Perinatal Exposure to Low-Dose Methoxychlor Impairs Testicular Development in C57BL/6 Mice

Methoxychlor (MXC), an organochlorine pesticide, has adverse effects on male reproduction at toxicological doses. Humans and wild animals are exposed to MXC mostly through contaminated dietary intake. Higher concentrations of MXC have been found in human milk, raising the demand for the risk assessment of offspring after maternal exposure to low doses of MXC. In this study, pregnant mice (F0) were given intraperitoneal daily evening injections of 1 mg/kg/d MXC during their gestational (embryonic day 0.5, E0.5) and lactational periods (postnatal day 21.5, P21.5), and the F1 males were assessed. F1 testes were collected at P0.5, P21.5 and P45.5. Maternal exposure to MXC disturbed the testicular development. Serum testosterone levels decreased, whereas estradiol levels increased. To understand the molecular mechanisms of exposure to MXC in male reproduction, the F1 testes were examined for changes in the expression of steroidogenesis- and spermatogenesis- related genes. RT-PCR analysis demonstrated that MXC significantly decreased Cyp11a1 and increased Cyp19a1; furthermore, it downregulated certain spermatogenic genes (Dazl, Boll, Rarg, Stra8 and Cyclin-a1). In summary, perinatal exposure to low-dose MXC disturbs the testicular development in mice. This animal study of exposure to low-dose MXC in F1 males suggests similar dysfunctional effects on male reproduction in humans.

Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane; MXC] is a chlorinated hydrocarbon pesticide in common, worldwide used as a replacement for DDT [1,1,1-trichloro-2,2bis(p-chlorophenyl)ethane]. MXC and its major metabolites, mono-desmethyl-MXC (OH-MXC) and bisdesmethyl-MXC (HPTE), possess ERa agonist, ERb antagonist and antiandrogenic activities [12,13,14]. Perinatal exposure to high-dose MXC is able to cause many reproductive abnormalities in adult male mice or rat [15,16,17,18,19,20,21]. Amstislavsky et al. reported that transient exposure to 166.7 mg/kg/d MXC at embryonic day (E) 2 to E4 significantly increased the seminal vesicles weight and decreased the testosterone (T) level in male mice at 6 months [15]. Perinatal and juvenile oral treatment of rats with 150 mg/kg/d or 60 mg/kg/d MXC reduced testicular size, decreased the number of spermatogonia and Sertoli cells and serum LH and FSH in those animals at adults [16,18]. Cupp et al. demonstrated that transient exposure to 150 mg/kg/d MXC from E7 through E15 reduced the number of germ cells in rat at postnatal-day (P) 17-P20 and increased the number of apoptotic elongating spermatids in rat at P60 [17]. Most previous studies were carried out using toxicological doses of MXC (over 100 mg/kg/d) [22,23], even though it is logically implausible that humans or wild life would be exposed under such conditions. Research on the effects on the male reproductive system of low doses or environmentally relevant doses of MXC (less than 2 mg/kg/d) [24,25] is urgently required. Clinically, impaired reproductive development has been demonstrated in the sons of female gardeners or farmers who have been exposed to pesticides [26,27,28,29,30,31]. Humans and wild animals are exposed to MXC mostly through the dietary intake of contaminated food. Higher concentrations of MXC have been found in human milk, elevating the demand for assessing the risk of offspring after maternal exposure to MXC. To clarify the molecular mechanisms of exposure to MXC on male reproduction, we conducted a study examining MXC exposure during the perinatal period to determine its effect on fetal development, as well as the long-term impacts on the male reproductive systems of C57BL/6 mice.

Ethics Statement
The Ethics Committee for Animal Experimentation of the China Agricultural University approved all of the animal experiments (Register No. SKLAB-2011-01-05). All surgeries were performed using sodium pentobarbital anesthesia, and efforts were made to minimize animal suffering.

