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Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Cannabis sativa has a complex history reflected in both selection on naturally occurring compounds and historical trade routes among humans. Iran is a rich resource of natural populationswhich hold the promise to characterize historical patterns of population structure and genetic diversity within Cannabis. Recent advances in high-throughput DNA sequencing technologies have dramatically increased our ability to produce information to the point that it is now feasible to inexpensively obtain population level genotype information at a large scale. We genotyped 98 cannabis samples 36 from Iranian locations and 26 accessions from two germplasm collections. However, single nucleotide variant analysis uncovered a relatively moderate level of variation among Iranian cannabis. Cannabis sativa L. Humans have cultivated the plant as a source of fiber, food, medicines, intoxicants and oils for thousands of years 1 , 2. This use and breeding has led to the selection of two distinct types of C. While these types are morphologically similar, they are distinguished by the type and level of cannabinoids produced. Levels of two types of cannabinoids in particular are used to distinguish marijuana and hemp C. First, Dtetrahydrocannabinol THC is a psychoactive compound 3 found in leaves and inflorescences but not seeds of juvenile and mature plants. The second compound, cannabidiol CBD , is an isomer of THC found in all plant tissues, however, this cannabinoid does not activate cannabinoid receptors 1 , 4 , 5. Marijuana varieties used for drug consumption are characterized by a high THC content, whereas fibre varieties hemp produce CBD as the predominant cannabinoid 6 , 7. Archaeological and palaeobotanical evidence supports the cultivation and use of Cannabis since the Neolithic period with subsequent secondary domestication events in geographical regions outside of the accepted native range 8 , 9 , 10 , 11 , 12 , 13 , 14 , For instance, archaeological evidence for the pharmaceutical or shamanistic use of Cannabis has been found in cave artifacts that include a large cache of Cannabis dating to ca. This long history of use has resulted in a complex biogeographical history for this species. Based on polymorphism in RAPD markers, the Eurasian Steppe region of Central Asia has been recognized as a putative center of origin for Cannabis , spreading from there to the Mediterranean as well as Eastern and Central European countries, in particular, Afghanistan and Pakistan However, the genus has also been described has having two centers of diversity, Hindustani and European—Siberian As with other cultivated plants it is difficult to pinpoint the exact place of origin for C. It is likely that Cannabis spread to ancient Persia very early, assisted by Aryan and Scythian tribes expanding westward from central Asia. Evidence for this early spread comes from archeological studies of the Scythians, who occupied an area encompassing large swathes of what is now northwest Iran from the 7th century BCE to the 4th century CE, this culture was known to use Cannabis for entertainment and spiritual purposes. While all Iranian cannabis has been described as a complex of landraces of C. Currently, the most important topics in C. A draft genome and accompanying transcriptome of C. Nevertheless, the phylogeography and domestication history of Cannabis remains poorly understood, in part due to limited access to genetic material from natural populations. Given that Cannabis is a native plant with a long history of cultural use in Iran, it is surprising that no studies of Cannabis diversity using molecular markers exist. Here we present an initial description of population structure and genetic diversity, between Iranian and global collections of Cannabis as well as within the Iranian collection. Specifically, we leverage genotyping-by-sequencing GBS 32 to generate single nucleotide polymorphisms SNPs across a large collection of Iranian cannabis. GBS provides a robust, cost-effective alternative to other approaches and provide greater power to detect genome wide patterns associated with population structure and demographics than other molecular markers 33 , In total 98 cannabis samples were digested, sequenced, and genotyped these included, 70 samples representing 35 locations in Iran Fig. For each location or accession one female and male plant was sampled. After quality filtering a total The remaining samples were represented by a mean of 4. These uniquely mapped sequence reads covered approximately 0. Geographical distribution of samples across Iran. This figure was produced using the R software version 3. Heterozygosity per location. Triangles represent male samples and circles represent female samples. These differences may arise from differences in sequencing depth across regions, excessive amplification in the PCR step, short read length, or problems with the sequencing platform The number of markers ranged from 2. After quality filtering, 24, high-quality SNPs were identified across all samples and 29, SNPs were identified for 68 Iranian individuals, including one Afghanistan sample. The transition:transversion ratio was 1. The majority of SNPs The ratio of transitions to transversions is consistent with other studies in various species 36 , 37 , 38 , S2 and a mean of 0. This pattern is common among groups that experience heterozygote advantage, wherein rare alleles are retained at low frequencies. Average heterozygosity was estimated at 0. This estimate of heterozygosity is similar to that found by Sawler et al. Population differentiation resulting from genetic structure was estimated using F ST. Low values indicate that genetic diversity is higher within individuals from these locations than between locations, a pattern consistent with gene flow between populations. F ST estimates above 0 indicate a reduction