Maternal Cypermethrin Exposure during the Perinatal Period Impairs Testicular Development in C57BL Male Offspring

<div><p>Numerous studies have demonstrated that endocrine-disrupting compounds (EDC) are a possible cause of male reproductive organ malfunction and malformation. Cypermethrin (CYP) is a widely used synthetic pyrethroid and a potential EDC. This study aimed to examine the effects of perinatal exposure to low-dose CYP on the development and function of the offspring testes. Pregnant mice were intragastrically administered 0.12 to 12 mg/kg/day CYP from embryonic day 0.5 (E0.5) to weaning (PD21.5, postnatal day 21.5). Maternal exposure to 0.12, 1.2, and 12 mg/kg/day CYP affected the body and organ weight of the offspring. Exposure of CYP led to a dose-dependent decrease in the male-to-female sex ratio. A histopathological analysis revealed a thinner seminiferous epithelium layer at PD21.5, interstitial hyperplasia at PD45.5, and germ cell vacuolization at PD90.5 in the 12 mg/kg/day CYP group. The TUNEL assay results revealed increased germ cell apoptosis in the 12 mg/kg/day CYP group. The serum testosterone (T) level decreased, whereas the estradiol level increased with age in the 1.2 and 12 mg/kg/day CYP groups. The RT-PCR analysis demonstrated decreased expression of T production-related, mitosis-related, and meiosis-related genes in the 1.2 and 12 mg/kg/day CYP groups. The <i>in vitro</i> experimental results demonstrated reduced expression of steroidogenesis genes and decreased T levels. It is concluded that perinatal exposure to low-dose CYP affects testes development and function in adults.</p></div

Effects of CYP-treatment on steroidogenesis-related genes and media T levels in mLTC-1 cells.

<p>(A) The mRNA levels of mLTC-1 steroidogenesis-related genes. (B) The protein levels of mLTC-1 steroidogenesis-related genes. (C) Quantitative analysis of scanning densitometry of protein levels of mLTC-1 steroidogenesis-related genes from (B). (D) Media T levels of mLTC-1 cells. The mRNA and protein levels of Star, 3β-HSD, Cyp17a1, and 17β-HSD3 were downregulated significantly compared with the control group. The mRNA and protein levels of Cyp19a1 were upregulated after CYP treatment. The media T levels were downregulated. LH was used as a positive control. The data represent the mean ± SEM. *<i>P</i><0.05. C, CYP.</p

3β-HSD, Pcna immunohistochemistry and TUNEL assay of testes.

<p>Slides of (A, B, C) control and (D, E, F) CYP-treated offspring at PD21.5, PD45.5, and PD90.5, respectively, were immunolocalized with antibody for 3β-HSD. Slides of (G, H, I) control and (J, K, L) CYP-treated offspring at PD21.5, PD45.5, and PD90.5, respectively, were immunolocalized with antibody for Pcna. Slides of (M, N, O) control and (P, Q, R) CYP-treated offspring at PD21.5, PD45.5, and PD90.5, respectively were stained using the TUNEL method. All of the images were taken at 400× magnification. Scale bars, 40 µm. (S) Quantification of 3β-HSD-positive Leydig cells in 5–20 related slides of A–F. (T) Quantification of Pcna-positive germ cells in 5–20 related slides of G–L. (U) Quantification of apoptotic germ cells in 5–20 related slides of M–R. The data represent the mean ± SEM. *<i>P</i><0.05.</p

Effects of maternal perinatal CYP-exposure on testicular steroidogenesis-related genes in offspring.

