Impact of cigarette‐smoking on sperm DNA methylation and its effect on sperm parameters

DNA methylation is an epigenetic modification of the genome. The purpose of this study was to determine the influence of cigarette‐smoking on sperm DNA methylation from a genomewide survey of sperm samples and to ascertain its effect on sperm parameters. Twenty‐eight sperm DNA samples (from 14 fertile smokers as a case study and 14 proven fertile nonsmokers as controls) were subjected to Infinium 450K BeadChip arrays to identify the changes in the DNA methylation level between the two groups. Then, deep bisulphite sequencing was used to validate five CpGs on 78 samples. The results from the Infinium 450K found that only 11 CpGs showed a significant difference in DNA methylation between the case and the control groups. Five CpGs of the eleven (cg00648582, cg0932376, cg19169023, cg23841288 and cg27391564) underwent deep bisulphite sequencing where cg00648582, related to the PGAM5 gene, and the cg23841288 CpGs, related to the PTPRN2 gene amplicons, showed a significant increase in their DNA methylation level in more than one CpG in the case group. In contrast, a significant decrease was found at cg19169023 and at its various neighbouring CpGs in the TYRO3 gene‐related amplicons. Furthermore, this study demonstrated a significant correlation between the variation in sperm DNA methylation level and standard sperm parameters in the case group.     

增精颗粒对大鼠附睾功能性指标GPC及SA的影响

目的探讨中药“增精颗粒”对大鼠附睾功能性指标精子膜甘油-3-磷酸胆碱(GPC)和附睾液唾液酸(SA)的作用。方法采用雷公藤多甙(GTW)灌胃造成大鼠不育模型,观察增精颗粒(ZJKL)对GPC和SA含量的影响。同时对相应精液指标进行检测。结果ZJKL对GPC及SA含量有明显影响。与模型组比较,ZJKL各组均可降低精子膜GPC含量(P<0.05),增加附睾液SA含量(P<0.05),以低剂量组作用最明显(P<0.01),与正常组比较无显著差异(P>0.05)。且精子密度、精子活率、精子畸形率指标有明显改善(P<0.05)。结论ZJKL对大鼠附睾功能性指标作用显著。提示ZJKL可调节附睾生理功能治疗男性不育。     

The effects of oxytocin and arginine vasopressin in vitro on epididymal contractility in the rat

Oxytocin (OT) and arginine vasopressin (AVP) have been found to be present in the reproductive system of the mammalian male, but their function in this system is unclear. This study examined the effects of these two hormones on contractions of the rat caput epididymis, as well as the hormones' effects on epididymal diameter during contractions. The initial segments of caput epididymides were observed in vitro in a solution of modified Tyrode's solution, with a test solution containing a concentration of either OT or AVP being added after a 5-min period. The frequency and diameter of the epididymal contractions were measured before and after addition of each test solution. Also observed was the effect of magnesium concentrations on the ability of OT to induce changes in epididymal contractility. This study found that the frequency of epididymal contractions increase and that the diameter of the epididymal tubules decrease proportionally during contractions with the addition of either OT or AVP. Furthermore, we noted that Mg++ has an inverse correlation with the frequency of contractions of the caput epididymis both in the presence and absence of OT. These findings suggest that both OT and AVP may have a role in regulating epididymal contractility and thus, perhaps, sperm transport through that organ.     

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human‐mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation.     

Induction of oxidative stress by organic hydroperoxides in testis and epididymal sperm of rats in vivo

The present study describes the extent and pattern of oxidative stress induction in testis and epididymal sperm of rats following in vivo exposure to repeated sublethal doses of 2 model pro-oxidants, namely, t-butyl hydroperoxide (tbHP) and cumene hydroperoxide (cHP). Single sublethal (1/40, 1/20, and 1/10 LD(50)) doses of hydroperoxides (HP) administered intraperitoneally to male rats (CFT-Wistar strain) failed to induce any significant increase in malondialdehyde or reactive oxygen species (ROS) levels in testis or epididymal sperm. However, repeated doses for 1 or 2 weeks induced a marked dose-related enhancement of lipid peroxidation (LPO) and ROS levels in both testis and epididymal sperm. Further evidence, such as significant perturbations in both enzymic and nonenzymic antioxidants and enhanced levels of protein carbonyls in testis, suggested induction of oxidative stress. In testis, moderate depletion in reduced glutathione levels and marked diminution in ascorbic acid and alpha-tocopherol content were accompanied by increased activities of various antioxidant enzymes, namely glutathione peroxidase, glutathione-S-transferase, and catalase, in both the HP treatments. Furthermore, significant alterations in the specific activities of testicular enzymes such as LDH-X, G-6-PDH, and SDH indicated altered testicular physiology. Both HP at higher doses induced significant DNA damage (determined by fluorimetric analysis of DNA unwinding assay) in testis and epididymal sperm. Increased total iron levels in testis of HP-treated rats are indicative of the possible involvement of iron-mediated free radical reactions in this model. These findings provide an account of early oxidative damage in testis and epididymal sperm following short-term exposure to HP in vivo, and this model is being further exploited for understanding the consequences of chronic oxidative stress-mediated alterations for the physiology of male reproductive system and its implications for fertility.     

