Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity.

Inheritance of 5-methylcytosine from one cell generation to the next by DNA methyltransferase 1 (DNMT1) plays a key role in regulating cellular identity. While recent work has shown that the activity of DNMT1 is imprecise, it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides to find that the fidelity of DNMT1-mediated maintenance methylation is related to the local density of DNA methylation and the landscape of histone modifications. To gain deeper insights into methylation/demethylation turnover dynamics, we first extended Dyad-seq to quantify all combinations of 5-methylcytosine and 5-hydroxymethylcytosine at individual CpG dyads. Next, to understand how cell state transitions impact maintenance methylation, we scaled the method down to jointly profile genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from single cells (scDyad&T-seq). Using scDyad&T-seq, we demonstrate that, while distinct cell states can substantially impact the activity of the maintenance methylation machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.

Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity.

Transmission of 5-methylcytosine (5mC) from one cell generation to the next plays a key role in regulating cellular identity in mammalian development and diseases. While recent work has shown that the activity of DNMT1, the protein responsible for the stable inheritance of 5mC from mother to daughter cells, is imprecise; it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method that combines enzymatic detection of modified cytosines with nucleobase conversion techniques to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides. We find that the fidelity of DNMT1-mediated maintenance methylation is directly related to the local density of DNA methylation, and for genomic regions that are lowly methylated, histone modifications can dramatically alter the maintenance methylation activity. Further, to gain deeper insights into the methylation and demethylation turnover dynamics, we extended Dyad-seq to quantify all combinations of 5mC and 5-hydroxymethylcytosine (5hmC) at individual CpG dyads to show that TET proteins preferentially hydroxymethylate only one of the two 5mC sites in a symmetrically methylated CpG dyad rather than sequentially convert both 5mC to 5hmC. To understand how cell state transitions impact DNMT1-mediated maintenance methylation, we scaled the method down and combined it with the measurement of mRNA to simultaneously quantify genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from the same cell (scDyad&T-seq). Applying scDyad&T-seq to mouse embryonic stem cells transitioning from serum to 2i conditions, we observe dramatic and heterogenous demethylation and the emergence of transcriptionally distinct subpopulations that are closely linked to the cell-to-cell variability in loss of DNMT1-mediated maintenance methylation activity, with regions of the genome that escape 5mC reprogramming retaining high levels of maintenance methylation fidelity. Overall, our results demonstrate that while distinct cell states can substantially impact the genome-wide activity of the DNA methylation maintenance machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.

Spatial analysis of future climate risk to stormwater infrastructure.

Climate change is expected to result in more intense precipitation events that will affect the performance and design requirements of stormwater infrastructure. Such changes will vary spatially, and climate models provide a range of estimates of the effects on events of different intensities and recurrence. Infrastructure performance should be evaluated against the expected range of events, not just rare extremes. We present a national-scale, spatially detailed screening assessment of the potential effects of climatic change on precipitation, stormwater runoff, and associated design requirements. This is accomplished through adjustment relative to multiple future climate scenarios of precipitation intensity-duration-frequency analyses presented in NOAA Atlas 14, which are commonly used in infrastructure design. Future precipitation results are estimated for each Atlas 14 station (these currently omit the Pacific Northwest). Results are interpolated using a geographically conditioned regression kriging approach to provide information about potential climate change impacts in a format more directly useful to local stormwater managers. The intensity of 24-h events with 2-year or greater recurrence is likely to increase in most areas of the United States leading to increased runoff and potential need for increased storage volumes. Changes in more frequent events (e.g., the 90th percentile event) commonly used in design of green infrastructure are relatively less.

Polymorphisms in the HSF2, LRRC6, MEIG1 and PTIP genes correlate with sperm motility in idiopathic infertility.

