Bisphenol-A affects male fertility via fertility-related proteins in spermatozoa.
ABSTRACT: The xenoestrogen bisphenol-A (BPA) is a widespread environmental contaminant that has been studied for its impact on male fertility in several species of animals and humans. Growing evidence suggests that xenoestrogens can bind to receptors on spermatozoa and thus alter sperm function. The objective of the study was to investigate the effects of varying concentrations of BPA (0.0001, 0.01, 1, and 100 ?M for 6 h) on sperm function, fertilization, embryonic development, and on selected fertility-related proteins in spermatozoa. Our results showed that high concentrations of BPA inhibited sperm motility and motion kinematics by significantly decreasing ATP levels in spermatozoa. High BPA concentrations also increased the phosphorylation of tyrosine residues on sperm proteins involved in protein kinase A-dependent regulation and induced a precocious acrosome reaction, which resulted in poor fertilization and compromised embryonic development. In addition, BPA induced the down-regulation of ?-actin and up-regulated peroxiredoxin-5, glutathione peroxidase 4, glyceraldehyde-3-phosphate dehydrogenase, and succinate dehydrogenase. Our results suggest that high concentrations of BPA alter sperm function, fertilization, and embryonic development via regulation and/or phosphorylation of fertility-related proteins in spermatozoa. We conclude that BPA-induced changes in fertility-related protein levels in spermatozoa may be provided a potential cue of BPA-mediated disease conditions.
Project description:Conventional semen analyses are used to evaluate male factor fertility/infertility in humans and other animals. However, their clinical value remains controversial. Therefore, new tools that more accurately assess male fertility based on sperm function and fertilization mechanism are of interest worldwide. While protein markers in spermatozoa that might help differentiate fertile and infertile sperm have been identified, studies are in their infancy, and the markers require validation in field trials. In the present study, to discover more sensitive biomarkers in spermatozoa for predicting male fertility, we assessed protein expression in capacitated spermatozoa. The results demonstrated that cytochrome b-c1 complex subunit 2 (UQCRC2) was abundantly expressed in high-litter size spermatozoa (>3-fold). On the other hand, equatorin, beta-tubulin, cytochrome b-c1 complex subunit 1 (UQCRC1), speriolin, Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and seminal plasma sperm motility inhibitor were abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A and UQCRC1 expression negatively correlated with litter size, while UQCRC2 expression positively correlated with litter size. Finally, the putative biomarkers predicted litter size in field trials. Our study suggests that biomarkers present in spermatozoa after capacitation can help differentiate superior male fertility from below-average fertility with high sensitivity.
Project description:Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood.The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice.Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 ?g/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease.BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases.Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa. Citation: Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238-245;?http://dx.doi.org/10.1289/EHP378.
Project description:Peroxiredoxins (PRDXs) are important antioxidant enzymes reported to have a role in sperm function and male fertility. However, how PRDXs affects male fertility remain fundamental unanswered questions. We therefore sought to investigate the role of these enzymes in sperm function and fertilisation. In this in vitro trial, mouse spermatozoa were incubated with different concentrations of conoidin A (1, 10, or 100?µM), a specific inhibitor of PRDXs. Our results demonstrated that inhibition of PRDXs by conoidin A significantly decreased the oxidized form of peroxiredoxins (PRDXs-SO3) in spermatozoa. Decreased PRDX activity was associated with a significant reduction in sperm motility parameters, viability, and intracellular ATP, whereas ROS levels, DNA fragmentation, and loss of mitochondrial membrane potential were increased. Simultaneously capacitation and the acrosome reaction were also significantly inhibited perhaps as a consequence of decreased tyrosine phosphorylation and protein kinase-A activity. In addition, fertilisation and early embryonic development were adversely affected following PRDXs inhibition in spermatozoa. Taken together, our data demonstrate that decreased PRDX activity directly affects male fertility due to negative effects on important functions and biochemical properties of spermatozoa, ultimately leading to poor fertilisation and embryonic development.
Project description:Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans.
Project description:Background:The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process. Results:Our strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa (higher % cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization. Conclusions:These results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.
Project description:Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing.Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate?<?1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions.Despite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.
Project description:The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.
Project description:Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p?>?0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility.
Project description:BACKGROUND: Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. RESULTS: Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. CONCLUSION: This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.
Project description:Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = -0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = -0.49, P<0.006 and r = -0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1-3 vs. Bull 4-5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.