in genetic exchange between population with a value of 1 indicating complete isolation. Across all individuals the maximum F ST , 0. CAN37 was previously described as hemp type and originating in France, however, Sawler et al. We also estimated genetic differentiation among marijuana and hemp accessions and Iranian samples and found a larger F ST across hemp 0. Similar to Sawler et al. Marijuana and Iranian cannabis clustered together with genetic distances of 0. Overall, these results suggest that Iranian collections are more genetically similar to marijuana collections than hemp. It is important to note that neither of the reference genomes used in this study were from a male plant. Our approach failed to identify sex specific alleles at high frequency outside of the sex determining region. Previous analyses have shown that marijuana and fibre types differ across the genome and not just at specific loci. Our approach failed to identify positions with significant deviations in allele frequency among 19, SNPs between types. Sawler et al. Our reanalysis of these data identified 9 SNPs with allele frequencies of 1 for hemp and 0 for marijuana and 92 SNPs with allele frequency 0 for hemp and 1 for marijuana. All positions and their frequencies are supplied in Table S3. An initial analysis of population structure was performed using individual-based principal component analysis PCA. This plot revealed two nonconforming individuals CANM and ArdF that failed to group with the two main clusters. Previous outliers from Sawler et al. Visualisation of DAPC results using the first 22 principal components clearly clusters, marijuana, hemp, germplasm collections, and Iranian collections Fig. Principle components analysis of 95 samples from Iranian collection, 43 hemp and 71 marijuana samples using 13, SNPs. Hemp samples are colored blue and marijuana samples are colored red. D stand for New Data and P. D stand for previously analyzed data. Discriminant analysis of principal components DAPC results. B Scatterplot based on the DAPC output for four assigned genetic clusters, each indicated by different colours. Dots represent different individuals. PCA within the Iranian collection identified two primary clusters Fig. This pattern is consistent with reduced gene flow from cluster 1 which includes 18 samples Fig. S4 , Table S5. According to these results we can define distinct genetic clusters for locations Neyriz, Piranshahr, Gahwareh, Arak, Urmia and Abhar. This pattern is consistent with perennial dioecious plants wherein the majority of variation is harbored within populations Together these suggest that Iranian cannabis populations tend to share more DNA with geographically proximate populations where may have genomes made up of mixtures of inferred source populations, while our simulation incorporated drift between locations, but not admixture. Individual-based principal components analysis for 35 Iranian regions and Afghanistan using 29, SNPs. Male plants are colored blue and female plants are colored red. Cannabis , both marijuana and fibre types, is a globally important plant, driving a multi-billion dollar industry. Unraveling the population genomic parameters of natural populations can help identify sources of genetic diversity, as well as describing patterns of domestication for this widely used plant. In this study, we have found that natural populations of Cannabis in Iran are more closely related to marijuana than hemp, and that these populations harbor unique pools of genetic diversity. Taken together these data support the hypothesis that reduced diversity across fibre types suggests that hemp cultivars are derived from marijuana Population analyses among all accessions sampled defined 4 distinct genetic clusters Figs 3 , 4 and 5. These analyses support previous findings Sawler et al. This evidence provides support for the hypothesis that Iranian cannabis harbors unique genetic diversity and may represent a distinct genetic lineage of marijuana. Heterozygosity indicates levels of genetic diversity within populations, and has also been used to estimate genetic distance between populations 49 , Consistent with genetic diversity levels in the present study, previous estimates of heterozygosity across diverse marker types e. However, it should be noted that one study found lower levels of heterozygosity in hemp varieties across samples and SNPs It has been suggested that this may result from limited hemp sample representation in the collection Heterozygosity estimates within our Iranian collection were similar to those found by Sawler et al. If, as we surmise, Iranian cannabis are marijuana accessions, then these accessions likely represent remnants of cultivated germplasm from the other regions, possibly through migration of Cannabis from neighboring countries like Afghanistan and Pakistan into Iran. These results demonstrate that Iran is a public repository of marijuana genetic diversity; however, the loss of this unique germplasm is of great concern as there are no breeding programs and growing Cannabis is associated with strict legal penalties. These observations reveal that Iranian cannabis, despite clear evidence of admixture likely the result of breeding , harbors distinguishable pools of genetic diversity. The lack of strong population differentiation is unsurprising since, all known cultivars of Cannabis are wind-pollinated and highly heterozygous confirmed by AMOVA, Table S6. Population structure is further complicated by the fact that marijuana cultivars are clonally propagated in order to retain high-levels of THC production. Intentionally growing Cannabis plants in Iran is punishable by prison sentence, populations of plants are more likely to have arisen from seed and therefore represent more natural populations. Although Iranian cannabis is not likely a subspecies it does represent a genetically unique variety of marijuana, and thus provides a novel source of genetic