Coenzyme Q10 improves the quality and in vitro fertility of post-thawed buffalo (Bubalus bubalis) semen via its antioxidative effect.

This study aimed to determine the effects of Coenzyme Q10 (CoQ10) in the freezing medium on functional and oxidative stress parameters and in vitro fertilization (IVF) rate of buffalo sperm. Collected samples were relocated to the laboratory for initial evaluation, gentle dilution in extenders, cooling (4°C, 2 h), equilibration (4°C, 4 h), packaging (straws, 0.5 mL), programmable freezing, and thawing (37°C, 30 s). Statistical analysis depicted that adding CoQ10 (100 µM) in a freezing medium caused a significant augmentation in total motility (%), average path, and straight-line velocities (µm/sec) of buffalo sperm than control. Adding CoQ10 (100 µM) improved sperm progressive motility, rapid velocity, and functional parameters (%) compared to the control and 10 µM of CoQ10. Moreover, CoQ10 in a freezing medium caused a significant augmentation in seminal plasma catalase (U/mL) and glutathione reductase (GSH; nmol/109 ) at 100 µM than control and other treatments. CoQ10 inclusion (100 µM) ameliorates seminal plasma superoxide dismutase (U/mL), glutathione-S-transferase (GST; nmol/mL/min) fructose (µg/mL), and ATP (nmol/million) than control. Furthermore, CoQ10 at 100 µM improved seminal plasma glutathione peroxidase (µM) levels than control, 10 µM, and 20 µM. Lastly, hydrogen peroxide (H2 O2; nM) production was significantly lower at 100 µM than at control and 10 µM. CoQ10 (100 µM) caused a significant augmentation in the un-capacitated pattern followed by a reduction in the capacitated pattern, and apoptosis-like changes (%) than control, and other treatments, whereas viability was increased than control and other treatments. CoQ10 (100 µM) significantly improved the IVF rate in comparison with control, CoQ10 at 10 µM, and 20 µM groups. In conclusion, the addition of CoQ10 (100 µM) in the freezing medium can improve the quality and in vitro fertility of post-thawed buffalo semen via its antioxidative effect. Further studies are needed to evaluate the effect of CoQ10 on the in vivo fertility of buffalo bull semen.

Mutation pattern of cancer suppressor p53 gene in factory workers exposed to polypropylene and impact on their reproductive hormones.

BACKGROUND: Polypropylene is a thermoplastic polymer playing the role of an endocrine disruptor that interferes with the union, emission, transport or elimination of normal hormones. Epidemiological information indicated the relation of endocrine-disturbing chemicals with prostate cancer, testis tumor and diminished fertility. p53 is a key tumor silencer gene. The present study aimed to evaluate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels and the risk of p53 mutations as a result of exposure to polypropylene in non-tumorous adult male factory workers. METHODS: In total, 150 (controls = 35, workers = 115) subjects were recruited. Groups were maintained according to the tenure of exposure G1 (1-5 years), G2 (6-10 years), G3 (11-15 years) and G4 (16-20 years). Concentrations of LH and FSH were determined through an enzyme-linked immunosorbent assay. Genotyping analysis was performed by polymerase chain reaction based gel electrophoresis followed by DNA sequencing. The structural and functional impact of the mutation on the p53 structure was evaluated using 50-ns molecular dynamics (MD) simulations and protein-DNA docking. RESULTS: Mean plasma LH levels were significantly decreased in G1 (p > 0.05) as well as the G2, G3 and G4 (p > 0.001) groups. Similarly, FSH levels were significant decrease in G1 (p > 0.05), G2 (p > 0.01), G3 (p > 0.001) and G4 (p > 0.001) compared to the control group. Sequencing results found three variants i.e. g.13450 T>G, g.13430C>T and g.13737G>A. One of them was predicted to be disease-causing others are polymorphisms. MD simulation of missense mutation R273H showed no structural impact on the protein structure in MD simulation, but it resulted in weaker binding of p53 with the DNA that might lower the gene expression of cell cycle regulatory proteins. CONCLUSIONS: These findings predict decreased fertility and risk of malignancies in the future. The spectrum of p53 mutations as a result of polypropylene exposure in the Pakistani population has not been investigated before. Further studies and meta-analyses are required to elucidate the role of different plasticizers in reproduction and cancer-causing risk factors in a larger population.

