In Utero Exposure to Metformin Reduces the Fertility of Male Offspring in Adulthood.

Metformin is a drug used for the treatment of type 2 diabetes and disorders associated with insulin resistance. Metformin is also used in the treatment of pregnancy disorders such as gestational diabetes. However, the consequences of foetal exposure to metformin on the fertility of exposed offspring remain poorly documented. In this study, we investigated the effect of in utero metformin exposure on the fertility of female and male offspring. We observed that metformin is detectable in the blood of the mother and in amniotic fluid and blood of the umbilical cord. Metformin was not measurable in any tissues of the embryo, including the gonads. The effect of metformin exposure on offspring was sex specific. The adult females that had been exposed to metformin in utero presented no clear reduction in fertility. However, the adult males that had been exposed to metformin during foetal life exhibited a 30% reduction in litter size compared with controls. The lower fertility was not due to a change in sperm production or the motility of sperm. Rather, the phenotype was due to lower sperm head quality - significantly increased spermatozoa head abnormality with greater DNA damage - and hypermethylation of the genomic DNA in the spermatozoa associated with lower expression of the ten-eleven translocation methylcytosine dioxygenase 1 (TET1) protein. In conclusion, while foetal metformin exposure did not dramatically alter gonad development, these results suggest that metabolic modification by metformin during the foetal period could change the expression of epigenetic regulators such as Tet1 and perturb the genomic DNA in germ cells, changes that might contribute to a reduced fertility.

Caffeine induces sperm detachment from sperm head-to-head agglutination in bull.

Sperm head-to-head agglutination is a well-known known phenomenon in mammalian and non-mammalian species. Although several factors have been reported to induce sperm agglutination, information on the trigger and process of sperm detachment from the agglutination is scarce. Since hyperactivated motility is involved in bovine sperm detachment from the oviduct, we focused on caffeine, a well-known hyperactivation inducer, and aimed to determine the role of caffeine in sperm detachment from agglutination. Agglutination rate of bovine sperm was significantly decreased upon incubation with caffeine following pre-incubation without caffeine. Additionally, we observed that bovine sperm were detached from agglutination only when the medium contained caffeine. The detached sperm showed more asymmetrical flagellar beating compared to the undetached motile sperm, regardless of whether before or after the detachment. Intriguingly, some sperm that detached from agglutination re-agglutinated with different sperm agglutination. These findings indicated caffeine as a trigger for sperm detachment from the agglutination in bull. Furthermore, another well-known hyperactivation inducer, thimerosal, also significantly reduced the sperm agglutination rate. Overall, the study demonstrated the complete process of sperm detachment from sperm head-to-head agglutination and proposed that hyperactivated motility facilitates sperm detachment from another sperm. These findings would provide a better understanding of sperm physiology and fertilization process in mammals.

Impact of chronic vitamin A excess on sperm morphogenesis in mice.

BACKGROUND: The increasing availability of fortified foods and supplements has caused an overconsumption of vitamin A (VA), above the recommended level. To date, the effects of chronic VA excess (VAE) on spermatogenesis remain unclear. OBJECTIVE: This study aims to investigate the long-term excessive intake of VA effects on spermatogenesis in mice. MATERIALS AND METHODS: Dams were initially fed a control diet (4 IU/g) or a VAE diet (250 IU/g), 4 weeks prior to mating and during pregnancy. Dams and their male pups continued this diet regimen until the offspring reached 12 weeks of age. At 12 weeks of age, epididymis caudal spermatozoa and testes were collected. For histological analysis, sections were stained with periodic acid-Schiff-hematoxylin, and quantitative PCR was used to detect changes in gene expression in the testes of the VAE mice. Sperm motility and morphology were evaluated to detect the endpoint of VAE toxicity. RESULTS: Body weights were not significantly different between the control and VAE groups. Testicular cross-sections from the control and VAE mice contained a normal array of germ cells, and the daily sperm production was similar between the two groups. However, the percentage of seminiferous tubules in stages VII and VIII was significantly lower in the VAE mice than in the control. In addition, significant changes in the expression of genes involved in retinoid metabolism, spermatogenesis, and spermiogenesis were detected in the testes of the VAE mice. Consistently, sperm motility and head morphology were significantly impaired in the VAE mice. DISCUSSION AND CONCLUSION: Our findings suggest that long-term dietary intake of VAE was able to influence both pre- and post-meiotic spermatogenesis. As a result of testicular toxicity, we demonstrated, to the best of our knowledge, for the first time that long-term VAE caused sperm-head abnormalities.