material for cultivar development. In plants, the sex determination system is important for two reasons; first, understanding the role of sex determination in shaping plant evolution, and second, diversity in the mechanisms through which sex is determined. There have been many studies on gender in Cannabis , including whether a plant should be classified as female or male, and in addition to the identification of sex chromosomes 21 , some male-specific DNA markers have been identified in C. Sex determination in Cannabis is a complex process and can be modified or reversed by environmental factors and chemical treatment 55 , Additionally, male flowers are able to develop on female plants under extreme conditions Because confirmed sex-associated DNA markers such as MADC2 sometimes fail to discriminate sex phenotype 22 , we attempted to identify sex associated markers from autosomal regions. While our study generated thousands of differentiating markers, we failed to find sex locus specific SNPs. This is likely because no male reference genome is available and the proportion of coding regions covered by the GBS derived SNPs. Future studies can capitalize on the utility of high-throughput sequencing technologies to look for markers associated with sex-determining loci, in particular coding derived SNPs e. We were able, however, to identify marijuana and fibre type specific markers through reanalysis of previously published data. Our conclusions, consistent with previous studies, show that genetic differences between hemp and marijuana accessions are widely distributed across the genome Comparative analysis of Purple Kush marijuana and Finola fibre genomes revealed highly discriminative SNPs that are distributed across the genome and are not restricted to particular loci e. In this study, we identified SNPs that appear to be tightly linked to type, and are outside of cannabinoid genes, which should prove useful for future research. More immediately, these markers can be validated for early and rapid identification of marijuana and fibre type plants for current breeding programs. Natural populations of Cannabis in Iran were identified and seeds were collected for growing in the field in university of Tehran. Sex identities were verified using taxonomic keys. Figure 1 was produced using the R software version 3. Additionally Dplyr version 0. DNA was extracted using a Qiagen DNeasy plant mini-kit, from leaf tissue of one female and one male plant from each location. We performed in silico digestion of the Cannabis genome sequence with Pst I and Apek I to select the best restriction enzyme library preparation. Libraries were prepared using the GBS protocol published by Sonah et al. High-throughput was performed on an Illumina Hiseq. After unzipping fastq. To elucidate the relationship of Iranian cannabis with marijuana and fibre type accessions, we merged our data with marijuana and hemp data prepared by Sawler et al. In a high-throughput genotyping workflow, alignment of short reads to a reference genome is the first step after read processing and filtering. BWA 62 was used to map reads of the individual genotypes to the reference genome with the default parameters. Reads mapped to Purple Kush canSat3: a special variety of hemp and Finola finola1: a special variety of marijuana C. The mapping outputs were used for removing unmapped reads to produce BAM files using Samtools 63 and only reads mapping to a unique location in the genome were retained. FreeBayes was run using default parameters. This was performed for or males and females and drug and non-drug types separately to find positions linked to gender and type. Bi-allelic, missingness, quality, and depth were filtered. Bi-allelic markers were identified by a command-line written in our lab. This package can filter each position for each individual. After screening a few markers we found that read depth and quality were not being appropriately filtered for our data set and therefore we opted to use vcflib. Finally, summary statistics were collected using vcf-stats before and after data filtering. Identification of DNA markers associated with gender and type was carried out based on comparison of SNP allele frequency differences between each group female-male and marijuana-fibre. To do this, we called SNPs for sample pairs female and male, marijuana and fibre, separately using FreeBayes We computed the fixation index F ST using VCFtools 66 among all wise locations in the Iranian collection and also between marijuana and hemp types. Estimation of heterozygosity for each individual was conducted with custom command-line scripts by dividing the number of heterozygous sites by the number of non-missing genotypes. The number of heterozygous sites was counted by vcflib tools. Plotting PCA results was completed via the ggplot2 59 package in Rstudio version 0. We also applied discriminant analysis DA of principal components 44 using the adegenet package Discriminant analysis can ascribe relationships for pre-defined groups without relying on a particular population genetics mode Files were read using the function read. In DAPC, data is first transformed using a principal components analysis PCA and subsequently the number of genetic clusters was assessed using the find. For k-means clustering, all of the principal components were retained. The K value with the lowest BIC was selected as the optimal number of clusters. DAPC was implemented using the optimized number of principal components as determined by the optim. Other values of K were tested not shown , but did not provide further optimization or descriptive value. MIGRATE-N was implemented with following parameters: the Bayesian inference strategy, for number of recorded steps in chain, a burn-in of for each chain and a full migration model with two population sizes and two migration rates. Significance levels for variance components and F-statistics were estimated using permutations. Small, E. A practical and natural taxonomy for Cannabi s. Article Google Scholar. Adams, I. Cannabis : pharmacology and toxicology in animals and humans. Gaoni, Y. Isolation, structure and partial synthesis of an active constituent of hashish. Journal of the American Chemical Society. Siniscalco Gigliano, G. Forensic Science Review. Google Scholar. Taura, F. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. Federation of European Biochemical Societies. Broseus, J. Forensic Science International. Hillig, K. Genetic evidence for speciation in Cannabis Cannabaceae. Genetic Resources and Crop Evolution. Bradshaw, R. New fossil evidence for the past cultivation and processing of hemp Cannabis sativa L. New Phytologist. Duvall, C. Drug laws, bioprospecting and the agricultural heritage of Cannabis in Africa. Space Polity. Herbig, C. Palaeobotanical evidence for agricultural activities in the Eifel region during the Holocene: plant macro-remain and pollen analyses from sediments of three maar lakes in the Quaternary Westeifel Volcanic Field Germany, Rheinland-Pfalz. Vegetation History and Archaeobotany. Li, H. The origin and use of cannabis in eastern asia linguistic-cultural implications. Economic Botany. Murphy, T. Hemp in ancient rope and fabric from the Christmas Cave in Israel: talmudic background and DNA sequence identification. Journal of Archaeological Science. Piluzza, G. Differentiation between fiber and drug types of hemp Cannabis sativa L. Rivoira, G. Patron, Bologna Janick and A. Russo, E. Phytochemical and genetic analyses of ancient cannabis from Central Asia. CAS Google Scholar. Faeti, V. Plant Breeding. Zeven, A. In: Dictionary of cultivated plants and their centres of diversity. Green, G. Mandolino, G. Theoretical and Applied Genetics. Sakamoto, K. Plant cell physiology. Gilmore, S. Isolation of microsatellite markers in Cannabis sativa L. Molecular Ecology Notes. Pacifico, D. Genetics and marker-assisted selection of the chemotype in Cannabis sativa L. Molecular Breeding. Alghanim, H. Development of microsatellite markers in Cannabis sativa for DNA typing and genetic relatedness analyses. Analytical and Bioanalytical Chemistry. Hakki, E. Inter simple sequence repeats separate efficiently hemp from marijuana Cannabis sativa L. Electronic Journal of Biotechnology. Lynch, R. Genomic and chemical diversity in Cannabis. Critical Reviews in Plant Sciences. Sawler, J. The Genetic Structure of Marijuana and Hemp. PLoS One. The draft genome and transcriptome of Cannabis sativa. Genome Biology 12 Elshire, R. A robust, simple genotyping-by-sequencing GBS approach for high diversity species. Deschamps, S. Genotyping-by-Sequencing in Plants. Soorni, A. DNA fingerprinting of Leonurus cardiaca L. Biochemical Systematics and Ecology. Bailey, T. Practical guidelines for the comprehensive analysis of ChIP-seq data. Batley, J. Plant Physiol. Coulondre, C. Molecular basis of base substitution hotspots in Escherichia coli. Pootakham, W. Shearman, J. SNP identification from RNA sequencing and linkage map construction of rubber tree for anchoring the draft genome. Tajima, F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. De Meijer, E. Variation of Cannabis with reference to stem quality for paper pulp production. Industrial Crops and Products. Nei, M. Genetic distance between populations. The American Naturalist. Diversity of cannabis. Wageningen: Wagenigen University. Jombart, T. Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet. Beerli, P. Comparison of Bayesian and maximum-likelihood inference of population genetic parameters. New York: Cambridge University Press. Excoffier, L. Arlequin version 3. Evolutionary Bioinformatics Online. Sheng, Y. Annals of Botany. Chakraborty, R. Relationship between heterozygosity and genetic distance in the three major races of man. American Journal of Physical Anthropology. Guerreiro, J. Effect of average heterozygosity on the genetic distance of several Indian tribes from the Amazon region. Annals of Human Biology. Gao, C. Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers. Hu, Z. Journal of Plant Genetic Resources. Zhang, L. Genetics and Molecular Research. Chailakhyan, M. Genetic and hormonal regulation of growth, flowering and sex expresion in plants. American Journal of Botany. Mohan Ram, H. Sex reversal in the female plants of Cannabis sativa by cobalt ions. Proceedings of the Indian Academy of Sciences. Clarke, R. Aarau, Schweiz: AT Verlag Onofri, C. Sequence heterogeneity of cannabidiolic- and tetrahy- drocannabinolic acid-synthase in Cannabis sativa L. Phytochemistry , 57—68 Wickham, H. Springer-Verlag New York, Herten, K. GBSX: a toolkit for experimental design and demultiplexing genotyping by sequencing experiments. BMC Bioinformatics. Aronesty, E. Comparison of Sequencing Utility Programs. The Open Bioinformatics Journal 7 , 1—8 Fast and accurate long-read alignment with Burrows—Wheeler transform. Garrison E. Haplotype-based variant detection from short-read sequencing. Quinlan, A. BEDTools: a flexible suite of utilities for comparing genomic features. Danecek, P. The variant call format and VCFtools. Bradbury, P. Knaus, B. Molecular Ecology Resources. Pre print, Pembleton, L. StAMPP: an R package for calculation of genetic differentiation and structure of mixed-ploidy level populations. Raj, A. Lawson, D. Inference of population structure using dense haplotype data. PLoS Genetics. Download references. The authors are grateful to the Ministry of Science, Research and Technology of Iran as a funding source of this project. Special thanks to Dr. You can also search for this author in PubMed Google Scholar. All the authors participated in the discussion of the results and writing of the article. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and permissions. Sci Rep 7 , Download citation. Received : 23 May Accepted : 01 November Published : 15 November Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Genetic Resources and Crop Evolution Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. Download PDF. Subjects Natural variation in plants Plant domestication. Abstract Cannabis sativa has a complex history reflected in both selection on naturally occurring compounds and historical trade routes among humans. Genetic insights into agronomic and morphological traits of drug-type cannabis revealed by genome-wide association studies Article Open access 22 April Genome-wide diversity analysis to infer population structure and linkage disequilibrium among Colombian coconut germplasm Article Open access 22 February Introduction Cannabis sativa L. Results Sequencing and mapping In total 98 cannabis samples were digested, sequenced, and genotyped these included, 70 samples representing 35 locations in Iran Fig. Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Discussion Cannabis , both marijuana and fibre types, is a globally important plant, driving a multi-billion dollar industry. Materials and Methods Collection of Genetic Material Natural populations of Cannabis in Iran were identified and seeds were collected for growing in the field in university of Tehran. Mapping, SNPs Discovery and filtering In a high-throughput genotyping workflow, alignment of short reads to a reference genome is the first step after read processing and filtering. Scan for Identification of SNPs associated with gender and type Identification of DNA markers associated with gender and type was carried out based on comparison of SNP allele frequency differences between each group female-male and marijuana-fibre. Analysis of population structure We computed the fixation index F ST using VCFtools 66 among all wise locations in the Iranian collection and also between marijuana and hemp types. References Small, E. Article Google Scholar Adams, I. Google Scholar Taura, F. Article Google Scholar Duvall, C. Article Google Scholar Herbig, C. Article Google Scholar Li, H. Article Google Scholar Murphy, T. Article Google Scholar Piluzza, G. Article Google Scholar Rivoira, G. Article Google Scholar Zeven, A. Article Google Scholar Lynch, R. Article Google Scholar Sawler, J. Article Google Scholar Nei, M. Article Google Scholar Chailakhyan, M. Google Scholar Clarke, R. Acknowledgements The authors are grateful to the Ministry of Science, Research and Technology of Iran as a funding source of this project. Haak Authors Aboozar Soorni View author publications. View author publications. Ethics declarations Competing Interests The authors declare that they have no competing interests. Additional information Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic supplementary material. Supplementary Information. Supplementary Table S1. Supplementary Table S2. Supplementary Table S3. About this article. Cite this article Soorni, A. Copy to clipboard. Publish with us For authors Language editing services Submit manuscript. Search Search articles by subject, keyword or author. Show results from All journals This journal. Advanced search. Close banner Close. Email address Sign up. Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing.
Assessment of Genetic Diversity and Population Structure in Iranian Cannabis Germplasm
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Metrics details. The potentially adverse effects of cannabis marijuana , a common leisure compound, on male reproductive performance are a reason for concern. The main objective of this study was to investigate the possible mechanism underlying the toxic effects of THC with a mechanistic insight into Sertoli cell-based reproductive dysfunction. The Mus musculus Sertoli cell line TM4 was cultured and exposed to different concentrations of THC and, MTT 3- 4, 5-Dimethylthiazolyl -2,5-diphenyltetrazolium bromide assay was then performed for evaluating cell viability. The expression of caspase-3 gene and genes related to growth factors were analyzed by real-time RT-PCR. Western blotting was performed for evaluating protein expression level. There was also a significant reduction in related protein levels in THC group. Administration of the THC promotes cytotoxic and apoptotic effects on TM4 cells partly through down-regulation of growth factors expression. Increased apoptosis, over expression of caspase-3, and down-regulation of growth factors expression in Sertoli cells exposed to THC may be a reflection of THC-induced testicular toxicity, which may be partly involved in infertility associated with marijuana smoking or medical cannabis use. Peer Review reports. Substance use among men of reproductive age remains a significant health concern, as that users of most addictive drugs show hypogonadism and impaired fertility. Emerging evidence has demonstrated that marijuana impairs male reproductive activity so that regular marijuana use has been linked to a lower semen quality and testosterone level \[ 2 , 3 , 4 , 5 \] as well as a higher risk of testicular cancer \[ 6 , 7 \]. The effects of THC on male fertility are still a topic of ongoing research. Some studies have reported no impact of THC exposure on sperm concentration or germ cell lineage in human \[ 9 \] and mice \[ 10 \] testis tissue. However, many other studies have shown that THC affects male fertility and causes gonadal dysfunction mainly at the testis and sperm levels. Accordingly, THC alters serum testosterone level and decreases sperm count, motility, normal morphology and acrosome reaction \[ 2 , 11 , 12 , 13 , 14 \]. In spite of these reports indicating that THC is involved in testicular toxicity and reproductive dysfunction, the underlying cellular and molecular mechanisms remain incomplete. Impaired spermatogenesis and irreversible infertility can result from alterations in Sertoli cell function and loss \[ 18 , 19 \]. Although toxicants, such as THC, are known to disrupt Sertoli cells, the detailed molecular events and underlying mechanisms involved in this process remain largely unknown. It has been suggested that