The influence of paternal overweight on sperm chromatin integrity, fertilization rate and pregnancy outcome among males attending fertility clinic for IVF/ICSI treatment.

BACKGROUND: Low and middle-income countries are facing a rapid increase in obesity and overweight burden, particularly in urban settings. Being overweight in men is associated with infertility and a higher risk to have a low sperm count or no sperm in their ejaculate. Despite potential limitations, this is one of few studies conducted to determine the potential risk of paternal overweight on sperm standard parameters, sperm chromatin integrity and assisted conception outcome including fertilization, embryo quality, cleavage rate, reduce blastocyst development, implantation, and cumulative live birth rate (CLBR). METHODS: A cross-sectional study of 750 infertile couples undergoing assisted reproduction technique at a single reproductive medicine center of Salma Kafeel Medical Centre Islamabad. Sperm from men undergoing ART were analyzed for chromatin integrity using sperm chromatin dispersion assay (SCD), Chromomycin A3 staining (CMA3), and toluidine blue (TB) staining, while other semen parameters were assessed on same day includes; standard semen parameters, reactive oxygen species (ROS), sperm deformity index (SDI), teratozoospermic index (TZI), and hypo-osmatic swelling test (HOST). Paternal body mass index (BMI) < 24.5-20 kg/m2 served as the reference group, while the male patients with BMI > 24.5-30 kg/m2 were considered to be overweight. RESULTS: In the analysis of the percentage of spermatozoa with chromatin maturity (CMA3) and chromatin integrity (TB) was reduced significantly in overweight men (p < 0.01) compared with a reference group. Increase in paternal BMI correlate with the increase in sperm chromatin damage (SCD r = 0.282, TB r = 0.144, p < 0.05), immaturity (CMA3, r = 0.79, p < 0.05) and oxidative stress (ROS) (r = 0.282, p < 0.001). Peri-fertilization effects were increased in oocytes fertilization in couples with overweight men (FR = 67%) compared with normal-weight men (FR = 74.8%), similarly, after univariant regression paternal weight remain predictor of sperm chromatin maturity, successful fertilization and CLBR. In the embryo, developmental stage number of the embryo in cleavage was higher in normal weight men, while day 3 (D3) embryos, percent good quality embryo D3, and blastocyst formation rate were compared able between the groups. The paternal overweight group had significant (p < 0.001) increased neonatal birth weight (2952.14 ± 53.64gm; within normal range) when compared with the reference group (2577.24 ± 30.94gm) following assisted reproductive technology (ART). CLBR was higher (p < 0.05) in normal weight men compared to couples with overweight male partners. CLBR per embryo transfer and per 2PN was a statistically significant (p < 0.05) difference between the two groups. An inverse association was observed in the linear regression model between paternal BMI with fertilization rate and CLBR. CONCLUSION: The present study demonstrated the impact of paternal overweight on male reproductive health, as these patients had a higher percentage of immature sperm (CMA3) with impaired chromatin integrity (SCD, TB) in their semen and had decreased fertilization rate, CLBR following assisted reproductive treatments. The present study supports that paternal overweight should be regarded as one of the predictors for fertilization, CLBR and useful for counseling, to consider body mass index not only in women but also for men, in those couples opting for ART treatment, and warrant a poor reproductive outcome in overweight men.

The ameliorating effects of crocetin on frozen-thawed quality, and fertility via attenuating oxidative status of bubaline spermatozoa.

Current study was conducted to appraise the cryoprotective influence of crocetin on quality, oxidative status, and fertility potential of bubaline spermatozoa. Collected semen from four bulls was diluted in five aliquots with (10 µM, 5 µM, 2 µM, 1 µM, and control [0 µM] supplementation of crocetin). After gentle dilution (37 °C), cooling (4 °C, in 2 h), equilibration (4 °C, for 4 h) and packaging of samples was done in straws (polyvinyl French, 0.5 ml), and then frozen (programmable cell freezer). This study established that crocetin supplementation significantly (p < 0.05) improves CASA (Computer Assisted Sperm motion Analyzer) total motility (%), rapid velocity (%), average-path, and curved-line velocities (µm/sec, 10 µM vs. control), and progressive motility (%), straight-line velocity (µm/sec), total antioxidant capacity (TAC, µMol/l), ATP concentrations (nmol/million), and fertility potential (%) (10 µM vs. control, and 1 µM), and mitochondrial potential (%) of buffalo spermatozoa (5, and 10 µM vs. control). Crocetin supplementation significantly (p < 0.05) alleviates DNA fragmentation, seminal plasma ROS (104 RLU/20/25 million, RLU = Relative light unit) levels, and lipid peroxidation (LPO, µMol/ml) in buffalo spermatozoa (10 µM vs. control). In a nutshell, crocetin supplementation improves post-thaw quality by means of motility parameters, motion kinematics, TAC, and ATP concentrations, and fertility potential, and abolished DNA fragmentation parameters, seminal plasma ROS, and LPO concentrations of buffalo spermatozoa. The exact mechanism by which crocetin acts are not fully elucidated; however, it is probable to speculate that the reduction in ROS, and LPO recorded in this study may be related to scavenging ability of this antioxidant during cryopreservation.