Epididymal obstruction results in isolated sperm heads in post-vasectomy rats

The aim of this study was to determine if, following vasectomy, epididymal obstruction resulted in changes in vasal stump fluid using a rat vasectomy model. One hundred and twenty-two mature male rats underwent bilateral surgical vasectomy and subsequent unilateral epididymal obstruction. Animals were randomly assigned to one of the five cohorts, which determined the time to kill and vasal fluid assessment. Numbers of whole sperm and sperm heads were compared between the obstructed and non-obstructed sides. Parametric analysis of microscopic vasal fluid findings was performed using a paired t-test. Whole sperm and sperm heads were detected bilaterally among the initial five cohorts. On the obstructed epididymis side, percentage of whole sperm dropped from 36.9% to less than 1% and sperm heads increased from 63.2 to 99.7% at 12 weeks post-obstruction (p < 0.05 at each time interval). On the unobstructed side, percentage of whole sperm rose from 66.3 to 89.5% and sperm heads dropped from 33.7 to 10.5% (p < 0.05 at each time interval). At 12 weeks, the difference between the obstructed and non-obstructed sides for both percentage and quantity of whole sperm and heads was significant with a p value of <0.001. In this rat model, following vasectomy and subsequent epididymal obstruction, testicular vasal stump fluid will contain progressively diminishing numbers of whole sperm and increases in the percentage and absolute numbers of sperm heads.     

Successful lowering of epididymal carnitine by administration of pivalate to rats

This study was designed to lower the epididymal content of carnitine in male rats and to examine subsequent effects on fertility and sperm motility. As carnitine is transported from serum into the epididymal lumen a method to lower serum carnitine was sought. Administration of 20 mmol/L sodium pivalate in the drinking water for up to 5 weeks substantially lowered serum carnitine (to 20% of control levels within 1 week) and reduced epididymal carnitine content (to 25% of control levels in the proximal and 52% of control in distal regions) within 2 weeks. Carnitine in distal cauda epididymal fluid was also reduced (to 30% of control levels) but no changes were observed in the sperm carnitine content. The percentage motility and kinematic parameters of spermatozoa released from four epididymal regions and diluted into artificial medium were unaltered by the treatment, and all males retained their fertility in mating tests performed at weekly intervals. Increasing the dose of sodium pivalate administered to 60 mmol/L for 2 weeks lowered serum carnitine concentration more but did not further decrease epididymal carnitine content and altered neither sperm motility nor male fertility. The rat epididymis secretes an excessive amount of carnitine into its lumen so that substantially lowering the tissue content does not reduce sperm carnitine or affect their motility or fertilizing ability.     

Effects of α-Interferon on Sperm Production, Concentration, and Motility in the Rat

Background :
We evaluated possible effects of α-interferon (α-IFN) on testicular spermatogenesis and epididymal sperm quality in the nude rat.
Methods :
Nude male rats were administered subcutaneous injections of human α-IFN daily for 3 months. The luminal content of the cauda epididymidis was collected by micropuncture. Daily sperm production was determined by Amann's method and sperm concentrations were determined by microassay. Progressive motility was judged by evaluating the linear distance traveled by the sperm in a diluent. Serum levels of testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were also measured at the end of the experiment.
Results :
Daily sperm production and epididymal sperm concentrations were significantly increased after administration of α-IFN, while progressive motility of the spermatozoa was not altered. α-IFN significantly increased serum testosterone levels, while it decreased serum LH levels and left serum FSH levels unchanged.
Conclusion :
α-IFN may improve testicular spermatogenesis and increase the epididymal sperm concentration in the rat. These promising results with α-IFN may pave the way for a new approach to treating male infertility.     