Distribution of DNA damage in the human sperm nucleus: implications of the architecture of the sperm head.

The sperm nucleus is prone to sustain DNA damage before and after ejaculation. Distribution of the damage is not homogeneous, and the factors determining differential sensitivity among nuclear regions have not yet been characterized. Human sperm chromatin contains three structural domains, two of which are considered the most susceptible to DNA damage: the histone bound domain, harboring developmental related genes, and the domain associated with nuclear matrix proteins. Using a quantitative polymerase chain reaction (qPCR) approach, we analyzed the number of lesions in genes homeobox A3 (HOXA3), homeobox B5 (HOXB 5), sex-determining region Y (SRY)-box 2 (SOX2), ß-GLOBIN, rDNA 18S, and rDNA 28S in human sperm after ultraviolet irradiation (400 µW cm-2, 10 min), H2O2treatment (250 mmol l-1, 20 min), and cryopreservation, which showed differential susceptibility to genetic damage. Differential vulnerability is dependent on the genotoxic agent and independent of the sperm nuclear proteins to which the chromatin is bound and of accessibility to the transcription machinery. Immunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that the highest level of oxidation was observed after H2O2treatment. The distribution of oxidative lesions also differed depending on the genotoxic agent. 8-OHdG did not colocalize either with histone 3 (H3) or with type IIα + ß topoisomerase (TOPO IIα + ß) after H2O2treatment but matched perfectly with peroxiredoxin 6 (PRDX6), which is involved in H2O2metabolism. Our study reveals that the characteristics of the sperm head domains are responsible for access of the genotoxicants and cause differential degree of damage to nuclear areas, whereas chromatin packaging has a very limited relevance. The histone-enriched genes analyzed cannot be used as biomarkers of oxidative DNA damage.

The potential effect of methylseleninic acid (MSA) against γ-irradiation induced testicular damage in rats: Impact on JAK/STAT pathway.

This study suggested that methylseleninic acid (MSA) could respond to the inflammatory signaling associated with ionizing radiation-induced testicular damage. Mature male rats were divided into four groups: negative control, whole body γ-irradiated (IRR) (5 Gy), MSA (0.5 mg/kg, daily for nine consecutive days), and MSA+ IRR groups. MSA increased serum testosterone level and testicular glutathione peroxidase (GPx) as well as decreased the percentage of sperm abnormalities. Radiation prompted inflammatory signaling in the testes through increasing phospho-janus kinase1 (p-JAK1), phospho-signal transducers and activators of transcription 3 (p-STAT3) protein expressions. This induced increment in the inflammatory markers including nuclear factor- kappa B (NF-κB) and interleukin-1beta (IL-1ß) levels. Also, radiation induced elevation of nitric oxide (NO) and malondialdhyde (MDA) levels with consequent reduction in testicular reduced glutathione level (GSH) and superoxide dismutase (SOD) activity. MSA significantly counteracted the radiation effect on testicular nuclear factor erythroid-2-related factor-2 (Nrf2) and suppressor of cytokine signaling (Socs3) protein expressions. In summary, this investigation proposed that MSA preserved spermatogenesis through increasing testosterone levels and GPx activity. Additionally, it diminished testicular inflammation by increasing of Nrf2 and Socs3 levels leading to reducing of p-JAK1, p-STAT3 and NF-κB levels. Histopathological examination results of testicular tissues showed a coincidence with the biochemical analysis.

Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles: Influence of surface modification on biodistribution, retention, DNA damage and oxidative stress.