Protamines and DNA integrity as a biomarkers of sperm quality and assisted conception outcome.

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome.

Assessment of antiandrogenic and antispermatogenic activity of Hedera nepalensis in adult male rats.

The use of medicinal plants for fertility regulation has been prevalent worldwide for many centuries. They possess natural substances having antiandrogenic properties and can be used as source of contraception. In the current study, methanolic leaf extract of Hedera nepalensis was evaluated for antiandrogenic and antispermatogenic activity in adult male rats through various reproductive parameters. Experimental findings showed significantly increased oxidative stress with reduced antioxidant activity at highest dose regimens in both in vitro and in vivo studies. Increased ROS generation and lipid peroxidation lead to DNA damage in rat sperm. In vivo determination of sperm parameters exhibited notable reduction in sperm motility, viability and DSP in dose-treated animals. Histopathological observations revealed reduced epithelial height and wider lumen having less number of spermatozoa in high-dose-treated groups. Additionally, a marked decline noted in Testosterone concentration in all extract treated groups, while plasma LH and FSH levels only in high-dose-treated groups were noted. The findings of the current study conclude that methanolic leaf extract of H. nepalensis has the potential to disturb male fertility by generating oxidative stress and hormonal imbalance leading to histological alterations and sperm DNA damage.

Association of endothelial nitric oxide synthase gene variants with preeclampsia.

BACKGROUND: Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology. The endothelial nitric oxide synthase (eNOS) gene and nitric oxide (NO) levels has been reported to be associated with PE predisposition in various populations. Therefore, present study was designed to investigate the role of NO levels and eNOS gene variants in preeclamptic women in Pakistan. METHODS: A total of 600 women were evaluated, 188 of PE with mild features, 112 of PE with severe features and 300 normotensive pregnant women. NO levels were detected by Greiss reaction method and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. RESULTS: Reduced concentrations of NO were reported in all PE groups (p < 0.05) as compared to controls. The frequency of c.894 T (p.298Asp) and g.-786C alleles were significantly associated with PE. In addition, novel homozygous variant g.2051G > A was also significantly associated with PE when compared to normotensive women. Dynamic simulation studies revealed that Glu298Asp mutation destabilize the protein molecule and decrease the overall stability of eNOS protein. Molecular docking analysis of mutant promoter with transcription factors STAT3 and STAT6 proposed changes in protein regulation upon these reported mutations in upstream region of the gene. CONCLUSION: Considering the results of current study, the functional alterations induced by these variants may influence the bioavailability of NO and represents a genetic risk factor for increased susceptibility to PE. However, large studies or meta-analysis are necessary to validate these findings.

Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology characterized by increased hypertension and proteinuria after 20 weeks of gestation. The present study was directed to determine the role of eNOS in susceptibility to PE and the association of c.894G > T (p.(Glu298Asp), intron 4b/4a, g.-786 T > C and other possible variants of eNOS gene with preeclampsia in Pakistani population. Computational analysis of identified variants in the coding and non-coding region of the eNOS gene was also conducted to determine the change in gene regulation and further protein stability. A total of 600 women were evaluated, 188 with mild and 112 with PE with severe features PE with 300 normotensive pregnant women. NO levels and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. Data from the current study suggest that there might be other risk variants of the eNOS gene (g.2051G > A and g.1861G > A) and lower levels of serum NO that confers in an increased risk of PE. The detailed computational investigation further confirmed the deformities and changes in protein flexibility upon Glu298Asp. These structural alterations might be associated with preeclampsia. Variants in the promoter region of the eNOS gene further validate the change in gene regulation for the onset of disease. Identification of key structural and functional features in eNOS protein and gene regulatory region might be used for designing specific drugs for therapeutic purpose.

