 2.7 mmol of DEHP or 2-EH/kg body weight daily for five days. In animals which received corresponding doses of MEHP the number of degenerated spermatocytes and spermatids was increased, whereas no such effects were found in animals given the MEHP-derived metabolites. MEHP was also the only compound that enhanced germ cell detachment from mixed primary cultures of Sertoli and germ cells.

Effect of some phthalate esters and other testicular toxins on primary cultures of testicular cells

Mixed cultures of Sertoli and germ cells were prepared from rat testes and their response to some model testicular toxins was studied. Cultures consisted of a monolayer of Sertoli cells to which clusters of spermatocytes and spermatogonia adhered. With time in culture, germ cells progressively detached from the Sertoli cells into the medium. Addition of mono-(2-ethylhexyl) phthalate (MEHP) to the culture medium resulted in a concentration-dependent increase in the rate of germ-cell detachment over the range 10 −7–10 −4 m. No such effect was produced by di-(2-ethylhexyl) phthalate or 2-ethylhexanol. An increased rate of germ-cell detachment was also produced by other phthalate monoesters known to cause testicular damage in vivo, whereas similar concentrations of a number of monophthalates not known to affect the testis in vivo had no such effect on the cultures. Known age and species differences in the testicular toxicity of di-(2-ethylhexyl) phthalate could be reproduced in cultures treated with MEHP. There was little effect on the viability of either germ cells or Sertoli cells at concentrations of MEHP that caused marked germ-cell detachment, but there were changes in Sertoli-cell morphology. Increased germ-cell detachment was also observed in cultures treated with 10 −7–10 −4 m-AF1312/TS, a compound that affects Sertoli cells in vivo, but was not seen in cultures exposed to a range of other testicular toxins with different target cells in the testis. Thus, the effects produced by phthalate monoesters in vitro may reflect damage to the Sertoli cells. Testicular cell cultures could be of value both for screening compounds and for studying underlying mechanisms of testicular toxicity.