Changes during puberty in chromatin condensation, morphology and fertilizing ability of epididymal spermatozoa of the golden hamster

Summary. Male golden hamster sperm acquire complete fertilizing ability at about 48 days of age. In this study hamsters, 27–130 days of age were killed and their male reproductive tracts examined. Sperm were found in the caudae epididymides from 37 days onward. None of the sperm from animals younger than 41 days were capable of fertilizing ova when placed in the uteri of superovulated females. Using flow cytometry of acridine-orange-stained cells, the chromatin condensation in cauda epididymal sperm was investigated. It was seen that DNA from sperm from the younger animals (under 40 days of age) was less tightly bound to protamine than that obtained from mature animals. In summary, the earliest sperm produced by pubertal hamsters were immature with regard to chromatin condensation, morphology, motility, and ability to fertilize ova, and they developed mature characteristics in the period between 40–48 days of age.     

DNA methylation level of spermatozoa from subfertile and proven fertile and its relation to standard sperm parameters

The epigenetic mechanism plays an important role in spermatogenesis such as DNA methylation where this episode is represented by either switching genes on or off. Twenty‐eight samples (14 case and 14 controls) were subjected to Infinium 450K BeadChip arrays to identify genomic regions that differ in sperm DNA methylation patterns in the subfertile compared to the proven fertile group. Then two CpGs were validated by deep bisulphite sequencing on 82 sperm samples. The results screening study revealed eight CpGs were significantly different in their sperm DNA methylation levels between cases and control group. The results of the validation study for the two CpGs (cg19779893 and cg19406113) showed that a significant variation in the methylation level at 2 CpGs of 3 CpGs related to cg19779893 site amplicon in cases compared to the controls. Moreover, six CpGs related to the cg19406113 site amplicon showed significant differences in sperm DNA methylation between the cases and the control group. Furthermore, there was a significant decrease in the sperm parameters in the cases compared to the control group. This study found two CpGs altered in their sperm DNA methylation levels. In addition, a strong association was found between changes in the sperm DNA methylation levels in these CpGs sites and sperm parameters.     

Effect of different doses of Malaysian honey on reproductive parameters in adult male rats

The aim of this study was to evaluate the effect of different doses of Malaysian honey on male reproductive parameters in adult rats. Thirty-two healthy adult male Sprague-Dawley rats were randomly divided into four groups (eight rats per group). Group 1 (control group) was given 0.5 ml of distilled water. Groups 2, 3 and 4 were given 0.2, 1.2 and 2.4 g kg(-1) body weight of honey respectively. The rats were treated orally by gavage once daily for 4 weeks. Honey did not significantly alter body and male reproductive organs weights. The rats in Group 3 which received honey at 1.2 g kg(-1) had significantly higher epididymal sperm count than those in Groups 1, 2 and 4. No significant differences were found for the percentage of abnormal sperm, elongated spermatid count, reproductive hormonal levels as well as the histology of the testis among the groups. In conclusion, Malaysian honey at a dose of 1.2 g kg(-1) daily significantly increased epididymal sperm count without affecting spermatid count and reproductive hormones. These findings might suggest that oral administration of honey at this dose for 4 weeks may enhance spermiogenesis in adult rats.     

Fluorescence microscopy and flow cytometry in measuring activated caspases in human spermatozoa

Staining of spermatozoa with the fluorescein-Val-Ala-Asp-fluoromethylketone has already been performed on ejaculated sperm samples, using fluorescence microscopy (FM) or flow cytometry (FCM) in order to score activated caspases. This assay may help in assessing apoptosis and its role in male fertility. The present study compares the above two techniques in order to adopt the most accurate method for detection in human frozen-thawed testicular, epididymal and ejaculated spermatozoa. The analyses were carried out on frozen/thawed testicular (n = 14), epididymal (n = 8) and ejaculated (n = 10) sperm samples. Activated caspases were detected in living spermatozoa using fluorescein-labelled inhibitors of poly-caspases (FLICA). For the measurements by FM, the same-observer and different-observer reliability were assessed in testicular and epididymal sperm samples. The inter-method (FM and FCM) reliability was assessed both in epididymal and ejaculated sperm samples. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements from the same sample were tested by Wilcoxon test for matched pairs. For the same-observer and the different-observer data, the ICC were 0.980 and 0.986. In testicular suspensions, the presence of different types of germinal and somatic cells hampers the differentiation of stained spermatozoa by FCM. For the inter-method reliability, the ICC was 0.903. A lower proportion of the viable spermatozoa stained with FLICA was observed by using FM (-6.60 +/- 7.38 %, mean +/- SD; p = 0.003) compared with FCM. To measure the proportion of spermatozoa with activated caspases by this test, FM is a highly accurate and reliable method whatever the sperm origin (ejaculate, epididymis, and testis). FCM cannot be used for testicular samples but seems to be more appropriate for analysis of epididymal and ejaculated sperm samples. The systematic lower proportion by FM in measuring the proportion of stained viable spermatozoa with FLICA involves that only the data measured by the same method (FM or FCM) may be compared.     