Superparamagnetic iron oxide nanoparticles (SPION) require stable surface modifications to render safe nanocapsules for biomedical applications. Herein, two types of surface modified poly(lactic-co-glycolic acid)-encapsulated SPION were synthesized using either α-tocopheryl-polyetheleneglycol-succinate (TPGS) or didodecyl-dimethyl-ammonium-bromide (DMAB) as surfactants by emulsification. SPION-TPGS (180 nm) was larger than SPION-DMAB (25 nm) and uncoated SPION (10 nm). Both formulations were positively charged and induced lower cyto-genotoxicity and ROS generation than uncoated SPION in human lymphocytes. SPION-DMAB was least cyto-genotoxic among the three. Based on these results, mice were gavaged with the formulations for 5 consecutive days and biocompatibility studies were performed on the 7th and 21st days. ICP-AES and Prussian blue staining revealed the internalization of SPION-DMAB in brain and spleen, and SPION-TPGS in liver and kidney on day 7. This was correlated with high DNA damage and oxidative stress in the same organs. Substantial clearance of Fe was accompanied by reduced genotoxicity and oxidative stress on day 21. Therefore, SPION-DMAB can be further studied for oral drug delivery to the brain and imaging of cerebral tissue without any functional ligand or external magnetic field.

Morphometry of Sperm Head in Rats Treated with an Antifungal Medication.

The parameters of sperm head in Wistar male rats orally treated with antifungal agent for 48 days during spermatogenesis were studied by the method of image analysis. The degree of roundness (roughness) of sperm head was calculated. Significant differences in morphometric parameters of sperm head, such as length, width, head angle, and roundness were revealed between the treatment and control group. The index of deformation of sperm head in the treatment group rats was 4.93 arb. units. These data indicated microcephaly accompanied by the enlargement of the head, transition of an acute angle to a right angle, and acquiring of a round form. Potential gonadotoxicity was confirmed by the analysis of the functional activity of spermatozoids of male rats (increased count of spermatozoa with head pathology), fertilization ability (enhanced fertility index), and genotoxicity (increased number of chromosomal aberrations polychromatophilic erythrocytes of murine bone marrow). These changes can be responsible for reduced fertility.

Preconception seminal plasma concentrations of endocrine disrupting chemicals in relation to semen quality parameters among male partners planning for pregnancy.

BACKGROUND: Some non-persistent endocrine disruptors (EDCs) are adversely associated with semen quality and few studies have measured those EDCs in seminal plasma. OBJECTIVE: To find an association between EDCs in seminal plasma and semen quality parameters. METHODS: Five chemical classes of non-persistent EDCs were quantified in seminal plasma from 339 male partners who participated in a prospective pregnancy study. Bisphenols, benzophenone UV-filters, antimicrobials and phthalate diesters and their monoester metabolites were measured using high performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Semen samples underwent next day analysis using a standardized protocol for the quantification of 35 endpoints. Linear mixed-effects models of EDCs that were log transformed and rescaled by their standard deviations or dichotomized at the 75th percentile for each exposure and outcomes with covariate adjustment were performed. EDCs in seminal plasma were also assessed relative to clinical reference values of semen quality endpoints using logistic regression or generalized estimating equations. RESULTS: The most consistent findings supporting adverse associations between seminal EDCs and semen quality were observed for some phthalate metabolites. For example, seminal plasma mono-ethyl, mono-n-butyl, mono-2-isobutyl and mono-benzyl phthalate concentrations were associated with decreased odds of having semen volume above clinical reference values (mEP: aOR=0.46; 95%CI= 0.32, 0.66; mBP: aOR=0.40; 95%CI= 0.28, 0.57; miBP: aOR=0.39; 95%CI= 0.27, 0.56), and mBzP: aOR= 0.34; 95%CI= 0.24, 0.49). CONCLUSIONS: Environmentally relevant concentrations of specific phthalates in seminal plasma were associated with diminished semen volume, sperm motility, viability, and morphological alterations in sperm heads such that semen volume and sperm viability fall below reference values.

Effects of In Utero and Lactational Exposure to New Generation Green Plasticizers on Adult Male Rats: A Comparative Study With Di(2-Ethylhexyl) Phthalate.