The aim of this study was to investigate the association of 24 functionally important single nucleotide polymorphisms (SNPs) with male infertility. In this cross-sectional study, we genotyped 24 functionally important single nucleotide polymorphisms in 24 infertility candidate genes in 500 oligo-/astheno-/oligoastheno-/normo-zoospermic infertile men with idiopathic infertility. Sequenom iPlex gold assay was used for genotyping. Sperm count and motility were compared between prevalent genotypes at each test locus. We did not observe any significant difference in the average sperm count between the alternate genotypes for the loci in the KLK3, LRRC6, MEIG1, HSF2, ESR2 and PTIP genes. However, we observed a significant difference in sperm motility between the alternate genotypes for the loci in the LRRC6, MEIG1, HSF2 and PTIP genes. Polymorphisms in the LRRC6 (rs200321595), MEIG1 (rs150031795), HSF2 (rs143986686) and PTIP (rs61752013) genes show association with sperm motility.

Histone Methylation Regulates Gene Expression in the Round Spermatids to Set the RNA Payloads of Sperm.

Gene expression during spermatogenesis undergoes significant changes due to a demanding sequence of mitosis, meiosis, and differentiation. We investigated the contribution of H3 histone modifications to gene regulation in the round spermatids. Round spermatids were purified from rat testes using centrifugal elutriation and Percoll density-gradient centrifugation. After enzymatic chromatin shearing, immuno-precipitation using antibodies against histone marks H3k4me3 and H3K9me3 was undertaken. The immunoprecipitated DNA fragments were subjected to massive parallel sequencing. Gene expression in round spermatids and sperm was analyzed by transcriptome sequencing using next-generation sequencing methods. ChIP-seq analysis showed significant peak enrichment in H3K4me3 marks in active chromatin regions and H3K9me3 peak enrichment in repressive regions. We found 53 genes which showed overlapping peak enrichment in both H3K4me3 and H3K9me3 marks. Some of the top H3K4me3-enriched genes were involved in sperm tail formation (Odf1, Odf3, Odf4, Oaz3, Ccdc42, Ccdc63, and Ccdc181), chromatin condensation (Dync1h1, Dynll1, and Kdm3a), and sperm functions such as acrosome reaction (Acrbp and Fabp9), energy generation (Gapdhs), and signaling for motility (Tssk1b, Tssk2, and Tssk4). Transcriptome sequencing in round spermatids found 64% transcripts of the H3K4me3-enriched genes at high levels and of about 25% of H3K9me3-enriched genes at very low levels. Transcriptome sequencing in sperm found that more than 99% of the ChIP-seq corresponding transcripts were also present in sperm. H3K4me3 enrichment in the round spermatids correlates significantly with gene expression and H3K9me3 correlates with gene silencing that contribute to sperm differentiation and setting the RNA payloads of sperm.

Forest riparian buffers reduce timber harvesting effects on stream temperature, but additional climate adaptation strategies are likely needed under future conditions.

Stream water temperature imposes metabolic constraints on the health of cold-water fish like salmonids. Timber harvesting can reduce stream shading leading to higher water temperatures, while also altering stream hydrology. In the Pacific Northwest, riparian buffer requirements are designed to mitigate these impacts; however, anticipated future changes in air temperature and precipitation could reduce the efficacy of these practices in protecting aquatic ecosystems. Using a combined modeling approach (Soil and Water Assessment Tool (SWAT), Shade, and QUAL2K), this study examines the effectiveness of riparian buffers in reducing impacts of timber harvest on stream water temperature in Lookout Creek, Oregon across a range of potential future climates. Simulations assess changes in riparian management alone, climate alone, and combined effects. Results suggest that maximum stream water temperatures during thermal stress events are projected to increase by 3.3-7.4 °C due to hydroclimatic change alone by the end of this century. Riparian management is effective in reducing stream temperature increases from timber harvesting alone but cannot fully counteract the additional effects of a warming climate. Overall, our findings suggest that the protection of sensitive aquatic species will likely require additional adaptation strategies, such as the protection or provisioning of cool water refugia, to enhance survival during maximum thermal stress events.

Array-based DNA methylation profiling reveals peripheral blood differential methylation in male infertility.