Chronic exposure of bisphenol S (BPS) affect hypothalamic-pituitary-testicular activities in adult male rats: possible in estrogenic mode of action.

BACKGROUND: The industrial revolution has resulted in increased synthesis and the introduction of a variety of compounds into the environment and their potentially hazardous effects have been observed in the biota. The present study was aimed to evaluate the potential endocrine-disrupting effects of chronic exposure to the low concentrations of bisphenol S (BPS) in male rats. METHODS: Weaning male Sprague-Dawley rats (22 days old) were either exposed to water containing 0.1% ethanol for control or different concentrations of BPS (0.5, 5, and 50 µg/L) in drinking water for 48 weeks in the chronic exposure study. After completion of the experimental period, animals were dissected and different parameters (hormone concentrations, histology of testis and epididymis, oxidative stress and level of antioxidant enzymes in the testis, daily sperm production (DSP), and sperm parameters) were determined. RESULTS: Results of the present study showed a significant alteration in the gonadosomatic index (GSI) and relative reproductive organ weights. Oxidative stress in the testis was significantly elevated while sperm motility, daily sperm production, and the number of sperm in epididymis were reduced. Plasma testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations were reduced and estradiol levels were high in the 50 µg/L-exposed group. Histological observations involved a significant reduction in the epithelial height of the testis along with disrupted spermatogenesis, an empty lumen of the seminiferous tubules, and the caput region of the epididymis. CONCLUSION: These results suggest that exposure to 5 and 50 µg/L of BPS for the chronic duration started from an early age can induce structural changes in testicular tissue architecture and endocrine alterations in the male reproductive system which may lead to infertility in males.

Epigallocatechin-3-gallate (EGCG) addition as an antioxidant in a cryo-diluent media improves microscopic parameters, and fertility potential, and alleviates oxidative stress parameters of buffalo spermatozoa.

The disparity between the endogenous antioxidants concentration and free radicals in spermatozoa results in reactive oxygen species (ROS) generation. In this prospect, epigallocatechin-3-gallate (EGCG) preserves vigorous antioxidant features. Current study explored the influence of EGCG in a cryo-diluent media on microscopic parameters, oxidative stress parameters, and fertility potential of buffalo spermatozoa during cryopreservation. Concisely, collected semen from three donor bulls for four times were then evaluated for volume, motility, concentrations and then dilution in a cryo-diluent media with different concentrations of EGCG (EGCG-0 = control; EGCG-50 = 50 µM, EGCG-100 = 100 µM, EGCG-200 = 200 µM, and EGCG-300 = 300 µM) at 37 °C, cooled to 4 °C in 2 h, equilibrated for 4 h at 4 °C, and cryopreserved. At post-thawing, Computer-Assisted Sperm motion Analysis motilities (total and progressive, %) and rapid velocity (%), plasma membrane functionality, supravital plasma membrane integrity, and mitochondrial potential (%) were found higher (P < 0.05) in EGCG-200, and EGCG-300 than control, whereas average-path, straight-line, and curved-linear velocities (µm/sec), and acrosome integrity (%) were recorded higher in EGCG-300 than control. Further, comet length (µm), and tail length (µm), LPO (lipid peroxidation, µM/mL), and apoptosis-like changes (%) in spermatozoa were significantly decreased in EGCG-300 than control. Seminal plasma antioxidant enzymes activities (glutathione peroxidase, U/mL, and superoxide dismutase, U/mL) were increased with EGCG-300 than control. Moreover, EGCG-300 addition in a cryo-diluent media improves the fertility potential (%) of buffalo spermatozoa. In a nutshell, the inclusion of EGCG-300 in a cryo-diluent media enhances post-thaw microscopic parameters, and fertility potential, whereas decreases oxidative stress parameters in buffalo spermatozoa.

Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility, plasmalemma, mitochondrial membrane potential, acrosome, and DNA integrities, and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa.