Di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, is a ubiquitous environmental contaminant and may act as an endocrine disruptor. Early life exposures to DEHP may result in anti-androgenic effects, impairing the development of the male reproductive tract. However, data on the long-lasting consequences of such DEHP exposures on adult male reproductive function are still rare and discrepant. Previously, we identified 2 novel plasticizers, 1,4-butanediol dibenzoate (BDB) and dioctyl succinate (DOS), as potential substitutes for DEHP that did not reproduce classically described endocrine disrupting phenotypes in prepubertal male offspring after maternal exposure. Here, we investigated the consequences of in utero and lactational exposure to BDB and DOS on adult male rat reproductive function in a comparative study with DEHP and a commercially available alternative plasticizer, 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH). Timed pregnant Sprague Dawley rats were gavaged with vehicle or a test chemical (30 or 300 mg/kg/day) from gestation day 8 to postnatal day 21. While DEHP exposure (300 mg/kg/day) significantly increased epididymal weight in the adult, exposure to DINCH, BDB, or DOS did not affect reproductive organ weights, steroid levels, or sperm quality. Using a toxicogenomic microarray approach, we found that adult testicular gene expression was affected by exposure to the higher dose of DEHP; transcripts such as Nr5a2, Ltf, or Runx2 were significantly downregulated, suggesting that DEHP was targeting estrogen signaling. Lesser effects were observed after treatment with either DINCH or BDB. DOS exposure did not produce such effects, confirming its potential as a responsible substitute for DEHP.

Expression and localisation of RXFP3 in human spermatozoa and impact of INSL7 on sperm functions.

Insulin-like peptide 7 (INSL7) or relaxin-3 is a member of the insulin superfamily that is recently discovered. This hormone interacts with relaxin family peptide receptor 3 (RXFP3). Although recent studies of INSL7 have focused on its function in the brain as a neuropeptide, spermatozoa may be a candidate target of INSL7 due to its detection in testes and contains binding sites. Therefore, this study aims to analyse the expression and localisation of RXFP3 on human spermatozoa and to assess the effect of INSL7 on human sperm motility. We have incubated normal semen samples in different doses of INSL7. Sperm motility was analysed by Computer Assisted Sperm Analysis. Moreover, localisation and expression of RXFP3 were assessed in human spermatozoa by immunofluorescence and RT-PCR respectively. This study indicated that RXFP3 mainly localised in the post-acrosomal region of sperm head and neck. However, we did not observe expression of RXFP3 mRNA in human spermatozoa. This study showed that INSL7 alleviated the natural decline in sperm motility after a 4-hr incubation period. This was particularly observed in the 1.8 pmol/L treated samples. These data suggested that most likely expression of RXFP3 arrested in spermiogenesis, but the RXFP3 peptide existed on the surface of mature spermatozoa.

Male urinary paracetamol and semen quality.

The endocrine-disrupting properties of paracetamol have been previously demonstrated in rodent studies of abnormal sperm morphology and diminished testosterone production, in addition to epidemiologic studies of diminished couple fecundity. In this study, we examined the relationship between paracetamol and its metabolite p-aminophenol quantified in a single spot urine and semen quality among 501 male partners of couples planning for pregnancy. Men provided a urine specimen and two fresh semen samples collected approximately one month apart and underwent 24-h analysis for 35 semen quality parameters. Paracetamol and p-aminophenol were quantified in urine by ultra-high-performance liquid chromatography coupled with an electrospray triple quadrupole mass spectrometry. The relationship between natural-log-transformed urinary paracetamol and p-aminophenol rescaled by their standard deviation and 21 Box-Cox-transformed, 14 non-transformed semen parameters was assessed using linear mixed-effects models. The median concentrations (IQR) of urinary paracetamol and p-aminophenol were 15.5 ng/mL (5.44, 73.5) and 978 ng/mL (500, 1596), respectively. Following adjustment for creatinine and age, a 1-standard deviation increase in log-transformed urinary paracetamol was associated with a reduction in beat cross-frequency and an increase in DNA fragmentation [ß (95% CI): -0.59 Hz (-1.16, -0.03) and 0.05% (0.01, 0.09), respectively]. These findings were corroborated in models of categorical chemical concentrations; higher concentrations of paracetamol remained associated with reduced beat cross-frequency and increased DNA fragmentation. A 1-standard deviation increase in log-transformed urinary p-aminophenol was associated with a reduction in sperm head area [ß (95% CI): -0.1 µm2 (-0.18, -0.02) and width -0.02 µm (-0.04, -0.01)]. However, only the association with sperm head area remained statistically significant in models of p-aminophenol quartiles. Our findings suggest that adult male urinary paracetamol is associated with sperm motility and DNA fragmentation, while the metabolite, p-aminophenol, is predominantly associated with sperm head morphometry.

