Chronic Exposure to Diesel Exhaust Particulate Matter Impairs Meiotic Progression during Spermatogenesis in a Mouse Model

50 Background: Exposure to air pollutants represented by diesel exhaust PM 2.5 (DEP) correlates 51 with the decline of semen quality, but the underlying biological mechanism has not been fully 52 understood. In the present study, mice were intratracheally instilled with DEP for around 7 53 months, and the effects of PM 2.5 exposure on the spermatogenic process as well as the 54 alterations of testicular gene expression profile were assessed. 55 Results: Our results show that chronic exposure to DEP significantly impairs the fertility of male 56 mice without influencing their libido. Compared with Vehicle-exposed group, the sperm count 57 and motility from DEP-exposed mice were significantly decreased. In addition, 58 immunohistological staining of γH2AX and DMC1, biomarkers for meiotic double strand breaks 59 (DSBs), demonstrated that chronic exposure to DEP comprised the repair of meiotic DSBs, thus 60 disrupts the spermatogenesis. Deep RNA sequencing test shows massive altered expressions 61 of testicular genes including the GnRH signaling pathway. 62 Conclusion: In summary, our research demonstrates that chronic exposure to PM 2.5 disrupts 63 spermatogenesis through targeting the meiotic recombination, providing a new perspective for 64 the research on the male reproductive system damage caused by air pollution. 65 division produces haploid spermatids, and the spermiogenesis that transforms spermatids into sperms. Notably, despite the above-mentioned considerable evidence for the disruption of spermatogenesis by PM2.5 exposure, how PM2.5 exposure affects the spermatogenic process has hardly been investigated, except for the histological analysis of seminiferous tubules Therefore, the present study exploits the mouse model of intratracheal instillation of diesel exhaust PM2.5 (DEP) to examine the effects of PM2.5 exposure on the spermatogenic process and also thoroughly document the alterations of testicular gene expression profile induced by DEP exposure. Our results show that DEP exposure decreased the number of advanced spermatogenic cells but not spermatogonia, paralleled by marked increase in meiotic double strand breaks (DSBs) in pachytene but not leptotene spermatocytes, strongly suggesting that DEP exposure disrupts spermatogenesis through specifically targeting the repair of meiotic DSBs.


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Epidemiological studies demonstrate a global uncontrolled decline in the semen quality and 70 male fertility rate over the past several decades [1,2]. The reasons for this decline has not yet 71 been well established. As this is a global decline, genetics apparently may not be its main 72 reasons. The male reproductive system is well known to be vulnerable to various environmental 73 stressors. Thus, environmental pollution is believed to probably account for the major decline in abnormalities induced by PM2.5 exposure. In addition, exposure to PM2.5 was shown to evoke 94 reactive oxygen species (ROS) production [12,17], inflammation [18], and endoplasmic 95 reticulum (ER) stress [14] in the testes, suggesting that these may also be local mediators for 96 the spermatogenic abnormalities induced by PM2.5 exposure. 97 Given that the major components of inhaled PM2.5 may not enter the systemic circulation, a 98 mechanism linking PM2.5 inhalation to the pathology in the testis is clearly needed. . We recently demonstrated that exposure to concentrated ambient PM2.5 110 (CAP) influences not only circulating FSH and testosterone but also the hypothalamic 111 expression of GnRH [15]. These studies strongly suggest that the HPG axis may be a crucial 112 mediator for those adverse testicular effects of PM2.5 exposure.

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Spermatogenesis is a complicated process that includes the mitotic division that produces 114 type A (self-renewal of stem cells) or B (committed to spermatocytes) spermatogonia, the 115 6 meiotic division that produces haploid spermatids, and the spermiogenesis that transforms 116 spermatids into sperms. Notably, despite the above-mentioned considerable evidence for the 117 disruption of spermatogenesis by PM2.5 exposure, how PM2.5 exposure affects the 118 spermatogenic process has hardly been investigated, except for the histological analysis of 119 seminiferous tubules [9,15]. Therefore, the present study exploits the mouse model of