<p>(A) The mRNA levels of <i>Star</i>, <i>Cyp11a1</i>, <i>3β-HSD</i>, <i>Cyp17a1</i>, <i>17β-HSD3</i>, and <i>Cyp19a1</i> in the testes at PD21.5 using RT-PCR. <i>Star</i>, <i>Cyp17a1</i>, and <i>17β-HSD3</i> were significantly downregulated in the CYP exposure groups. <i>Cyp11a1</i> displayed a downward trend, but this trend was not significant. However, <i>Cyp19a1</i> was upregulatedand, and there was no change in <i>3β-HSD</i>. (B) mRNA levels of steroidogenesis-related genes at PD45.5. <i>Star</i>, <i>3β-HSD</i>, and <i>17β-HSD3</i> were significantly downregulated in the CYP exposure groups. <i>Cyp19a1</i> was upregulated, and there were no changes in the <i>Cyp11a1</i> and <i>Cyp17a1</i> expression levels. (C) mRNA levels of steroidogenesis-related genes at PD90.5. <i>Star</i>, <i>3β-HSD</i>, and <i>17β-HSD3</i> were downregulated significantly in the CYP exposure groups, and <i>Cyp19a1</i> was upregulated significantly. The data represent the mean ± SEM. *<i>P</i><0.05.</p

Effects of CYP-treatment on AR, Erα, and Pcna in mLTC-1cells.

<p>(A) The mRNA levels of <i>AR</i>, <i>ERα</i>, and <i>Pcna</i> in mLTC-1 cells. (B) The protein levels of AR, Erα, and Pcna in mLTC-1 cells. (C) Quantitative analysis of scanning densitometry of protein levels from (B). The mRNA and protein levels of AR were downregulated after CYP treatment, which is consistent with the T levels. In contrast, the ERα mRNA and protein levels were upregulated. The Pcna mRNA and protein levels suggest that CYP treatment may promote the proliferation of Leydig cells. The data represent the mean ± SEM. *<i>P</i><0.05. C, CYP.</p

Effects of maternal CYP-exposure on the AR, ERα, Pcna, and E2/T levels of offspring.

<p>(A) The mRNA level of <i>AR</i> was not affected at PD21.5. The <i>ERα</i> level was upregulated significantly in the 12 mg/kg/day-exposure group, and there was an increasing trend in the other exposure group. <i>Pcna</i> expression was not altered. (B) <i>AR</i> expression was downregulated significantly in the exposure groups, and <i>ERα</i> and <i>Pcna</i> were upregulated, which may explain the hyperplasia of interstitial cells. (C) <i>AR</i> and <i>Pcna</i> were downregulated significantly, and <i>ERα</i> was upregulated, as observed at PD21.5 and PD45.5. (D) Serum T and E₂ levels and the E₂/T ratio at the three time points. The T level was lower, and the E₂ level was higher in the CYP exposure groups at PD45.5 and PD90.5. The data represent the mean ± SEM. *<i>P</i><0.05.</p

Offspring body and organs weight.

<p>Con, control group. Different superscripts (^a, ^b) depict significant differences among body or organs weight (<i>P</i><0.05).</p

Effects of maternal perinatal CYP-exposure on the testicular histology of offspring.

<p>(A) Testicular histology of the control group offspring at PD21.5. (B) Testicular histology of the 12 mg/kg/day CYP-treated group offspring at PD21.5: the seminiferous epithelium layer was thinner than that of the control. (C) Testicular histology of the control group offspring at PD45.5: normal spermatogenesis was observed. (D) Testicular histology of the 12 mg/kg/day CYP-treated group offspring at PD45.5. Some of the germ cells displayed vacuolation (arrow) and hyperplasia of interstitial cells (arrowhead). (E) Testicular histology of the control group offspring at PD90.5. The lumen of the seminiferous tubules was filled with spermatozoa. (F) Testicular histology of the 12 mg/kg/day CYP-treated group offspring at PD90.5. More severe vacuolation of germ cells and destruction of the seminiferous epithelium were observed. All of the images were taken at 400× magnification. Scale bars, 40 µm. (G) Quantification of germ cells in 5–20 related slides of A–F. (H) Quantification of germ cell vacuolation in 5–20 related slides of A–F. The data represent the mean ± SEM. *<i>P</i><0.05.</p

The sequences of primers, annealing temperature and amplified products of PCR.

<p>F, forward; R, reverse.</p

Number of offspring and sex ratio of male to female.

<p>Number of offspring and sex ratio of male to female.</p