OBJECTIVE: To study peripheral blood DNA differential methylation in oligozoospermic infertile men in comparison with normozoospermic fertile controls. DESIGN: Case-control study. SETTING: Reproductive biology laboratory. PATIENTS(S): Azoospermic and oligozoospermic infertile patients (n = 6) and normozoospermic fertile controls (n = 6) in the discovery phase, and oligo/asthenozoospermic infertile men (n = 11) and normozoospermic fertile controls (n = 10) in the validation phase. INTERVENTION(S): Blood samples drawn from all participants, DNA isolation and methylation analysis. MAIN OUTCOME MEASURE(S): DNA methylation values analyzed using genomewide methylation 450K BeadChip array, followed by deep sequencing of selected regions for methylation analysis in the neighborhood regions of differentially methylated CpGs. RESULT(S): We found 329 differentially methylated CpG spots, out of which 245 referred to the genes, representing 170 genes. Deep-sequencing analysis confirmed the methylation pattern suggested by 450K array. A thorough literature search suggested that 38 genes play roles in spermatogenesis (PDHA2, PARP12, FHIT, RPTOR, GSTM1, GSTM5, MAGI2, BCAN, DDB2, KDM4C, AGPAT3, CAMTA1, CCR6, CUX1, DNAH17, ELMO1, FNDC3B, GNRHR, HDAC4, IRS2, LIF, SMAD3, SOD3, TALDO1, TRIM27, GAA, PAX8, RNF39, HLA-C, HLA-DRB6), are testis enriched (NFATC1, NMNAT3, PIAS2, SRPK2, WDR36, WWP2), or show methylation differences between infertile cases and controls (PTPRN2, RPH3AL). CONCLUSION(S): We found a statistically significant correlation between peripheral blood DNA methylation and male infertility, raising the hope that epigenome-based blood markers can be used for screening male infertility risk. The study also identified new candidates for spermatogenesis and fertility.

Genome-wide differential methylation analyses identifies methylation signatures of male infertility.

STUDY QUESTION: Do methylation changes in sperm DNA correlate with infertility? STUDY ANSWER: Loss of spermatogenesis and fertility was correlated with 1680 differentially-methylated CpGs (DMCs) across 1052 genes. WHAT IS KNOWN ALREADY: Methylation changes in a number of genes have been correlated with reduced sperm count and motility. STUDY DESIGN, SIZE, DURATION: This case-control study used spermatozoal DNA from 38 oligo-/oligoastheno-zoospermic infertile patients and 26 normozoospermic fertile men. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Genome-wide methylation analysis was undertaken using 450 K BeadChip on spermatozoal DNA from six infertile and six fertile men to identify DMCs. This was followed by deep sequencing of spermatozoal DNA from 32 infertile patients and 20 fertile controls. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1680 DMCs were identified, out of which 1436 were hypermethylated and 244 were hypomethylated. Classification of DMCs according to the genes identified BCAN, CTNNA3, DLGAP2, GATA3, MAGI2 and TP73 among imprinted genes, SPATA5, SPATA7, SPATA16 and SPATA22 among spermatogenesis-associated genes, KDM4C and JMJD1C, EZH2 and HDAC4 among genes which regulate methylation and gene expression, HLA-C, HLA-DRB6 and HLA-DQA1 among complementation and immune response genes, and CRISPLD1, LPHN3 and CPEB2 among other genes. Genes showing significant differential methylation in deep sequencing, i.e. HOXB1, GATA3, EBF3, BCAN and TCERG1L, are strong candidates for further investigations. The role of chance was ruled out by deep sequencing of select genes. LARGE-SCALE DATA: N/A. LIMITATIONS, REASON FOR CAUTION: Genome-wide analyses are fairly accurate, but may not be exactly validated in replication studies across all DMCs. We used the 't' test in the genome-wide methylation analysis, whereas other tests could provide a more robust and powerful analysis. WIDER IMPLICATIONS OF THE FINDINGS: DMCs can serve as markers for inclusion in infertility screening panels, particularly those in the genes showing differential methylation consistent with previous studies. The genes validated by deep sequencing are strong candidates for investigations of their roles in spermatogenesis. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Council of Scientific and Industrial Research (CSIR), Govt. of India with grant number BSC0101 awarded to Rajender Singh. None of the authors has any competing interest to declare.

