The effects of nicotine exposure on the testis morphology and germ cell number
To explore the effects of maternal nicotine exposure on testis morphology of the progeny we compared hematoxylin and eosin-stained frozen testis sections in 11-week-old male animals born to the nicotine-exposed or control mothers. We examined the tubules at stage VI-VII where all cell types are known to be present to focus on homogenous cell populations between groups. We counted all the cells in a seminiferous tubule. The spermatogonia and early meiotic cells (leptotene-zygotene stage) are normally localized proximal to membrane of seminiferous tubule cells, whereas more advanced pachytene and haploid cells advance closer to the lumen (Fig. 1a-b). We found that the number of cells appears to decrease as a result of nicotine exposure by 0.7-fold (Fig. 1c). Similarly, our analysis of the seminiferous tubules thickness at stage VI-VII showed the decrease in exposed animals by 0.78-fold (Fig. 1d).
Next, we analyzed the number of germinal meiotic cells using γH2AX mark. Non-canonical phosphorylated histone variant H2AX replaces the canonical histone H2A during formation of double strand breaks (DSBs), thus allowing the chromosomes to become more condensed (reviewed in [21]). DSBs formation occurs throughout the whole genome and at each chromosome thus γH2AX is widely spread. At early stages of meiosis (leptotene-zygotene) when DSBs start to form, γH2AX appears as a very bright staining all over the nucleus. At a later stage (pachytene-diplotene), when autosomes segregate and DSBs are getting repaired, γH2AX is retained only at the sex-chromosomes as a bright spot (e.g., [22]). The γH2AX staining at cell types such as Sertoli cells as well as spermatocyte type 2, spermatogonia and spermatids is normally not strong and generally only appears as foci in case of random DNA damage. We performed the immunofluorescence assay (Fig. 2a) and calculated γH2AX positive cells as well as counted all cells in tubules. Our analysis revealed that there is 0.76-fold decrease in γH2AX positive cells suggesting possible decrease in the meiotic cell population or delay in the initiation of the meiosis in the testis of the nicotine-exposed offspring (Fig. 2b).
To reveal which cell populations might we impacted by nicotine, we decided to analyze the expression of genes which are specifically expressed in each cell population using as a source of information a recently published dataset [23]. To this end, we extracted total RNA and performed quantitative RT-qPCR analysis. Our analysis revealed that genes that are specifically expressed in spermatogonia, Kit and Lgr4, have decreased their expression by 0.16 and 0.15- folds, respectively. In contrast the expression of Stra8 had tendency to increase 1.5-fold (Fig. 2c). We determined that several genes that are normally expressed in spermatocytes, Piwil1, Pttg1, H2ax and Rad51, have all decreased their expression by 0.15, 0.4, 0.1 and 0.27-folds respectively. The Kiss1 gene that is specific for Sertoli cells have increased the expression by 2.13-fold, in contrast, Ctsl and Amhr2 genes that also are expressed in Sertoli cells have decreased their expression by 0.6 and 0.08-folds respectively (Fig. 2c). Finally, Prm1 and Prm2, specific for spermatid fraction have decreased and increased their expression by 0.48 and 1.2 -folds respectively (Fig. 2c).
In conclusion our morphology analysis demonstrated global decrease in germ cell population. We also observed that gene expression specific for each cell type was also perturbed suggesting that prenatal exposure to nicotine notably alters gene expression in almost all cell types of the testis.
Effects on meiosis
To further explore effects of nicotine exposure on meiosis we opted to analyze the formation of H3K9me3 marks in the nuclei. During meiosis H3K9me3 is normally localized all over the nucleus during leptotene- zygotene stages, and the H3K9me3 marks become concentrated at the nuclear periphery at pachytene-diplotene stages [24]. H3K9me3 is normally enriched at the regions of compact heterochromatin during meiosis (reviewed in [25]). During normal meiosis, H3K9me3 is observed mainly at the nuclear periphery in form of patches, and in some pathological conditions the formation of H3K9me3 patches could become perturbed. This perturbation, associated with alteration of telomere attachment and reduction of H3K9me3, leads to a loss of chromatin repression at telomeres [26]. We obtained testis spreads and immunostained the sections for H3K9me3 (Fig. 3a) followed by a quantitative analysis of mark’s intensity. Our analysis showed that in PNE cells there is 1.7-fold increase in H3K9me3 staining, suggesting possible impact of nicotine and this marks on normal chromosome segregation (Fig. 3b). One of the known functional consequences for H3K9me3 increase is a decrease in TERRA transcript and reduced telomere stability [27].