Assessment of nuclear DNA integrity of epididymal spermatozoa following experimental chronic spinal cord injury in the rat

Infertility is considered as one of the major problems associated with spinal cord injury (SCI). However, the exact underlying mechanism is still unknown. Therefore, the main objective of this experimental study was to evaluate the effect of chronic SCI on sperm parameters as well as chromatin integrity and DNA of spermatozoa aspirated from cauda epididymis of rats. Forty-five adult Wistar rats were divided into three groups - SCI, sham, and control. Following laminectomy, SCI was induced onto exposed dura matter (T10). The sham group underwent laminectomy of T10 only, while the control rats were not exposed to any type of injury or medication. The cauda epididymal sperms were aspirated after 8 weeks for analysis of sperm parameters and sperm chromatin integrity with aniline blue (AB), chromomycin A3 (CMA3), sodium dodecyl sulphate (SDS), and acridine orange (AO) tests. The sperm progressive motility and normal morphology of SCI rats were significantly changed when compared with other groups (p < 0.05). In addition, AB as well as CMA3 tests were insignificantly increased in the SCI group when compared with the sham and control groups. However, SDS and AO tests were significantly changed in SCI samples when compared with the sham and control groups (p < 0.001). The results showed that chronic SCI in rat disturbs sperm parameters as well as nuclear maturity and DNA integrity of sperms. Therefore, sperm chromatin structure is compromised in SCI animals as revealed by chromatin structural probes. These alterations may reduce the fertility potential of the male gamete following SCI.     

Rose oil inhalation protects against formaldehyde-induced testicular damage in rats

In this experimental study, harmful effects of formaldehyde (FA) inhalation on sperm concentration, sperm quality, serum testosterone levels and the rat testes were investigated. In addition, the possible protective effects of rose oil against to these harmful effects were evaluated. For this purpose, 21 albino-Wistar rats were used. The rats in Group I were used as control group. When the rats of Group II were exposed FA (10 ppm/1 h) for 35 days, the rats of Group III inhalated rose oil (1 ml/1 h) after FA. The epididymal tissues were taken for sperm analysing and the testes were removed for histological examination. In addition, testosterone levels were determined from the blood samples. Although the testosterone levels, the epididymal sperm concentration, and the progressive sperm motility significantly decreased, the abnormal sperm rate significantly increased in the Group II when compared to Group I. In the Group III, these damages were seen less. When the rats in the Group II compared with the control group, there were serious histological damages. In the Group III, it was determined that the histological changes were less than group II. It can be expressed that serious damages occurred via formaldehyde exposure in male reproductive system and that the rose oil had protective effects against these damages.     

Effects of altered epididymal sperm transit time on sperm quality

The epididymal sperm transit time seems to have an important role in the process of sperm maturation, and it seems that alterations to the transit can harm the process. The aim of the present work was to evaluate the influence of altered sperm transit time through the epididymis on sperm parameters and fertility of rats, as well as the role of testosterone in the alterations. Sprague–Dawley adult male rats were randomly assigned to four different groups and were treated for 12 days: (i) 10 μg/rat/day DES, to accelerate the transit; (ii) 6.25 mg/kg/day guanethidine sulphate, to delay the transit; (iii) same treatment as group 1, plus androgen supplementation; (iv) control animals received the vehicles. Guanethidine treatment delayed the sperm transit time through the epididymal cauda, provoking increased sperm reserves in this region. Animals exposed to DES showed an acceleration of sperm transit time in the epididymis, and consequently decreased sperm density in both epididymal regions, the caput-corpus and cauda, and diminished sperm motility. In both cases sperm production was not altered. Testosterone supplementation was able to restore the transit time to values close to normality, as they were higher than in the control rats. The same occurred in relation to sperm motility. Rats exposed to DES presented lower fertility after in utero artificial insemination using sperm collected from the proximal cauda epididymis. Therefore, it was concluded that the acceleration of rat sperm transit time appeared to harm normal sperm maturation, thus decreasing sperm quality and fertility capacity, in an androgen-dependent way.     