Experimental design: In vivo mouse studies
Female C57BL/6 mice were purchased from Weitonglihua, Beijing (under the license of Charles River, US). To test the appropriate perinatal exposure dose of MXC, a pilot study was carried out on gestating C57BL/6 mice (F0 generation). The female mice were given intraperitoneal daily (at 20:00) injections of MXC (0.25 mg/kg/d, 2.5 mg/kg/d, 20 mg/kg/d, 1 mg/kg/d) from plug date (E0.5) to weaning (P21.5), and the male offspring (F1) were assessed (n = 6-8/group). The control group received the vehicle alone (sesame oil). To determine the effects of different ways of MXC exposure, intraperitoneal injection or intragastrical administration of 1 mg/kg/d MXC were studied. There was no significant difference between the intraperitonally injected and intragastrically administrated plasma or adipose tissue MXC concentrations (Fig. S1). Thus for further experiments we have used intraperitoneal MXC injections. The concentrations of MXC in plasma and adipose tissue in female mice are comparable to the concentrations in women (2.23 ng/ml and 24.38 ng/g in mice, 0.38 ng/ml and 29.86 ng/g in women) [32]. Moreover, as we obtained significant results from the 1 mg/kg/d MXC-treated group, the later analyses were conducted in this group. The majority of animals were tested by P45.5 of age. The testes from control and MXC F1 generation mice were collected at three time points (P0.5, P21.5 and P45.5), and the body and testes weights were recorded simultaneously.

Intraperitoneal injection and intragastrical administration of MXC
To determine the effects of different ways of MXC exposure, female C57BL/6 mice were given daily (at 20:00) intraperitoneal injections or intragastrical administration of 1 mg/kg/d MXC for 5 days (n = 4/group). Then the mice were anesthetized, and blood and abdominal adipose tissue were colleted for MXC detection. Detection of MXC with ELISA Concentration of MXC in the plasma and adipose tissue were measured by ELISA following the manufacturer's protocol (R&D Systems, US). Briefly, adipose tissue was homogenated and extracted with 70% Methanol (2 ml/g adipose tissue), then filtered with Whatman No 1. filter paper. After proper dilution, standards and samples were added to a 96-well plate (Coated with MXC coupling antigen). The antibody-enzyme conjugate was then added, and the plate was incubated for 30 min at 37uC and washed. Next the color-reagents A and B were added to the well and incubated for 10 min at 37uC. The stop solution was then added and the absorbance value at 450 nm was read. In order to calculate the sample's concentration, the standard curve was generated according to the standards.

Sperm count and motility
Epididymis sperm was obtained as previously described [33]. Caudae epididymidis and vasa deferentia were excised and then rinsed with medium containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 30 mM HEPES, 10 mM glucose, 10 mM lactic acid, and 1 mM pyruvic acid (pH 7.4). After transfer to 1 ml of medium supplemented with 5 mg of bovine serum albumin per ml and 15 mM NaHCO 3 , semen was allowed to exude (15 min at 37uC, 5% CO 2 ) from three to five small incisions. Total sperm number was determined by using a Neubauer hemocytometer as previously described [34]. The percentage of motile sperms was recorded using a phase contrast microscope at a magnification of 4006. To determine sperm motility, 100 sperm each were observed in 3 different fields, and classified into motile and non-motile sperms, and the motility was expressed as percentage incidence.

Histology analyses and quanlification of germ and Leydig cell number
Testes were fixed in 10% neutral buffered formalin or Bouin's (Sigma-Aldrich, US), embedded in paraffin, sectioned in 5 mm, and then stained with hematoxylin and eosin (H&E), according to standard procedures. Quantification of the number of germ cells at different stages was carried out as described previously (n = 8/ group) [35]. Briefly, the nuclei of different germ cells were counted in 100 round seminiferous tubules cross sections, chosen at random for each mouse. The counts were subjected to Abercrombie correction for section thickness and differences in the nucleus or nucleolar diameter [36], as modified by Amann [37]: average number of cells = [number of nuclei counted6thickness (mm) of section]/[average diameter of nuclei (mm)+thickness (mm) of section]. The number of Leydig cells was measured, as described previously (n = 7/group) [38].

RNA extraction, RT-PCR and semi-quantitative RT-PCR
Total RNA was extracted from frozen testicular tissues or cells using the acid guanidinium method [40] and stored at 280uC until assayed. RT-PCR was performed as previously [41], with one microgram of total RNA being incubated with 10 IU avian myeloblastosis virus reverse transcriptase (Promega, US) at 37uC for 1 h. The cDNAs were then denatured at 95uC for 5 min and amplified through 25 cycles of PCR, using the following conditions: 94uC for 30 sec, 56uC to 61uC for 45 sec, and 72uC for 45 sec. An aliquot of the RT-PCR product was subjected to agarose gel electrophoresis and visualized by ethidium bromide staining (n = 6/group). Each sample was run in triplicate, and three individual experiments were conducted. Ribosomal protein L19 gene was amplified as an internal control. The primers used for the various RT-PCR analyses were given in Table 1. The gel bands were quantified using ImageJ software version 1.42 (Image processing and analysis in Java, NIH, Bethesda, MD; http://rsb. info.nih.gov/ij/download.html).