Perinatal exposure to 2,2',4'4' -Tetrabromodiphenyl ether induces testicular toxicity in adult rats.

Since 1965, polybrominated diphenyl ethers (PBDEs) have been used internationally as flame-retardant additives. PBDEs were recently withdrawn from commerce in North America and Europe due to their environmental persistence, bioaccumulative properties and endocrine-disrupting effects. Generations exposed perinatally to the highest environmental doses of PBDE account for one-fifth of the total United States population. While, toxicity of PBDE for the male reproductive system has been demonstrated in several human and animal studies, the long-lasting effects of perinatal exposures on male reproduction are still poorly understood. In this study, pregnant Wistar rats were exposed to 0.2mg/kg 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) from gestation day 8 until postnatal day 21. Male reproductive outcomes were analyzed on postnatal day 120 in offspring. Exposed animals had significantly smaller testes, displayed decreased sperm production per testis weight, had significantly increased percentage of morphologically abnormal spermatozoa, and showed an increase in spermatozoa head size. Perinatal BDE-47 exposure led to significant changes in testes transcriptome, including suppression of genes essential for spermatogenesis and activation of immune response genes. In particular, we observed a 4-fold average decrease in expression of protamine and transition protein genes in testes, suggesting that histone-protamine exchange may be dysregulated during spermatogenesis, resulting in an aberrant sperm epigenome. The possibility of long-lasting effects of developmental PBDE exposures calls for additional studies to build a foundation for the development of preventive and protective interventions against the environmentally-induced decline in fertility.

Role of potassium and pH on the initiation of sperm motility in the European eel.

The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing K+ from this media, and by testing the use of K+ channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K+ and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K+ from the seminal plasma and by treatment with the K+ ionophore valinomycin. This therefore indicates that a reduction of K+ levels in the quiescent stage inhibits further motility. The normal decrease in sperm head area induced by seawater activation was altered by the removal of K+ from the seminal plasma, and an increase in the pHi in the quiescent stage was also induced. Intracellular pH (pHi) was quantitatively measured for the first time in European eel spermatozoa, being 7.2 in the quiescent stage and 7.1 post-activation. Intracellular and external pH levels influenced sperm motility both in the quiescent stage and at activation. The alkalinization of the pHi (by NH4Cl) inhibited sperm motility activation, while acidification (by Na-acetate) did not have any effect. Our results indicate that a pH gradient between the sperm cell and the seminal plasma is necessary for sperm motility activation. The presence of the ion K+ in the seminal plasma (or in the extender medium) is necessary in order to maintain sperm volume, intracellular pH and sperm motility.

The control of male fertility by spermatid-specific factors: searching for contraceptive targets from spermatozoon's head to tail.

Male infertility due to abnormal spermatozoa has been reported in both animals and humans, but its pathogenic causes, including genetic abnormalities, remain largely unknown. On the other hand, contraceptive options for men are limited, and a specific, reversible and safe method of male contraception has been a long-standing quest in medicine. Some progress has recently been made in exploring the effects of spermatid-specifical genetic factors in controlling male fertility. A comprehensive search of PubMed for articles and reviews published in English before July 2016 was carried out using the search terms 'spermiogenesis failure', 'globozoospermia', 'spermatid-specific', 'acrosome', 'infertile', 'manchette', 'sperm connecting piece', 'sperm annulus', 'sperm ADAMs', 'flagellar abnormalities', 'sperm motility loss', 'sperm ion exchanger' and 'contraceptive targets'. Importantly, we have opted to focus on articles regarding spermatid-specific factors. Genetic studies to define the structure and physiology of sperm have shown that spermatozoa appear to be one of the most promising contraceptive targets. Here we summarize how these spermatid-specific factors regulate spermiogenesis and categorize them according to their localization and function from spermatid head to tail (e.g., acrosome, manchette, head-tail conjunction, annulus, principal piece of tail). In addition, we emphatically introduce small-molecule contraceptives, such as BRDT and PPP3CC/PPP3R2, which are currently being developed to target spermatogenic-specific proteins. We suggest that blocking the differentiation of haploid germ cells, which rarely affects early spermatogenic cell types and the testicular microenvironment, is a better choice than spermatogenic-specific proteins. The studies described here provide valuable information regarding the genetic and molecular defects causing male mouse infertility to improve our understanding of the importance of spermatid-specific factors in controlling fertility. Although a male contraceptive 'pill' is still many years away, research into the production of new small-molecule contraceptives targeting spermatid-specific proteins is the right avenue.

Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm-oviduct binding.

In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been characterized as a key molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca(2+) and HCO3(-) on sperm-oviduct binding in the horse. Carbohydrate-specific lectin staining indicated that N-acetylgalactosamine, N-acetylneuraminic acid (sialic acid) and D-mannose or D-glucose were the most abundant carbohydrates on equine oviduct epithelia, whereas D-galactose moieties were not detected. However, in a competitive binding assay, sperm-oviduct binding density was not influenced by any tested carbohydrates, glycosaminoglycans, lectins or D-penicillamine, nor did the glycosaminoglycans induce sperm tail-associated protein tyrosine phosphorylation. Furthermore, N-glycosidase F (PNGase) pretreatment of oviduct explants and APM did not alter sperm-oviduct binding density. By contrast, a combination of the sperm-capacitating factors albumin and HCO3(-) severely reduced (>10-fold) equine sperm-oviduct binding density by inducing rapid head-to-head agglutination, both of which events were independent of Ca(2+) and an elevated pH (7.9). Conversely, neither albumin and HCO3(-) nor any other capacitating factor could induce release of oviduct-bound sperm. In conclusion, a combination of albumin and HCO3(-) markedly induced sperm head-to-head agglutination which physically prevented stallion sperm to bind to oviduct epithelium.

An in vivo assay of the mutagenic potential of imidacloprid using sperm head abnormality test and dominant lethal test.

OBJECTIVE: To assess the mutagenic effects of imidacloprid in germ cells of Swiss albino male mice by sperm head abnormality (SHA) assay and dominant lethal test (DLT). METHODS: Swiss albino mice were exposed to imidacloprid (22, 11 and 5.5 mg/kg/day) along with 3% gum acacia as vehicle control through oral route for 7, 14 and 28 days for SHA assay and for 28 days for DLT. The epididymal sperm smear in 1% eosin stain was analyzed for SHAs. In DLT, male mice were allowed to mate with females after 1, 3 and 6 weeks of end of pesticide treatment. The uterine contents of the sacrificed females were observed for live and dead implants. The analysis of test and control groups data was done by one way ANOVA at p < 0.05. RESULTS: Exposure of all dose levels of imidacloprid (22, 11 and 5.5 mg/kg/day) for seven days did not induce significant SHAs while they induced significant SHAs compared with the control group following exposure for 14 and 28 days. The analysis of uterine content revealed a significant increase in the number of dead implants/female compared with the vehicle control in only those females which were mated with male mice after six weeks of treatment of highest dose level of imidacloprid. The dominant lethal mutations were observed only at spermatogonial stage. CONCLUSIONS: Long-term exposure of pesticide generated SHAs even at lowest dose level (5.5 mg/kg/day for 14 days) and mutagenic effects at spermatogonial stage at highest dose level (22 mg/kg/day for 28 days).

Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to the procedures of fish sperm cryopreservation.

Ameliorative effect of grapefruit juice on amiodarone-induced cytogenetic and testicular damage in albino rats.