Next, we sought to analyze the expression of meiotic GRR genes given their importance for normal meiosis functioning. Our analysis showed that most of tested GRR genes increased their expression including Brdt, Dazl, Ddx4, Tdrd1 by 1.5, 1.6, 1.7, 5 -fold respectively, suggesting potential perturbation of reprograming at GRR loci due to nicotine exposure (Fig. 3c). Hormad1 expression that is essential for meiosis [28] also had a tendency to increase by 1.2-fold (Fig. 3c).
In summary, meiosis and GRRs gene expression were perturbed in PNE male rats suggesting that PNE has a deleterious effect on the reproductive system in adults.
Analysis on protamine and regulatory histone marks
To determine the effect of nicotine on haploid cell population we analyzed the level of protamine proteins by WB (Fig. 4a). The protamines are encoded by Prm1 and Prm2 genes to replace histones during late stage of spermatogenesis and assist in forming a protected and compact structure of chromatin characteristic for spermatozoids. We determined that the PRM2 concentration decreased 2.4-times in exposed cells (Fig. 4b).
Next, we analyzed H3K4me3 (that marks open chromatin) and H3K9me3 that is associated with gene silencing abundant at compact heterochromatin. Our analysis showed no significant changes in the level of H3K4me3 at the global level although we cannot exclude the possibility of regional changes (Fig. 4c). On the contrary, the analysis of H3K9me3 showed a significant increase in this histone mark in nicotine-exposed samples (Fig. 4d). The results of WB of H3K9me3 are consistent with immunofluorescence analysis of H3K9me3 (Fig. 3A-B), which also showed higher level of H3K9me3 in spermatocytes suggesting global impact of nicotine exposure on H3K9me3.
In summary, the WB analysis shows that, in addition to a decrease in meiotic cell number, there is also a decrease in post-meiotic cells, which are normally enriched in PRM2 protein. The higher level of H3K9me3 during meiosis can also have an impact on compact heterochromatin and telomere functioning during meiosis.
Analysis of DNA methylation
To explore the impact of nicotine on DNA methylation we performed DNA methylation analysis using a genome-wide sequencing technique. In our study, nearly ~ 57K of CpG-rich regions were detected. We identified 366 regions that showed differential pattern of DNA methylation (FDR < 0.1) (Fig. 5a). Among differential peaks, 198 showed increased methylation and 165 regions showed decreased DNA methylation (Fig. 5b, Additional file S1). The analysis using ChIPseeker application showed that the vast majority of the differentially methylated regions (DMRs) are distally located regions. However, nearly ~ 14% of DMRs were found in introns (Fig. 5c). To determine differentially methylated genes based on the DMRs, we assigned DMRs to genes using Chipseeker. Functional annotation of differentially methylated regions (DMRs) by DAVID showed that they are enriched in genes that normally expressed in neuronal cell body (Dmd, Tgfb1) (Fig. 5d); they are also enriched in genes encoding transcription factors (Foxj3, Nfact2, Rfx3, Meis3, Rbpj, Foxs1, Dmtf1, Hoxb3, Hoxa2, Tead1, E2f7, Hoxa5, Hoxb5, Creb5), the glutamatergic synapse (Qsec2, Dtnbp1, Cabp1, Begain, Dnm2, Olfm1, Actc1, Slitrk2, Psd2, Prkar2a, Slitrk1, Syt10, Gpc6, Adgrl3), and GABAergic signaling factors (Slitrk2, Slitrk1, Sst, Iqsec3, Igsf9b) (Fig. 5e). In addition, the genes encoding estrous cycle related factors (Trpm2, Anxa1, Cyp1b1, Pgr) were determined to harbor DMRs.