On unilateral testicular and epididymal torsion: no effect on the contralateral testis

It is often stated that unilateral testicular torsion results in damage to the contralateral testis; however, there are a growing number of experimental and clinical papers which suggest this is not so. Conflicting results from experimental studies confuse the issue and may be due, among other things, to some specifics of the experimental model. In the present paper, we have examined bilateral rat testes 30 and 60 days after 720 degrees torsion to determine 1) the effect of unilateral testicular torsion with and without the inclusion of epididymal torsion, 2) the effect of relatively chronic torsion (24 hr., 10 day) versus relatively acute torsion (two hr., four hr.), and 3) the effect of establishing the model using scrotal surgery versus using an abdominal approach. Bilateral testicular histology, testis wt. (gm.), cauda epididymal sperm concentrations (sp./ml.), and cauda sperm motility scores (0-4) were examined. Ipsilateral testicular torsion or testicular plus epididymal torsion of two hr. or four hr. duration significantly reduced (p less than .05) ipsilateral testis weights, sperm concentrations, and motility scores, and disrupted normal tissue histology. Contralateral testicles were not altered. Epididymal ischemia alone produced no significant ipsilateral or contralateral effects. Chronic torsion (one day, 10 days) also destroyed ipsilateral testis function without altering the contralateral testicles. The occult cryptorchidism associated with the scrotal approach to establishing the torsion model had no effect on contralateral testicles. In no group, using either Lewis rats or Sprague-Dawley rats, were contralateral testicles altered by unilateral testicular torsion. These results plus recent clinical reports indicate that contralateral testicular damage due to ipsilateral torsion is hardly a proven phenomenon, let alone a significant factor contributing to male infertility.     

Cycloheximide prevents production of arresting, a fraction of 30-50 kDa obtained from seminiferous tubule conditioned medium

Aim: To evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermatogenesis and sperm count in male rats. Methods: The study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections. The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined. Results: The fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P＜0.01) and reduced the epididymal sperm count significantly. Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VⅡ in rats treated with arresting compared to that observed in controls. The length of stage VⅡ in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control. Conclusion: The difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules. (Asian J Androl 2004 Dec;6:359-364)     

Monoclonal antibodies to epididymis-specific proteins using mice rendered immune tolerant to testicular proteins

Monoclonal antibodies (mabs) have been used as a powerful tool for identification of newer sperm proteins. However, conventional hybridoma technology rarely provides chance to obtain mabs to epididymal proteins. To increase this chance, we have used an alternate method of neonatal tolerization. In this protocol, animals were tolerized at birth using testicular proteins followed by immunization with cauda epididymal sperm protein (which is a cocktail of proteins both from testicular and epididymal origin). This protocol induced a specific immune response to epididymal sperm proteins. Spleen from one of these animals was then used for preparation of mabs. This fusion resulted in a number of mabs reacting specifically to epididymal proteins. Although mabs identified a protein of approximately similar molecular weight on 1-dimensional Western blot analysis, there were differences in regional localization on rat sperm as seen by indirect immunofluorescence. Immunohistochemical localization of these proteins in rat epididymis showed region specific synthesis. The synthesis of proteins was seen in the distal caput epididymis, and maximum expression was seen in supranuclear region of corpus epithelium. The proteins were localized on sperm from corpus and cauda region. Epididymis specific synthesis of the proteins and agglutinating nature of the mabs to these underlines the functional importance of these proteins in sperm maturation in epididymis. These antibodies could therefore, be used as tools for understanding the physiology of maturation of sperm in epididymis and role of the epididymal protein in fertilization.     

Effects of cyclosporin A on male reproduction in rats

Effects of cyclosporin (Cs) on male reproduction in rats were examined. A dose-dependent decrease of the sperm counts in the cauda epididymis was observed 6 weeks after Cs was administered. A significant decrease of sperm motility was also observed in the each Cs-treated group in any observational period after Cs injection, which suggested an injury to epididymis by Cs. A slight damage of the seminiferous tubules was demonstrated 6 weeks after administration of 40 or 60 mg/kg of Cs. No change in serum levels of luteinizing hormone and testosterone was demonstrated throughout the experiment. But serum levels of follicle-stimulating hormone were significant high in any observational period except 6 weeks after Cs injection. It was concluded that Cs gave injuries to both spermatogonia and epididymal function in rat.     

Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment

DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome‐wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual‐specific effects that may be caused by other environmental factors.