OBJECTIVE: To evaluate the ameliorative role of grapefruit juice on the cytogenetic and testicular damage induced by the antiarrythmic drug amiodarone in albino rats. METHODS: Animals were divided into four groups. Group I was considered as control. Group II was given grapefruit juice at a dose level of 27 mL/kg body weight. Group III was orally administered amiodarone (18 mg/kg body weight) daily for 5 weeks. Animals were sacrificed after 5 weeks of treatment. Bone marrow was collected from the femurs for analysis of chromosomal aberrations and mitotic indices. Testes were removed and stained with H&E for histological examination. Sperms were collected from epidedymis for detection of sperm head abnormalities. Comet assay was used to detect DNA damage. RESULTS: Amiodarone treatment caused a significant increase in the percentage of chromosomal aberrations, decreased the mitotic index and increased DNA damage. The testis showed many histopathological alterations, inhibition of spermatogenesis and morphometric changes. The number of sperm head abnormalities was increased. Treating animals with amiodarone and grapefruit juice caused a reduction in chromosomal aberrations, mitotic index, DNA damage and testicular alterations caused by amiodarone. CONCLUSIONS: The results of this study indicated that grapefruit juice ameliorates the cytotoxicty and testicular alterations induced by amiodarone in albino rats and this is may be due to the potent antioxidant effects of its components.

Measuring the regulation of dynein activity during flagellar motility.

Flagellar and ciliary motility are driven by the activity of dynein, which produces microtubule sliding within the axonemes. Our goal is to understand how dynein motile activity is regulated to produce the characteristic oscillatory movement of flagella. Analysis of various parameters, such as frequency and shear angle in beating flagella, is important for understanding the time-dependent changes of microtubule sliding amounts along the flagellum. Demembranated flagella can be reactivated in a wide range of ATP concentrations (from 2 µM to several mM) and the beat frequency increases with an increase in ATP. By imposed vibration of a micropipette that caught a sperm head by suction, however, the oscillatory motion can be modulated so as to synchronize to the vibration frequency over a range of 20-70Hz at 2mM ATP. The time-averaged sliding velocity calculated as a product of shear angle and vibration frequency decreases when the imposed frequency is below the undriven flagellar beat frequency, but at higher imposed frequencies, it remains constant. In addition to the role of ATP, the mechanical force of bending is involved in the activation of dynein. In elastase-treated axonemes, bending-dependent regulation of microtubule sliding is achieved. This chapter provides an overview of several approaches, using sea urchin sperm flagella, to studying the measurements in the regulation of dynein activity with or without mechanical force.

Sperm head vacuoles are not affected by in-vitro conditions, as analysed by a system of sperm-microcapture channels.

Since the introduction of the motile sperm organelle morphology examination, there has been increasing recognition of the fact that the presence of large nuclear vacuoles might have deleterious effects on embryo development. Nevertheless, one fundamental question still being debated is whether specific in-vitro conditions during the handling of semen have an impact on vacuole formation. This study's objective was to analyse whether incubation temperature (20, 37°C) or oxidative stress stimulates the formation of nuclear vacuoles. Furthermore, it examined whether vacuoles disappear in the presence of an acrosome reaction inducer. Therefore, a system of sperm-microcapture channels was developed to permit the observation of the same living spermatozoa over a period of 24h. Neither incubation at 37°C nor induction of oxidative stress led to de-novo formation of nuclear vacuoles. Induction of the acrosome reaction using calcium ionophore A23587 did not lead to any modifications in the proportion of spermatozoa with vacuoles or to the disappearance of pre-existing vacuoles. According to these observations, it is concluded that nuclear vacuoles on the sperm head are already produced at earlier stages of sperm maturation and are not induced or modulated by routine laboratory environments. The examination of spermatozoa at very high magnification has led to the increasingly widespread recognition that the presence of large vacuoles in the human sperm head has deleterious effects on embryo development. One fundamental question, however, still remains: do specific conditions in the laboratory during the preparation and the handling of semen have an impact on vacuole formation? Our initial objective was to analyse whether different incubation temperatures (20, 37°C) and the induction of oxidative stress lead to the formation of sperm head vacuoles. Furthermore, we examined whether vacuoles disappear in the presence of an acrosome reaction inducer. In order to do this we developed a system of sperm-microcapture channels, which permits the observation of the same living spermatozoa over a period of 24h. Incubation at 37°C or induction of oxidative stress did not lead to the formation of any new vacuoles. After inducing the acrosome reaction, we did not detect any modification in the proportion of vacuolated spermatozoa. According to our observations, different temperatures or environmental conditions in the laboratory have no impact on the formation or disappearance of vacuoles. We conclude that sperm head vacuoles are already produced at earlier stages of sperm maturation.