The aromatic amino acid l-tryptophan is an essential and versatile molecule, acts by transferring an electron to free radicals and protects the plasma membrane from injuries. The aim of the present study was to investigate the effects of l-tryptophan in extender on semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during cryopreservation. Two ejaculates were collected from each bull (n = 2 ejaculates and n = 4 bulls) with artificial vagina at 42 °C followed by initial evaluation for volume, motility, concentrations and were diluted in five extenders (C = lacking l-tryptophan, D1 = 25 µ M l-tryptophan, D2 = 50 µ M l-tryptophan, D3 = 75 µ M l-tryptophan, and D4 = 100 µ M l-tryptophan) respectively, and cryopreserved. The experiment was repeated four times (n = 4 replicates). At post-dilution, sperm plasma membrane integrity (PMI, %), supravital plasma membrane integrity (SVPMI, %), hypo-resistivity (HR, %) and acrosome integrity (ACR-I, %) were significantly higher (P < 0.05) in extender supplemented with D4 than control. At post-thawing, progressive motility (PM, %), PMI, SVPMI, HR, ACR-I, and DNA-I of buffalo bull spermatozoa were significantly higher in D4 than control. Sperm in vitro longevity (%) assessed in terms of PM, SVPMI, and ACR-1 were significantly higher in D4 than control. Sperm mitochondrial membrane potential (%) was higher in treated groups than the control. The in vivo fertility rate was significantly higher in D4 than control (60.17% vs. 44.17%, P < 0.05). It is concluded that the supplementation of l-tryptophan in tris citric acid extender improves semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during freezing and thawing process.

Exposure of BPA and its alternatives like BPB, BPF, and BPS impair subsequent reproductive potentials in adult female Sprague Dawley rats.

Bisphenol A (BPA) and its alternatives are extensively used in household and industrial products. BPA and its alternatives have affinity for estrogen receptors and mimic its actions. The present study aims to examine the comparative effects of BPA and its alternatives bisphenol B (BPB), bisphenol F (BPF), and bisphenol S (BPS) on the reproductive health in adult female rats. One hundred and seventy post-weaning female rats (90 ± 25) were divided into 17 groups and assigned for different treatments. Control and treated groups were injected with concentrations of 50-500 µg/ml and 5-50 mg/kg of BPA, BPB, BPF, and BPS for 28 days. The results showed adverse morphological and histopathological alterations in rat ovaries in all treated groups. A remarkable decrease was observed in antral and corpus luteum follicles while rise in atretic and cystic follicles in the ovaries of BPA and its alternatives 5 and 50 mg/kg treated groups when compared with control. Significant decrease in catalase (CAT), super oxidase (SOD), and peroxidase (POD) levels was noted while increase in the values of thiobarbituric acid reactive substance (T-BARS) and reactive oxygen species (ROS) was observed when BPA and its alternatives groups were compared with the control. Hormones were also observed with alterations in their concentrations when treated groups were compared with the control. The current data suggest that BPA and its alternatives exposure during the pre-pubertal stage have the potential to induce oxidative stress and histopathological changes during follicular development in female rats.

Evaluation of coagulation factors and serum ferritin in preeclamptic Pakistani women.

Preeclampsia (PE) is a pregnancy-related disorder involving multiple organ systems and characterised by an increase in hypertension and proteinuria after 20 weeks of gestation. The study aimed to determine the role of coagulation factors and ferritin in relation to PE susceptibility in Pakistani women. Blood samples of 100 normotensive and 100 preeclamptic women, including 73 with mild PE and 27 with severe PE were taken for the study to evaluate activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalising ratio (INR), fibrinogen levels, platelet count (PLT) and ferritin levels. Prolonged aPTT, PT and INR were recorded in both PE groups with a decrease in platelets and fibrinogen levels, compared to the control groups. Ferritin levels were not significantly (p=0.23) different in any of the groups. In conclusion, coagulopathic disorder should be clinically suspected and the coagulating factors in PE patients should be examined for early detection, effective antenatal care and for the proper management of this disorder to decrease maternofoetal mortality, morbidity and perinatal mortality.

Screening and computational analysis of colorectal associated non-synonymous polymorphism in CTNNB1 gene in Pakistani population.