THC-induced testicular toxicity may happen partly through induction of the early apoptosis of testicular germ cells and somatic cells \[ 20 , 21 , 22 , 23 \]. THC-induced apoptosis was also related to cytochrome c release and caspase-3 activation in cultured neurons and Sertoli cells \[ 23 , 24 \]. Several previous reports suggest that altered secretion of growth factors, as essential regulators in the process of cell life and development in the testis, are important contributing factors for several chronic conditions attributed to testicular toxicity and infertility. The removal of VEGFA isoforms causes subfertility and a reduction in the quantity of sperm in mice \[ 29 \]. The glial cell-derived neurotrophic factor GDNF is also released by Sertoli and TM4 cells \[ 30 \] and may increase stem cell numbers and sperm production \[ 31 \]. Another common growth factor that is produced by Sertoli cells is the epidermal growth factor EGF which stimulates the proliferation of numerous cell types and appears to be involved in the formation of the testis and spermatogenesis \[ 32 , 33 \]. Fibroblast growth factor FGF is another Sertoli cell survival factor that plays a role in the proliferation and differentiation of testicular cells and spermatogenesis \[ 34 , 35 \]. Based on the previous reports, the production and secretion of growth factors and expression of their receptors can be impressed by cannabinoids in different cells and tissues \[ 36 , 37 , 38 , 39 \]. It has been shown that doses of THC equivalent to those found in the serum of cannabis users inhibit proliferation of different cells by affecting several genes that encode for growth and apoptosis \[ 40 , 41 \]. To the best of our knowledge, there are no studies examining the effects of THC on testicular growth factors and their expression profile. Therefore, this study aimed to determine through which mechanism s , an alteration of Sertoli cell death occurs as a result of THC exposure and investigate critical growth biomarkers that may link cannabinoid system components to apoptotic pathway activation. In the first phase of our study, we evaluated cell viability by examining a range of different concentrations of THC to determine the concentration-response relationship of THC and establish its toxicological thresholds. Accordingly, the cells were exposed to THC at the final concentrations of 0 cells exposed to a THC-free media, as the control group , 0. The selection of THC concentrations and duration of exposure was based on previous studies that employed similar grouping strategies and suggested that this range of concentrations is more likely to produce toxic effects \[ 23 , 42 , 43 \]. This process was carried out in two independent tests with duplicate cultures. The MTT 3- 4, 5-Dimethylthiazolyl -2,5-diphenyltetrazolium bromide assay was then performed to investigate the cell viability of different concentration groups. The following equation was used to determine the percentage of viable cells: O. Analyses of the data produced mean and standard error of means SEM , obtained from 2 determinations. On the other hand, experiments have shown that dead cells settle slower than live cells, and this opens up the possibility to bleed out dead cells in a continuous centrifuge. Tests in a cell separator prove that this is feasible, and a significant portion of the dead cells can be removed from the system \[ 46 , 47 \]. Cell pellets were used for RNA extraction. The expression of the caspase-3 gene as well as genes related to growth factors were investigated by real-time RT-PCR, using specific primers Table 1. After h of the last treatment, the cells were trypsinized and cell pellets were used for RNA extraction. The 2-duct method was used to figure out fold changes in gene expression as a ratio of the levels of expression in the THC-exposed group to the levels of expression in the control group. For protein extraction, a cell lysis buffer including protease and phosphatase inhibitors was utilized. The protein concentration in the cell lysates was determined using the Bradford reagent using bovine serum albumin BSA as the standard after cell lysis Bio-Rad, TX. Normally distributed data O. The data related to gene and protein expressions, which were not normally distributed, were analyzed by the Mann-Whitney U test. The MTT results indicated that the percentage of cell viability was reduced significantly with an increase in the concentration of THC. Stars show the statistical significance of change among the groups. The results showed that caspase-3 mRNA levels was significantly increased to 1. THC significantly increased caspase-3 expression level. Star shows the statistical significance of change between the groups. Following above mentioned MTT and caspase-3 evaluations, the next series of experiments were performed to investigate the possible mechanism of apoptotic effects of THC in TM4 Sertoli cells. THC significantly decreased growth factors expression levels. Stars show the statistical significance of change between the groups. FGF protein level was also significantly decreased 0. In spite of the considerable knowledge about THC-induced testicular toxicity, there is little available information regarding cellular basis and molecular mechanisms underlying this pathological process. Lack of such knowledge interrupts evidence-based development of pharmacological intervention to repair damage caused by THC. On the other hand, no clear correlation between marijuana abuse and reproductive dysfunction can be demonstrated in human studies, since drug-dependent men often abuse other substances such as tobacco, opioids and alcohol as well as marijuana. Moreover, marijuana abuse and its possible correlations to testicular damage and later infertility can usually be studied retrospectively in humans. In vitro models allow the investigation of such relations with prospective, well-controlled study designs and allow for meticulous