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Chronic exposure to DEP impairs the male fertility 130 To examine the effect of chronic DEP exposure on the fertility of males, male C57Bl/6J mice 131 were subject to 6-month intratracheal instillation of vehicle (PBS) or DEP, and then their 132 fertilities were assessed via an 18-day mating with normal female C57Bl/6J mice. As shown in 133 Figure 1A, these sires were continued with their intratracheal instillation of vehicle or DEP 134 throughout this 18-day mating and the following 1-week singly housing. Therefore, they had 135 been exposed to vehicle or DEP for approximately 7 months in total when euthanized. Figures   136   1B and 1C show that all the sires inseminated their dam during this 18-day mating and that the 137 times taken for Vehicle-or DEP-exposed sires to inseminate their dam were comparable, 138 suggesting that exposure to DEP may not impact the libido of male mice. In contrast, Figure 1D 139 reveals that while 100% Vehicle-exposed sires impregnated their dam during this 18-day mating, 140 70% DEP-exposed sires only impregnated their dam, revealing that DEP exposure markedly 141 impairs the fertility of male mice. All the dams were sacrificed on day E16.5. The outcomes of 142 these pregnancies are presented in Table 1. The paternal exposure to DEP did not significantly 143 influence the rates of stillbirth and absorption, implantations per dam, live fetuses per dam, live 144 fetus weight, placental weight and uterus weight.   of epididymal sperms showed that chronic exposure to DEP did not significantly influence the 154 rate of morphologically abnormal sperms (Figures 2A and 2D).

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Chronic exposure to DEP alters the testicular histology 157 The testis is the organ that produces sperms. Given the reduction in the epididymal sperm count 158 by chronic exposure to DEP, histological analyses were performed on the testes of Vehicle-or 159 DEP-exposed mice to assess the effects of chronic DEP exposure on testicular histology. In  (Figures 3A and 3C) in the testes of 163 DEP-exposed mice versus those of vehicle-exposed mice. Sperms are released in Stage VIII 164 seminiferous tubules; therefore, the proportion of Stage VIII seminiferous tubules somehow 165 represents the sperm production rate. We thus assessed the proportion of Stage VIII 166 seminiferous tubules in these testes. In line with the reduction in the epididymal sperm count by 167 chronic DEP exposure, Figure 3D reveals that DEP-versus Vehicle-exposed mice had 168 significantly reduced proportion of Stage VIII seminiferous tubules in the testes. In contrast,    To verify whether chronic exposure to DEP disrupts the spermatogenesis through impact on 213 the repair of DSBs, the sections of testes from the Vehicle-and DEP-exposed mice were 214 visualized using γH2AX antibody. Figure 6A shows that the γH2AX + cells in the advanced 215 spermatocytes (cells close to the lumen of seminiferous tubules) were markedly increased in the 216 testes of DEP-exposed mice versus those of Vehicle-exposed mice, strongly supporting the 217 impairment of repair of DSBs and thus disruption of the meiotic progression by chronic DEP 218 exposure. In normal testes such as the PBS-exposed in Figure 6A, most γH2AX + cells are the 219 early spermatogenic cells (those close to the base membrane), and the visualization of γH2AX 220 11 facilitates determining the meiotic stages of these early spermatogenic cells: the diffused 221 distribution pattern marked by the yellow arrows in Figure 6A represents the leptotene or 222 zygotene spermatocyte; and the focused distribution pattern marked by the red arrows in Figure   223 6A represents the pathytene or diplotene spermatocyte. Therefore, we analyzed the meiotic 224 stages of seminiferous tubules using the γH2AX distribution pattern of early spermatogenic cells. are 80 genes differentially expressed in the testes of DEP-exposed mice versus those of 236 Vehicle-exposed mice (p value of FDR < 0.05 and fold change < 0.5 or > 2): 56 genes were 237 under-expressed in the DEP-versus Vehicle-exposed testes, and 24 genes were over-238 expressed in the DEP-versus Vehicle-exposed testes. The relative expression levels of these 239 differentially expressed genes are presented in Figure 7B. To identify the biological processes 240 that are influenced by the chronic DEP exposure, gene ontology (GO) enrichment analysis 241 using the 80 differentially expressed genes was performed. Figure 7C shows that 8 GO terms 242 were significantly enriched, including our previously identified GnRH signaling pathway. The male reproductive system is vulnerable to environmental pollution, and published studies 246 have increasingly demonstrated that it may be targeted by PM2.5 exposure. However, the 247 biological mechanism by which PM2.5 exposure disrupts the male reproductive system and thus 248 the male fertility has not yet been fully understood. In the present study, we show that chronic 249 exposure to DEP, an important source for ambient PM2.5, 1) impaired the fertility of male mice  Epidemiological studies increasingly demonstrate that exposure to PM2.5 inversely correlates 258 with semen quality and male fertility rate [4-8]. The present study corroborates the impairment of 259 semen quality and thus male fertility in a mouse model. The sperm count, motility, and 260 morphology collectively determine the semen quality and thus male fertility. Notably, the present 261 study shows that chronic exposure to DEP decreased the epididymal sperm count and motility 262 but did not increase the rate of abnormal sperm (Figure 2). The lack of effect on the rate of 263 abnormal sperm is supported by our present data showing that chronic exposure to DEP did not 264 increase the rates of stillbirth and absorption (Table 1)