Simulated Sensitivity of Urban Green Infrastructure Practices to Climate Change.

Climate change is likely to alter the quantity and quality of urban stormwater, presenting a risk to water quality and public health. How might stormwater management practices need to change to address future climate? Answering requires understanding how management practices respond to climate forcing. Traditional "gray" stormwater design employs engineered structures, sized based on assumptions about future rainfall, which have limited flexibility once built. Green infrastructure (GI) uses vegetation, soil, and distributed structures to manage rainwater where it falls and may provide greater flexibility for adaptation. There is, however, uncertainty about how a warmer climate may affect performance of different types of GI. This study uses the hydrologic and biogeochemical watershed model, Regional Hydro-Ecologic Simulation System (RHESSys), to investigate sensitivity of different GI practices to climate. Simulations examine 36 urban "archetypes" representing different development patterns (at the city block scale) of typical U.S. cities, eleven regional climatic settings, and a range of mid-21st century scenarios based on downscaled climate model output. Results suggest regionally variable effects of climate change on the performance of GI practices for water quantity, water quality, and carbon sequestration. GI is able to mitigate most projected future increases in surface runoff, while bioretention can mitigate increased nitrogen yield at nine of eleven sites. Simulated changes in carbon balance are small, while local evaporative cooling can be substantial. Given uncertainty in the local expression of future climate, infrastructure design should emphasize flexibility and robustness to a range of future conditions.

Is MTHFR 677 C>T Polymorphism Clinically Important in Polycystic Ovarian Syndrome (PCOS)? A Case-Control Study, Meta-Analysis and Trial Sequential Analysis.

BACKGROUND: Optimum efficiency of the folate pathway is considered essential for adequate ovarian function. 677 C>T substitution in the 5, 10-methylene tertrahydrofolatereductase (MTHFR) gene compromises activity of the MTHFR enzyme by about 50%. The significance of correlation between 677C>T substitution and PCOS remains dubious due to the low power of published studies. METHODS AND RESULTS: We analyzed MTHFR 677 C>T site in ethnically two different PCOS case-control groups (total 261 cases and 256 controls) from India. The data analysis revealed a lack of association between this polymorphism and PCOS [OR = 1.11 (95%CI = 0.71-1.72), P = 0.66]. Group-wise analysis on the basis of ethnicity also revealed no association in any of the ethnic groups [Indo-Europeans, P = 1; Dravidians, P = 0.70]. Homocysteine levels did not differ significantly between cases (15.51 µmol/L, SD = 2.89) and controls (15.89 µmol/L, SD = 2.23). We also undertook a meta-analysis on 960 cases and 1028 controls, which suggested a significant association of the substitution with PCOS in the dominant model of analysis (OR = 1.47 (95%CI = 1.04-2.09), P = 0.032]. Trial sequential analysis corroborated findings of the traditional meta-analysis. However, we found that the conclusions of meta-analysis were strongly influenced by studies that deviated from the Hardy Weinberg equilibrium. A careful investigation of each study and a trial sequential analysis suggested that 677 C>T substitution holds no clinical significance in PCOS in most of the populations. CONCLUSION: In conclusion, MTHFR 677 C>T polymorphism does not affect PCOS risk in India. The association seen in the meta-analysis is due to an outlier study and studies showing deviation from the Hardy Weinberg equilibrium.

Disulfiram and its novel derivative sensitize prostate cancer cells to the growth regulatory mechanisms of the cell by re-expressing the epigenetically repressed tumor suppressor-estrogen receptor ß.