In vitro testicular cultures and treatments
The testes were dissected from new born C57BL/6 mice in PBS solution. Testes were cultured atop 1.5-to 1.7-ml agar blocks, as described [42]. All-trans Retinoic acid (RA, Sigma-Aldrich, US) was dissolved in ethanol, and Busulfan (Sigma-Aldrich, US) and MXC (Sigma-Aldrich, US) were dissolved in DMSO. For busulfan treatment, busulfan (40 mg/kg body weight) was injected intraperitoneally into pregnant female mice at 9.5 days post coitum [43]. The concentrations of the compounds in the culture media were as follows: all-trans RA, 0.7 M; and MXC, 10 26 M. Control cultures were treated with ethanol and/or DMSO, as appropriate.

In situ hybridization
For in situ hybridization, the testes were fixed in 4% paraformaldehyde at 4uC overnight. Whole-mount in situ hybridization was performed as reported [44]. The digoxigeninlabeled RNA probe for Stra8 was detected using an alkaline phosphatase-conjugated antidigoxigenin antibody and through staining with a BM purple alkaline phosphatase substrate (Roche, US) (n = 4/group). RA stimulates the expression of Stra8 [45] and the RA-treated testes served as a positive control. Busulfan could mediate the depletion of germ cell [46] and the busulfan-treated testes served as a negative control.

Statistical analysis
The data were analyzed for statistical significance with the SPSS 12.0.1 Package (SPSS Inc., US). Data for all groups were first tested for normality with the Shapiro-Wilk test. If the group's data were normally distributed, they were compared using a one-way analysis of variance. P values ,0.05 were regarded as statistically significant. All values are presented as the means 6 SEM (standard error of mean). All of the graphs were generated with GraphPad Prism 5.0 (GraphPad Software Inc., US).

MXC affects both the body and testicular weights of F1 generation male mice
Gestating C57BL/6 mice (F0 generation) were given MXC (0.25 mg/kg/d, 2.5 mg/kg/d, 20 mg/kg/d or 1 mg/kg/d) from E0.5 of gestation to P21.5 of weaning. Both the body and testicular weights of F1 males were recorded at the three time points (P0.5, P21.5 and P45.5). At P0.5, the exposure of pregnant mice to 20 mg/kg/d and 1 mg/kg/d MXC significantly decreased the neonatal weights of pups (Table 2). At P21.5, the body weights of all MXC-treated groups were not affected, compared with control group; however, the absolute and relative testicular weights of all MXC-treated groups increased significantly ( Table 2, 3). At P45.5, the body weights of the MXC-treated groups increased significantly compared to the control group, but the absolute testicular weights were not altered and the relative testicular weights were decreased significantly (

MXC alters the testicular histology and cell numbers of F1 generation mice
The image quantification analysis demonstrated that the numbers of pachytene spermatocyte and round spermatid were significantly decreased at P21.5 in the 1 mg/kg/d MXC-treated group, compared with vehicle-treated animals (Fig. 1A, B). And the number of Leydig cells at P45.5 in the 1 mg/kg/d MXCtreated group was increased significantly (Fig. 1A, C). No histomorphology abnormality was observed in other MXC dosage groups (Fig. S2). However, prenatal exposure to 1 mg/kg/d MXC did not significantly affect the epididymal sperm count and motility at P45.5 (Fig. S3).