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This also suggests that PM2.5 exposure impact the sperm count, motility and the rate of 272 abnormal sperm probably through different mechanisms.

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In the present study, we demonstrate that chronic exposure to DEP did not impact the  The present study shows that chronic exposure to DEP markedly influenced the processing of suggests that chronic exposure to DEP may not impact the initiation of spermatogenic meiosis, 306 as evidenced by the normal formation of DSBs in the leptotene spermatocytes ( Figure 5G) of 307 DEP-exposed testes and the normal number of spermatogonia of DEP-exposed testes ( Figure   308 4E). Along with the above-mentioned evidence for the disruption of repair of meiotic DSBs by 309 chronic DEP exposure, these results strongly suggest that the repair of meiotic DSBs is 310 precisely targeted for chronic DEP exposure to impair the spermatogenesis.

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The present study is also the first one using high-throughput technique to thoroughly  However, our testicular gene expression profiling did not identify any ROS-related genes 324 differentially expressed in DEP-versus Vehicle-exposed testes.

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Although the present study provides compelling evidence that long-term exposure to DEP 326 affects the male reproductive system by disrupting first meiosis, it has a range of important 327 limitations. This includes the time and dose-dependent data that we have not provided for any of 328 these adverse effects due to DEP exposure. Another limitation is that this study failed to 329 determine which protein or gene changes in meiosis caused meiosis abnormalities. Of course, 330 this in-depth discussion requires a deeper accumulation of expertise and more sensitive 331 technology. Furthermore, the present study did not provide any data on the causal relationship 332 between meiosis arrest and damage to spermatogenesis. Therefore, it is necessary to conduct 333 additional experiments to determine how the male reproductive system damage via affecting the 334 meiosis process due to DEP exposure.   On the day of experiment, after measurement of their body weight, all the mice were euthanized 384 and their blood was harvested from the orbital venous plexus. Fresh isolated testes, epididymis 385 and seminal vesicles were weighted, fixed in 4% paraformaldehyde for morphological analysis 386 and/or snap-frozen in liquid nitrogen and then stored at -80℃ for further use.

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The testicular histology and spermatogenetic parameters were analyzed as described were identified using cuffdiff with standard parameters and clustered by Genesis using a 421 hierarchical clustering method. Go enrichment was analyzed using Metascape (metascape.org).

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A hypergeometric test was performed using the default parameters to adjust the p value. In summary, the present results demonstrate that long-term exposure to DEP impact 432 spermatogenesis by disrupting meiotic prophase and thus impair the male reproductive function.

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To the best of our knowledge, our study is the first to use meiosis mechanism to analyze the   Chronic exposure to DEP impairs the quality of semen. A. Papanicolaou staining images of sperm morphology. a, normal sperm; b-d, abnormal sperm head; e-f, abnormal sperm tail. B. Sperm count in epididymis tissue of male mice after 6-month intratracheal instillation of PBS/DEP. n = 10/group, *p<0.05 versus PBS, student t test. C. Sperm motility of male mice after 7-month intratracheal instillation of PBS/DEP. n = 10/group, *p<0.05 versus PBS, student t test. D. Abnormal sperm percentage of male mice after 7-month intratracheal instillation of PBS/DEP. n = 10/group, *p<0.05 versus PBS, student t test. Percentage of stage VII seminiferous tubules. n = 10/group, *p<0.05 versus PBS, student t test. Figure 4