regulation over experimental conditions, such as the duration and concentration of toxicants exposure. On the other hand, while human and in vivo animal studies can indeed provide valuable insights into the overall impact of toxicants on spermatogenesis, identifying cellular and molecular changes, including the cascade of events, can be challenging when studying the testis as a whole \[ 48 \]. However, techniques such as sperm staining and RNA and protein extraction from testicular tissue for a range of different cellular and molecular investigation, can indeed provide valuable information in this regard. Therefore, while acknowledging the complementary role of animal experiments, we also emphasize the significance of in vitro models in offering new insights into the study of spermatogenesis, as they provide a practical approach to investigate the cellular and molecular mechanisms underlying testicular injury caused by toxicants \[ 48 , 49 \]. Previous researches show that the detrimental effects of environmental toxins on Sertoli cells, as observed in in vitro models, can be replicated in in vivo studies \[ 23 \]. The TM4 cell line is the most extensively researched Sertoli cell line and provides a readily available supply of cells with consistent and predictable properties and similar behavior to primary cultures of Sertoli cells \[ 50 \]. TM4 cells retained Sertoli cell-like characteristics, making them a valuable in vitro model for studying the effects of toxicants such as THC on Sertoli cells \[ 48 \]. Therefore, the present in vitro study was carried out to investigate cell viability and expression profile of caspase-3 and a number of key testicular growth factors in TM4 Sertoli cells exposed to THC to gain mechanistic insight into the THC-induced testicular toxicity. The current study consisted of two distinct phases. During first phase of the study, we conducted preliminary experiments to explore the impact of THC on Sertoli cell viability and apoptosis. Although this phase was not the primary focus of our research, it served two key objectives. Firstly, it allowed us to validate and confirm prior research on the effects of THC on Sertoli cells viability. Secondly, it enabled us to determine the effective concentration of THC IC25 or IC50 required for the subsequent phase of our investigation which can vary slightly across different research groups and laboratory conditions. The reduction of cell viability indicated by MTT was further confirmed by caspase-3 evaluation. These findings confirmed our previous results which demonstrated that THC significantly reduced the expression level of pro-caspase3 protein, while simultaneously increasing TUNEL positive apoptotic cells and the expression level of cleaved caspase3 protein in cultured TM4 Sertoli cells \[ 23 , 42 \]. This pro-apoptotic effect of THC was in line with the results of other studies that showed THC, at concentrations comparable to those used in this study, inhibits the proliferation of different cells and enhances apoptosis in different tissues \[ 51 , 52 , 53 , 54 \]. For example, Almadaa et al. Therefore, based on these explanations and given THC complex pharmacokinetics, indicating precise evidence-based blood levels for THC is challenging \[ 58 \]. On the other hand, a clear comparison between the concentration level of THC in human, in vivo and in vitro studies is not possible, since the in vitro experiments performed at non-physiological conditions do not necessarily correspond to in vivo results. All together, these data indicate that threshold concentration of THC to show a significant reduction of Sertoli cell viability seems to be higher than average THC blood level in marijuana users. However we have several explanations to claim that regular and prolonged cannabis consumers are at risk of Sertoli cells damage. Firstly, the oral administration of THC produces more active metabolite, which could more efficiently reach the effect site than THC \[ 59 , 60 , 61 \]. Secondly, the slow absorption kinetics of THC produces sustained plateau levels in the blood, which could influence the body and tissue distribution. Giroud et al. Thirdly, because cannabinoids accumulate in fat, chronic marijuana consumption may induce more blood THC level after a week or more of abstinence \[ 58 \]. And finally, we have recently shown that prolonged exposure to lower concentrations of THC led to significant reduction of Sertoli cell viability in an in vitro model \[ 23 \]. Accordingly, exposure to 0. THC exposure may interfere with Sertoli cell function, resulting in abnormalities in Sertoli cell markers. In the second phase of our study, which was the main objective of our study, we aimed to gain a deeper understanding of the mechanisms underlying THC-induced testicular toxicity, with a specific emphasis on THC-induced Sertoli cell apoptosis. Previous researches have shown that the influence of the cannabinoid system on the expression or production of growth factors in both normal cells with controlled proliferation activity and tumor cells with poor differentiation and previously enhanced proliferation activity is subject to variation based on differences in experimental conditions. According to previous reports, cannabis treatment decreased secreted protein and mRNA expression level of VEGF in prostate cancer cell lines \[ 63 \]. It has also been reported that high concentrations of cannabidiol decreased transforming growth factor TGF b production in human fibroblast extracellular matrix \[ 65 \]. Moreover, increased plasma levels of cannabinoids were associated with lower VEGF concentrations in medical cannabis users among chronic pain patients \[ 66 \] and with lower circulating levels of brain-derived neurotrophic factor \[ 67 \] and nerve growth factor \[ 68 \] in physically active cannabis users. These findings suggested that THC shows cytotoxic and apoptotic effects in TM4 Sertoli cells partly through down-regulation