Estrogen Receptor-ß (ER-ß), a tumor-suppressor in prostate cancer, is epigenetically repressed by hypermethylation of its promoter. DNA-methyltransferases (DNMTs), which catalyze the transfer of methyl-groups to CpG islands of gene promoters, are overactive in cancers and can be inhibited by DNMT-inhibitors to re-express the tumor suppressors. The FDA-approved nucleoside DNMT-inhibitors like 5-Azacytidine and 5-Aza-deoxycytidine carry notable concerns due to their off-target toxicity, therefore non-nucleoside DNMT inhibitors are desirable for prolonged epigenetic therapy. Disulfiram (DSF), an antabuse drug, inhibits DNMT and prevents proliferation of cells in prostate and other cancers, plausibly through the re-expression of tumor suppressors like ER-ß. To increase the DNMT-inhibitory activity of DSF, its chemical scaffold was optimized and compound-339 was discovered as a doubly potent DSF-derivative with similar off-target toxicity. It potently and selectively inhibited cell proliferation of prostate cancer (PC3/DU145) cells in comparison to normal (non-cancer) cells by promoting cell-cycle arrest and apoptosis, accompanied with inhibition of total DNMT activity, and re-expression of ER-ß (mRNA/protein). Bisulfite-sequencing of ER-ß promoter revealed that compound-339 demethylated CpG sites more efficaciously than DSF, restoring near-normal methylation status of ER-ß promoter. Compound-339 docked on to the MTase domain of DNMT1 with half the energy of DSF. In xenograft mice-model, the tumor volume regressed by 24% and 50% after treatment with DSF and compound-339, respectively, with increase in ER-ß expression. Apparently both compounds inhibit prostate cancer cell proliferation by re-expressing the epigenetically repressed tumor-suppressor ER-ß through inhibition of DNMT activity. Compound-339 presents a new lead for further study as an anti-prostate cancer agent. © 2015 Wiley Periodicals, Inc.

Comparison of preoperative nepafenac (0.1%) and flurbiprofen (0.03%) eye drops in maintaining mydriasis during small incision cataract surgery in patients with senile cataract: A randomized, double-blind study.

AIMS: This study compared the effectiveness of prophylactic administration of topical flurbiprofen 0.03% and nepafenac 0.1% in maintaining mydriasis during small incision cataract surgery (SICS). MATERIALS AND METHODS: This study was a prospective, randomized, double-blind comparative study in adult cataract patients given topical flurbiprofen or nepafenac prior to SICS and capsular bag intraocular lens (IOL) implantation at a tertiary care hospital. Horizontal and vertical diameters of pupil were measured at the beginning and end of surgery, and the mean values were compared across the two groups. Unpaired t-test and Fisher's exact test were used to analyse the results. RESULTS: A total of 70 eyes of cataract surgery patients, 33 males and 37 females, with a mean age of 58.5 ± 11.24 years, were included in the study. The mean horizontal and vertical diameters of the two groups were similar at the start of surgery. Significant differences were seen after IOL implantation, with the nepafenac group having the larger mean diameters in both horizontal (P = 0.03) and vertical (P = 0.04) pupillary measurements. CONCLUSIONS: Topical nepafenac has been shown to be a more effective inhibitor of meiosis during SICS and provides a more stable mydriatic effect compared to topical flurbiprofen.

M235T polymorphism in the AGT gene and A/G(I8-83) substitution in the REN gene correlate with end-stage renal disease.

BACKGROUND/AIMS: This study aimed at investigating if M235T polymorphism in the AGT gene and A/G(I8-83) polymorphism in the REN gene correlate with end-stage renal disease (ESRD). METHODS: We analyzed 173 ESRD patients and 329 individuals with normal kidney function for differences in the genotype distribution of AGT-M235T and REN-A/G(I8-83) polymorphisms between the two groups. The data for cases and controls were compared using the χ(2) test. RESULTS: We found significantly higher levels of serum creatinine and CRP in cases in comparison to controls (p < 0.0001). Data comparison showed a significant association of AGT M235T substitution with ESRD in the dominant model (p = 0.008) and in the comparison of the heterozygous substitution with the homozygous common genotype (p = 0.005). Similarly, REN A/G(I8-83) polymorphism showed a significant difference in the distribution of genotypes between cases and controls (p < 0.038) such that a heterozygous substitution was significantly more common in the ESRD cases in comparison to the homozygous common genotype (p = 0.023). CONCLUSION: We conclude that heterozygous substitutions at the AGT M235T and REN A/G(I8-83) loci correlate significantly with ESRD in a north Indian population.