Due to their importance for meiosis, we specifically examined DNA methylation status of 45 GRRs. We extracted the sequencing reads number of the methylated regions of GRRs and plotted them in Excel (Fig. 6a, Table S1). We found that DNA methylation marks are presents at ~ 30 genes. We observed that DNA methylation has tendency to decrease at the Hsf5 (p = 0.1) and is increased at Mov10l1 gene (p = 0.008) (Fig. 6b). Notably, the decreased DNA methylation at GRRs is negatively correlates with the GRRs gene expression (Fig. 3c) suggesting a possible impact of PNE on DNA methylation at GRRs during development on GRRs gene expression in adulthood.
We also decided to determine whether there is motif enrichment in DMRs in order to reveal a possible trigger mechanism induced by PNE. To this end, we extracted sequence information of DMRs in fasta format and performed motif search using MEME-ChIP. Our analysis determined that two common motifs are found in many sequences. The part of the first common motif is similar to binding site of the NRF1. Nrf1 encodes for a regulator of mitochondrial metabolism that plays a critical role in post-migrating primordial germ cells (PGC) development [29], suggesting possible role of this factor in nicotine-treated germ cells. The part of the second identified motif is similar to binding site of ETV4 (Fig. 6c). ETV4 regulates the cell proliferation [30].
To sum up, our DNA methylation analysis approach uncovered that upon nicotine exposure there is differential methylation of genes relevant to nervous system signaling and transcription factor activities. We propose that the alterations in activities in NRF1 and ETV4 transcription factors could trigger post-migrating primordial germ cells development and proliferation.
RNA-seq analysis of pituitary gland in nicotine-exposed animals reveals effects on protein-coding genes important for cell migration, cell adhesion and GABAergic signaling.
To explore the molecular mechanisms of nicotine exposure in the neuroendocrine system, we performed transcriptomic analysis of mRNA using paired-end stranded RNA sequencing using 3 biological replicates for the nicotine-treated and control pituitary glands. The samples showed some variations between replicates (Fig S1a-b) due to the complexity of cellular milieu in the pituitary. However, we were able to identify 60 differentially expressed genes (FDR < 0.1) (Fig. 7a, Fig. S1c, Table S3). The majority of differentially expressed genes (DEGs) were downregulated (Fig. 7a, Fig S1d). We performed a functional annotation of the DEGs by using the gene ontology program DAVID. Our analysis showed that the strongest enrichment in DEGs was found in the genes related to Tgfb1 signaling, regulation of cell migration, and cell adhesion among others (Fig. 7b). It is noteworthy, that Tgfb1 beta signaling factors encoding genes (Acta2, Col4a2) were upregulated in pituitary gland, in contrast to Tfgb1 DNA methylation decrease in the testis in exposed animal, suggesting thereby a possible link between brain and testis Tfgb1 signaling pathways.
Since we observed some alteration in DNA methylation of GABAergic signaling genes, we decided to closer look at these genes in our RNA-seq data. To this end, we determined the genes that are combined by gene ontology term “GABAergic synapse” using AMIGO database (https://amigo.geneontology.org/amigo). 136 genes were related to this term, with 121 out of them were expressed in the rat pituitary gland. We determined that Erbb4 and Slc6a6 were significantly upregulated upon nicotine exposure and three genes (Slc6a11, Slc32a1, Slc6a6) showed tendency to increase. Six genes (Gabra4, Gabrg2, Epha3, Gabra3, Gap43, Adra2a) showed tendency to decrease (Fig. 7C). These altered genes encode for proteins that are combined in network with central players GABRA2, GABRA3 and GABRA4, GABRG2 (Fig. 7d).
In summary, RNA-seq analysis showed that genes related to Tgfb1 signaling, cell migration, cell adhesion and GABAergic signaling were differential between control and nicotine groups, suggesting that prenatal exposure to nicotine affects the neuroendocrine system as well.