BACKGROUND: Colorectal cancer (CRC) is categorized by alteration of vital pathways such as ß-catenin (CTNNB1) mutations, WNT signaling activation, tumor protein 53 (TP53) inactivation, BRAF, Adenomatous polyposis coli (APC) inactivation, KRAS, dysregulation of epithelial to mesenchymal transition (EMT) genes, MYC amplification, etc. In the present study an attempt was made to screen CTNNB1 gene in colorectal cancer samples from Pakistani population and investigated the association of CTNNB1 gene mutations in the development of colorectal cancer. METHODS: 200 colorectal tumors approximately of male and female patients with sporadic or familial colorectal tumors and normal tissues were included. DNA was extracted and amplified through polymerase chain reaction (PCR) and subjected to exome sequence analysis. Immunohistochemistry was done to study protein expression. Molecular dynamic (MD) simulations of CTNNB1WT and mutant S33F and T41A were performed to evaluate the stability, folding, conformational changes and dynamic behaviors of CTNNB1 protein. RESULTS: Sequence analysis revealed two activating mutations (S33F and T41A) in exon 3 of CTNNB1 gene involving the transition of C.T and A.G at amino acid position 33 and 41 respectively (p.C33T and p.A41G). Immuno-histochemical staining showed the accumulation of ß-catenin protein both in cytoplasm as well as in the nuclei of cancer cells when compared with normal tissue. Further molecular modeling, docking and simulation approaches revealed significant conformational changes in the N-terminus region of normal to mutant CTNNB1 gene critical for binding with Glycogen synthase kinase 3-B (GSK3) and transducin containing protein1 (TrCp1). CONCLUSION: Present study on Pakistani population revealed an association of two non-synonymous polymorphisms in the CTNNB1 gene with colorectal cancer. These genetic variants led to the accumulation of the CTNNB1, a hallmark of tumor development. Also, analysis of structure to function alterations in CTNNB1 gene is crucial in understanding downstream biological events.

Comparative study of environmental pollutants bisphenol A and bisphenol S on sexual differentiation of anteroventral periventricular nucleus and spermatogenesis.

BACKGROUND: Bisphenol A is well known endocrine-disrupting chemical while Bisphenol S was considered a safe alternative. The present study aims to examine the comparative effects of xenobiotic bisphenol-A (BPA) and its substitute bisphenol-S (BPS) on spermatogenesis and development of sexually dimorphic nucleus population of dopaminergic neurons in the anteroventral periventricular nucleus (AVPV) of the hypothalamus in male pups. METHODS: Sprague Dawley rat's pups were administered subcutaneously at the neonatal stage from postnatal day PND1 to PND 27. Thirty animals were divided into six experimental groups (6 animals/group). The first group served as control and was provided with normal olive oil. The four groups were treated with 2 µg/kg and 200 µg/kg of BPA and BPS, respectively. The sixth group was given with 50 µg/kg of estradiol dissolved in olive oil as a standard to find the development of dopaminergic tyrosine hydroxylase neurons in AVPV regions. Histological analysis for testicular tissues and immunohistochemistry for brain tissues was performed. RESULTS: The results revealed adverse histopathological changes in testis after administration of different doses of BPA and BPS. These degenerative changes were marked by highly significant (p < 0.001) decrease in tubular and luminal diameters of seminiferous tubule and epithelial height among bisphenols treated groups as compared to control. Furthermore, significantly increased (p < 0.001) TH-ir cell bodies in the AVPV region of the brain with 200 µg/kg dose of BPA and BPS was evident. CONCLUSION: It is concluded that exposure of BPA and BPS during a critical developmental period can structural impairments in testes and affects sexual differentiation of a dimorphic dopaminergic population of AVPV region of hypothalamus in the male brain.

A biochemical and histological approach to study antifertility effects of methanol leaf extract of Asplenium dalhousiae Hook. in adult male rats.

The study was designed to investigate the antifertility properties of methanol leaf extract of Asplenium dalhousiae in adult male rats. Forty adult male Sprague Dawley rats (150 ± 10 g) divided into four groups (n = 10 animals/group) were administered with different doses (0, 50, 100, 150 mg/kg) of plant extract for 28 days. On day 29th, rats were decapitated, trunk blood and reproductive tissues were collected, and blood plasma was separated and stored until use for measuring reproductive hormones, while epididymis and testis were used for assessment of sperm parameters, oxidative stress status and morphometric analysis. Sperm motility, viability and sperm production rates were lowered in high dose treatment groups. Levels of catalase (CAT), sodium dismutase (SOD) and peroxidase (POD) decreased while stress biomarkers including reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) were increased among all treatment groups. Concentrations of plasma testosterone and follicle stimulating hormone (FSH) were decreased while levels of luteinizing hormone (LH) increased in high extract treated groups. Histological examination of testis showed disorganisation of seminiferous tubule and reduced spermatocytes number. The findings of current study revealed that methanol leaf extract of A. dalhousiae might induce antifertility effects via oxidative stress and interfering with testicular architecture leading to spermatogenic arrest.