of growth factors expression. If caused in vivo, this may manifest as increased testicular apoptosis and hence compromised testicular growth and function in marijuana smokers or medical cannabis users. Such findings suggest that marijuana exposure either recreationally or medicinally may increase the susceptibility to Sertoli cell-based reproductive dysfunction. However, the exact intracellular signaling pathways and molecular mechanisms through which the activation of the cannabinoid system leads to low expression of growth factors and apoptosis are not fully understood and may vary depending on the specific context and cell type involved. Several pathways and factors cyclooxygenase- and prostaglandin-mediated mechanisms, nitro-oxidative stress and immune-inflammatory signaling pathways could be implicated in this phenomenon \[ 43 , 69 \]. Accordingly, a recent research indicates that THC caused a reduction in the secretion of insulin growth factor 2 in human trophoblast cells, via oxidative stress responses \[ 43 \]. Cannabinoids could also potentially affect gene expression, transcription factors or nuclear receptors that regulate the production of growth factors. For example, peroxisome proliferator-activated receptors PPARs , which are a family of nuclear receptors and regulators of a plethora of target genes involved in cell differentiation, proliferation and growth factors production \[ 70 , 71 \], are activated by a large number of both phyto- and endo-cannabinoids \[ 70 \]. Likewise, growth factors can affect apoptotic pathways through different signaling mechanisms, depending on the specific type of growth factor and the type of cell being modified. It has been reported that THC-induced apoptosis was preceded by significant changes in the expression of genes involved in the MAPK signal transduction pathways in leukemic cell lines \[ 75 \]. Altogether, we hypothesize that activation of cannabinoid system by THC in TM4 cells may lead to low expression of growth factors which, in turn, triggers apoptotic pathways. However, further molecular and cellular researches and biochemical measurements are needed to fully understand the possible molecular connections and pathways between cannabinoid system, expression of growth factors and apoptotic mechanisms. Conducting research using cultured primary Sertoli cells and adult Sertoli cells, in vivo models of THC administration in animal models, and a seminiferous tubule culture approach could also help elucidate these relationships and the related hypotheses. In summary, exposure to THC significantly decreased cell viability and increased apoptosis in TM4 Sertoli cells, at least in part, through growth factors-dependent pathways. Decreased cell viability, over-expression of caspase-3 mRNA level and down-expression of growth factors mRNA and protein levels in Sertoli cells exposed to THC may be a reflection of THC-induced testicular injury resulting in enhanced Sertoli cell apoptosis, which may be partly involved in reproductive dysfunction associated with marijuana smokers or medical cannabis users. Simultaneous alcohol and marijuana use among young adults: A scoping review of prevalence, patterns, psychosocial correlates, and consequences. Alcohol res: curr rev. Article Google Scholar. Association between use of marijuana and male reproductive hormones and semen quality: A study among 1, healthy young men. Am J Epidemiol. Article PubMed Google Scholar. Hsiao P, Clavijo RI. Adverse effects of cannabis on male reproduction. 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Peroxisome proliferator-activated receptors and angiogenesis. Nutrit metab cardiovas diseases. Histol Histopathol. Understanding MAPK signaling pathways in apoptosis. Int J Mol Sci. Am J Transl Res. Cannabis-induced cytotoxicity in leukemic cell lines: The role of the cannabinoid receptors and the MAPK pathway. Activation of type 2 cannabinoid receptor CB2R by selective agonists regulates the deposition and remodelling of the extracellular matrix. Biomed pharmacother. Download references. This research was conducted as a part of a student thesis project for M. You can also search for this author in PubMed Google Scholar. All of the authors discussed the results and reviewed the manuscript. Correspondence to Shiva Roshan-Milani. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Reprints and permissions. Mohammadpour-Asl, S. In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Deltatetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity. BMC Pharmacol Toxicol 24 , 61 Download citation. Received : 26 April Accepted : 01 November Published : 09 November Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Research Open access Published: 09 November In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Deltatetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity Shadi Mohammadpour-Asl 1 , 2 , Shiva Roshan-Milani ORCID: orcid. Abstract The potentially adverse effects of cannabis marijuana , a common leisure compound, on male reproductive performance are a reason for concern. Introduction Substance use among men of reproductive age remains a significant health concern, as that users of most addictive drugs show hypogonadism and impaired fertility. Full size image. Discussion In spite of the considerable knowledge about THC-induced testicular toxicity, there is little available information regarding cellular basis and molecular mechanisms underlying this pathological process. Article Google Scholar Chayasirisobhon S. Acknowledgements This research was conducted as a part of a student thesis project for M. View author publications. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Supplementary Information. Additional file 1. Additional file 2. Additional file 3. About this article. Cite this article Mohammadpour-Asl, S. Copy to clipboard. Contact us General enquiries: journalsubmissions springernature.
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