MXC inhibits steroidogenesis in F1 generation mice testes
We observed a decrease in serum T and an increase in serum E 2 concentrations in MXC-treated males ( Fig. 2A, B). Also the steroidogenic genes were analyzed. We observed no changes in the mRNA levels of steroidogenic acute regulatory protein (Star) or 3 beta-hydroxysteroid dehydrogenase (Hsd3b), while cytochrome P450, family 11, subfamily a, and polypeptide 1 (Cyp11a1) was significantly decreased and Cytochrome P450, family 19, subfamily a, and polypeptide 1 (Cyp19a1) was significantly increased (Fig. 2C). Moreover, we achieved similar results in MXC-treated mLTC-1 Leydig cells (Fig. 2D). We also analyzed the mRNA expressions of hormone nuclear receptors. We did not observe any significant change in the mRNA expression of estrogen receptor a (Esr1), nuclear receptor subfamily 5, group A, member 1 (Nr5a1), Androgen receptor (Ar) or Insulin-like 3 (Insl3), whereas estrogen receptor b (Esr2) mRNA expression was significantly decreased (Fig. 2E). We also re-confirmed these testicular mRNA expression results in MXC-treated mLTC-1 Leydig cell line that showed very similar trend towards the in vivo data (Fig. S4).

MXC disturbs the meiosis in germ cells
At P21.5, the testes showed decreased numbers of pachytene spermatocyte and round spermatid in the 1 mg/kg/d MXC group (Fig. 1A, B). To determine the impact of MXC exposure on germ cell differentiation, we analyzed the expression of the undifferentiated spermatogonia specific-genes (Cyclin-d2, Nanos3 and Pou5f1), and no alteration was observed (Fig. 3A). We then checked the premeiotic germ cells marker in the testes and observed no change in the expression of Sohlh2 mRNA in MXCtreated testes (Fig. 3B). However, the expressions of the proliferation marker proliferating cell nuclear antigen (Pcna), stimulated by retinoic acid gene 8 (Stra8) and Cyclin-a1 mRNAs were significantly decreased in the MXC-treated testes (Fig. 3B). As Stra8 plays a critical role in meiosis, we also checked the protein expression in the testes. The protein level of Stra8 was decreased significantly (Fig. 3C). Moreover, we confirmed the effect of MXC on Stra8 with in vitro testicular culture and in situ hybridization. After treatment of 10 26 MXC for 48 h, the cultured testes revealed decreased Stra8 expression compared to control group (Fig. 3D). In addition, the Deleted in Azoospermia gene family (Dazl and Boll) and retinoic acid receptor gamma (Rarg) mRNA expressions were significantly decreased in the MXC-treated testes (Fig. 3E). We also re-confirmed these testicular mRNA expression results in MXC-treated GC-1 cell line that showed very similar trend towards the in vivo data (Fig.  S5).