Bisphenol A analogues bisphenol B, bisphenol F, and bisphenol S induce oxidative stress, disrupt daily sperm production, and damage DNA in rat spermatozoa: a comparative in vitro and in vivo study.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report the mechanisms by which BPA and three of its analogues bisphenol B (BPB), bisphenol F (BPF), and bisphenol S (BPS) cause generation of reactive oxygen species (ROS), sperm DNA damage, and oxidative stress in both in vivo and in vitro rat models. Sperm were incubated with different concentrations (1, 10, and 100 µg/L) of BPA and its analogues BPB, BPF, and BPS for 2 h. BPA and its analogues were observed to increase DNA fragmentation, formation of ROS, and affected levels of superoxide dismutase at higher concentration groups. In an in vivo experiment, rats were exposed to different concentrations (5, 25, and 50 mg/kg/day) of BPA, BPB, BPF, and BPS for 28 days. In the higher dose (50 mg/kg/day) treated groups of BPA and its analogues BPB, BPF, and BPS, DNA damage was observed while the motility of sperm was not affected.

Ameliorating potency of Chenopodium album Linn. and vitamin C against mercuric chloride-induced oxidative stress in testes of Sprague Dawley rats.

BACKGROUND: Mercury has been documented as an industrial risk that posed a serious danger to human health. Mercury exposure results in oxidative stress that may lead to the pathogenesis of male reproductive dysfunction. The present study investigated the ameliorating potential of Chenopodium album L. and vitamin C against mercuric chloride-induced oxidative deterioration of reproductive functions in adult male rats. METHODS: Group 1 (control) received saline. Group 2 received Mercury (0.15 mg/kg b.w, i.p) dissolved in distilled water. Groups 3 and 4 were given oral gavage of vitamin C (200 mg/kg b.w) and the ethanolic extract of C. album (200 mg/kg b.w) respectively, along with Mercury (0.15 mg/kg b.w, i.p). Group 5 was treated only with C. album (200 mg/kg b.w). After 30 days of the treatment, the rats were dissected and their testicular tissue and the cauda epididymis were used for biochemical analysis while blood plasma was used for protein determination. RESULTS: The applied dose-treatment of Mercury-induced oxidative stress in the testis and cauda epididymis tissues of the rats was apparent by a noteworthy decrease in total protein, CAT, SOD, POD, and GST values while there was increase in ROS and TBARS levels. Furthermore, Mercury decreases daily sperm production and enhanced sperm DNA damage as noticeable by an increase in the head and tail length of comets and decrease in intact DNA. There was no significant effect on the body weight and the weight of the reproductive tissues. Treatment with C. album significantly ameliorated the total protein, ROS, and TBARS content. Similarly, the level of CAT, SOD, POD, and GST was significantly improved and the daily sperm production was significantly increased. Furthermore, C. album administration significantly protected Mercury-induced sperm DNA damage. The results of the extract treatment group were compared with those of vitamin C in detoxifying the oxidative stress and restoring the sperm parameters. CONCLUSION: C. album showed protection against Mercury-induced oxidative stress by ameliorating antioxidant enzyme activity, daily sperm production, and DNA damage in rat testes. This suggests that C. album could be beneficial against toxicity induced by an environmental toxicant.

Effect of bisphenol F, an analog of bisphenol A, on the reproductive functions of male rats.