SRD5A2 gene polymorphisms affect the risk of breast cancer.

Androgens in breast cancer have been studied alone and in correlation with estrogens as estrogen to testosterone ratio. 5-α-reductase is one of the important enzymes participating in androgen metabolism, which affects androgen activity by affecting conversion of testosterone to dihydrotestosterone. We hypothesized that polymorphisms in the SRD5A2 gene (encoding 5-α-reductase) may affect breast cancer risk by affecting total androgen activity. Complete coding region of the SRD5A2 gene was sequenced in a group of 628 patients and 244 control samples from three southern states (Tamil Nadu, Andhra Pradesh, and Karnataka) of India. We observed three common polymorphisms in this gene; namely, A49T, V89L, and (TA)n repeats. A49T locus was monomorphic in the study population, but V89L showed a strong correlation with breast cancer (P = 0.03, OR = 1.40, CI = 1.02-1.91). (TA)0/(TA)9 and (TA)9/(TA)9 genotypes were at a lower risk of breast cancer (P = 0.01, OR = 0.64, CI = 0.46-0.90). We conclude that SRD5A2 genotypes significantly affect breast cancer risk in the South Indian populations.

Significant impact of the MTHFR polymorphisms and haplotypes on male infertility risk.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate and affects the activity of cellular cycles participating in nucleotide synthesis, DNA repair, genome stability, maintenance of methyl pool, and gene regulation. Genetically compromised MTHFR activity has been suggested to affect male fertility. The objective of the present study was to find the impact on infertility risk of c.203G>A, c.1298A>C, and c.1793G>A polymorphisms in the MTHFR gene. METHODS: PCR-RFLP and DNA sequencing were used to genotype the common SNPs in the MTHFR gene in 630 infertile and 250 fertile males. Chi-square test was applied for statistical comparison of genotype data. Linkage disequilibrium between the SNPs and the frequency of common haplotypes were assessed using Haploview software. Biochemical levels of total homocysteine (tHcy) and folic acid were measured. Meta-analysis on c.1298A>C polymorphism was performed using data from ten studies, comprising 2734 cases and 2737 controls. RESULTS: c.203G>A and c.1298A>C were found to be unrelated to infertility risk. c.1793G>A was protective against infertility (P = 0.0008). c.677C>T and c.1793G>A were in significant LD (D' = 0.9). Folic acid and tHcy level did not correlate with male infertility. Pooled estimate on c.1298A>C data from all published studies including our data showed no association of this polymorphism with male infertility (Odds ratio = 1.035, P = 0.56), azoospermia (Odds ratio = 0.97, P = 0.74), or oligoasthenoteratozoospermia (Odds ratio = 0.92, p = 0.29). Eight haplotypes with more than 1% frequency were detected, of which CCGA was protective against infertility (p = 0.02), but the significance of the latter was not seen after applying Bonferroni correction. CONCLUSION: Among MTHFR polymorphisms, c.203G>A and c.1298A>C do not affect infertility risk and c.1793G>A is protective against infertility. Haplotype analysis suggested that risk factors on the MTHFR locus do not extend too long on the DNA string.

Mucuna pruriens and its major constituent L-DOPA recover spermatogenic loss by combating ROS, loss of mitochondrial membrane potential and apoptosis.

BACKGROUND: The Ayurvedic medicinal system claims Mucuna pruriens (MP) to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD), and finding the possible mechanism of action thereof in a rat model. METHODOLOGY/FINDINGS: Ethinyl estradiol (EE) was administered at a rate of 3 mg/kg body weight (BW)/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15(th) day for a period of 56 days, and the results were compared with an auto-recovery (AR) group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP), recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility. CONCLUSION/SIGNIFICANCE: M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro-spermatogenic properties of M. pruriens. The manuscript bears CDRI communication number 8374.