Discussion
Although, it is widely accepted that EDCs have a great influence on human and animal reproduction at toxicological doses, the knowledge on the exposure of humans to lower or environmentally relevant doses of EDCs or of related effects on offspring remains limited. The present study was carried out to investigate the mechanisms responsible for the adverse effects of low-dose MXC exposure during the perinatal period, allowing for a better understanding of the mechanisms behind these histological abnormalities in the prepubertal period. Our results demonstrated that perinatal exposure to low-dose MXC could impair the testes development, not only by disturbing testicular Leydig cell proliferation but also by interrupting steroidogenesis and spermatogenesis.
Previous experiments have demonstrated that exposure to organochlorine pesticides might induce intrauterine growth retardation (IUGR) [38,39] and obesity in puberty [47,48,49,50]. Prenatal exposure to low doses of MXC reduces the postnatal growth [51]. Consistently, we found that the exposure to low doses of MXC perinatally decreased body weight at P0.5 and increased body weight at P45.5, thus we assumed obesity as one of the putative reasons for the differential body weights among the groups on P45.5. As IUGR has been linked to increased metabolic syndrome risk later in life [52,53], the study of low doses of MXC exposure leading to metabolic syndrome needs to be further investigated.
Many experiments have demonstrated that estrogen or estrogenic substances can conversely inhibit steroidogenic genes, such as Star, Cyp11a1 and Cyp17a1 [54]. HPTE, a metabolite of MXC, possesses ERa agonist activity [12,13]. In the present study, we found that Cyp11a1 and T levels were decreased, but Cyp19a1 and E 2 levels were increased. Therefore, MXC could regulate Cyp19a1 expression, and in turn leads to an increase in E 2 production. However, how the MXC disturbs the expression of steroidogenic genes is still unknown.
The balance of androgenic and estrogenic signals plays a key role in the male reproductive system. Our lab previous results demonstrated that the mice overexpressing human aromatase possessed a multiple structural and functional alterations in the reproductive organs [41,55,56].
Leydig cells are a major source of T, and the total number of Leydig cells is determined during pubertal development, as their proliferative activity is limited to the prepubertal period. Estrogen inhibits Leydig cell development via ERa [57] or ERb [38]. Hormonal regulation of the proliferative capacity of Leydig cells could be interfered by endocrine disruptors. The number and distribution of fetal Leydig cells are altered after in utero exposure to DBP or DEHP [58,59], genestin, resveratrol, or quercetin [60]. In the current study, we observed increased number of Leydig cells at P45.5. This may be caused by the ERb antagonist activity of HPTE [12,13], metabolites of MXC, and/or the reduction of ERb expression.
In this study, we demonstrated that exposure to low dose of MXC perinatally could induce the effects of decreasing or delaying of germ cell population in the testes of F1 males at onset of meiosis, as well as of downregulating the Rarg and Stra8, the key factors of meiosis [45,61,62]. The level of the meiotic marker, Cyclin-a1, was also reduced. Therefore, MXC was able to counteract the retinoic acid pathway, inhibit meiosis and disturb spermatogenesis.
It has been demonstrated that MXC can be metabolized into HPTE possessing antiandrogenic activity [14], T is a prerequisite for normal spermatogenesis and development [63,64]. Here, we found that MXC decreased the serum T. These results suggest that MXC could affect spermatogenesis through the downregulation of androgen signaling pathways by antiandrogenic activity and decrease in serum T.
Taken together, the current results indicate that perinatal exposure to a low dose of MXC perturbs the testicular development in mice. This is probably caused by the multiple actions of MXC during testicular development. Firstly, MXC is able to downregulate Cyp11a1 and upregulate Cyp19a1, leading to the decrease in serum T and increase in serum E 2 levels, consequently affecting the T/E 2 ratio balance. Secondly, the reduction of T, combined with the antiandrogenic activity of MXC metabolites, is likely to disturb spermatogenesis in P21.5 testes. Finally, the reduction of ERb expression and/or ERb antagonist activity of MXC metabolites may enhance the proliferation of Leydig cells in P45.5 testes. However, the exact mechanism, by which MXC disturbs the testicular steroidogenesis and spermatogenesis, is still unclear and requires further investigation. Our data provides a strong evidence of MXC effects on male mice fertility and forms the basis of a potential link between the exposure to EDCs and the increase in the incidence of male reproductive disorders for adult men. Figure S1 Concentrations of MXC in plasma and adipose tissue of mice treated with intraperitoneal injection or intragastrical administration of 1 mg/kg/ d MXC for 5 d (n = 4/group). Concentration of MXC was detected by ELISA. The data represent the mean 6 SEM. *P, 0.05. (TIF) Figure S2 Representative micrographs of H&E-stained testes of mice exposed to vehicle, 0.25 mg/kg/d, 2.5 mg/ kg/d or 20 mg/kg/d MXC (n = 6-8/group). Original magnification, 6100. (TIF) Figure S3 Epididymis sperm count and motility of mice exposed to vehicle or 1 mg/kg/d MXC at P45.5. (A) Epididymis sperm count was determined by hemocytometer (n = 5/group). (B) Epididymis sperm motility was recorded using a phase contrast microscope (n = 5/group). The data represent the mean 6 SEM. *P,0.05 versus vehicle. (TIF) Figure S4 The related mRNA expression of Esr1, Esr2, Ar, Nr5a1 and Insl3 was normalized to L19 level in mLTC-1 cell line cultured with DMSO or 10 26 M MXC for 24 h (n = 4/group). Vehicle-treated group was set at 1.0. The data represent the mean 6 SEM. *P,0.05 versus vehicle. (TIF) Figure S5 The related mRNA expression of Esr1, Esr2, Ar, Nr5a1, Insl3, Cyclin-d2, Nanos3, Pou5f1, Sohlh2, Pcna, Stra8, Cyclin-a1, Dazl, Boll and Rarg was normalized to L19 level in GC-1 cell line cultured with DMSO or 10 26 M MXC for 24 h (n = 4/group). Vehicle-treated group was set at 1.0. The data represent the mean 6 SEM. *P,0.05 versus vehicle. (TIF)