OBJECTIVE: Bisphenol A (BPA) is a monomer primarily used in the production of polycarbonate plastic and epoxy resins. Bisphenol F (BPF) is apparently the main BPA replacement that is used increasingly. BPF has been detected in canned food, thermal paper receipts, and soft drinks. In the present experiment, we did both in vitro and in vivo studies to evaluate the effect of low and high-dose BPF exposures on testosterone concentration, oxidative stress, and antioxidants activity in reproductive tissues of male rats. METHODS: Adult (80-90 days old) male Sprague Dawley rats (n = 36) obtained from the rodent colony of Animal Sciences Department of Quaid-i-Azam University. The direct effects of BPF on the antioxidant enzymes and testosterone secretion were measured in vitro and in vivo studies. In an in vivo experiment, adult male Sprague Dawley rats (n = 42) were exposed to different concentrations of bisphenol F (1, 5, 25, and 50 mg/kg/d) for 28 days. Various biochemical parameters were analyzed including the level of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), reactive oxygen species (ROS), and lipid peroxidation (LPO). Moreover, sperm motility, daily sperm production (DSP), comet assay, and histological analysis were performed. RESULTS: In vitro study showed that BPF exposure significantly (p < 0.05) induced oxidative stress biomarkers, i.e., ROS and LPO, while it did not change antioxidant enzyme and testicular testosterone concentration. Whereas, an in vivo study revealed that BPF induced dose-dependent effect and high-dose (100 mg/kg) exposure of BPF significantly reduced tissue protein (p < 0.05) content, CAT (p < 0.001), SOD (p < 0.05), and POD (p < 0.05) levels while significantly (p < 0.05) augmented ROS and lipid peroxidation. Furthermore, BPF reduces testosterone, LH, and FSH secretion in a dose-dependent manner. Significant (p < 0.001) reduction in plasma and intra-testicular testosterone, LH, and FSH was noticed at 100 mg/kg BFP dose. High-dose exposure reduces spermatogenesis. CONCLUSION: BPF showed an antagonistic effect on male reproductive hormones and induce alterations in testicular morphology. Increased oxidative stress and decreased testicular antioxidant status might be the underlying mechanism of BFP-induced testicular toxicity.

Self-Nanoemulsifying Drug Delivery System (SNEDDS) for Improved Oral Bioavailability of Chlorpromazine: In Vitro and In Vivo Evaluation.

Background and Objectives: Lipid-based self-nanoemulsifying drug delivery systems (SNEDDS) have resurged the eminence of nanoemulsions by modest adjustments and offer many valuable opportunities in drug delivery. Chlorpromazine, an antipsychotic agent with poor aqueous solubility-with extensive first-pass metabolism-can be a suitable candidate for the development of SNEDDS. The current study was designed to develop triglyceride-based SNEDDS of chlorpromazine to achieve improved solubility, stability, and oral bioavailability. Materials and Methods: Fifteen SNEDDS formulations of each short, medium, and long chain, triglycerides were synthesized and characterized to achieve optimized formulation. The optimized formulation was characterized for several in vitro and in vivo parameters. Results: Particle size, zeta potential, and drug loading of the optimized SNEDDS (LCT14) were found to be 178 ± 16, -21.4, and 85.5%, respectively. Long chain triglyceride (LCT14) showed a 1.5-fold increased elimination half-life (p < 0.01), up to 6-fold increased oral bioavailability, and 1.7-fold decreased plasma clearance rate (p < 0.01) compared to a drug suspension. Conclusion: The findings suggest that SNEDDS based on long-chain triglycerides (LCT14) formulations seem to be a promising alternative for improving the oral bioavailability of chlorpromazine.

Protective role of Monotheca buxifolia and Bosea amherstiana against H2O2 -induced DNA damage in human lymphocytes and its effect on oxidative enzymes.

Experimental based evidence suggests that most of the medicinal plants possess a wide-ranging pharmacological and biological activity that may possibly protect tissues against O2-induced damages. The objectives of the current study are: first, to investigate the effects of Monotheca buxifolia and Bosea amherstiana on H2O2 induced DNA damage in human lymphocytes and second, to determine its effect on oxidative enzymes. Cells were treated at concentration of 100µg/mL with both plants. Alkaline Single Cell Gel Electrophoresis/comet assay were used for DNA damage analysis. Activities of antioxidant enzymes TBARS, SOD, CAT and POD were assayed on treatment with the extracts. Both plants species possess the protective role against H2O2-induced lymphocytes DNA. Dichloromethane (DCM) fraction of Monotheca buxifolia (H DNA 94.79±0.29%) and methanolic fraction of Bosea amherstiana (H DNA 93.63±2.23%) possess high protection Significantly decrease occur in status of antioxidant enzymes. This study indicates that both plants have potential in preventing oxidative damages/stress related diseases and would be suitably used as supplements in combination with conventional drug for the treatment of cancer like diseases.