Alterations In Sperm Morphology Research Articles

Gold nanoparticle intratesticular injections as a potential animal sterilization tool: Long-term reproductive and toxicological implications

This study aimed to evaluate the gold nanoparticles (AuNPs) animal sterilizing potential after intratesticular injections and long-term adverse reproductive and systemic effects. Adult male Wistar rats were divided into control and gold nanoparticle (AuNPs) groups. The rats received 200 µL of saline or AuNPs solution (16 µg/mL) on experimental days 1 and 7 (ED1 and ED7). After 150 days, the testicular blood flow was measured, and the rats were mated with females. After mating, male animals were euthanized for histological, cellular, and molecular evaluations. The female fertility indices and fetal development were also recorded. The results indicated increased blood flow in the testes of treated animals. Testes from treated rats had histological abnormalities, shorter seminiferous epithelia, and oxidative stress. Although the sperm concentration was lower in the AuNP-treated rats, there were no alterations in sperm morphology. Animals exposed to AuNPs had decreased male fertility indices, and their offspring had lighter and less efficient placentas. Additionally, the anogenital distance was longer in female fetuses. There were no changes in the histology of the kidney and liver, the lipid profile, and the serum levels of LH, testosterone, AST, ALT, ALP, albumin, and creatinine. The primary systemic effect was an increase in MDA levels in the liver and kidney, with only the liver experiencing an increase in CAT activity. In conclusion, AuNPs have a long-term impact on reproduction with very slight alterations in animal health. The development of reproductive biotechnologies that eliminate germ cells or treat local cancers can benefit from using AuNPs.

Reproductive Development of Male Rats Exposed In Utero to Stress and/or Sertraline.

Despite increased prescription of sertraline during pregnancy, little is known about its action on reproductive development. Therefore, this study aimed to investigate the impact that stress, associated or not with sertraline, causes on the reproductive development of male rats. Pregnant Wistar rats were divided into 4 groups (n = 16/group): CO-received filtered water; SE-received 20 mg/kg sertraline; ST-submitted to restraint stress and received filtered water; SS-submitted to restraint stress and received sertraline. The treatment was carried out from gestational days (GDs) 13-20. The animals were euthanized on GD 20 (n = 8/group), postnatal day (PND) 45 (n = 8/group), and PND 110 (n = 8/group). The testes and epididymis were analyzed histologically, and immunohistochemistry was performed on the testes by proliferating cell nuclear antigen (PCNA) and the Wilms tumor protein (Wt1). Sperm quality was also analyzed on PND 110. The evolution of body weight, anogenital distance (AGD), and puberty installation day were also verified. Statistical analysis: 2-way ANOVA or Kruskal-Wallis test (p ≤ .05). Fetal testes presented a large number of acidophilic cells in the sertraline-exposed groups. The SS group also showed a decrease in the nuclear volume of Leydig cells. This same group showed low expression of PCNA and Wt1, decreased weight of the testes and epididymis, lower AGD, and delayed puberty installation. The adulthood groups exposed to sertraline presented alterations in sperm morphology and motility. The results demonstrated that prenatal exposure to sertraline compromises the development of the rat reproductive system.

Leaf and Fruit Methanolic Extracts of Azadirachta indica Exhibit Antifertility Activity on Rats' Sperm Quality and Testicular Histology.

The world's population is still growing, having an impact on the environment and the economic growth of developing countries; so that, there is a particular interest in the development of new fertility control methods, focused on male contraception. The objective of this study was to evaluate the effect of methanolic extracts of leaf and fruit of Azadirachta indica on sperm quality and testicular histology of Long Evans rats. Antifertility effects of a methanolic leaf and fruit extracts of A. indica on 24 male rats were investigated. The animals were randomly divided into two control groups and four treatment groups (n=4). Doses of the leaf and fruit extract were given at concentrations of 100 and 200 μg mL-1. A significant decrease in the viability of sperm cells was observed. The leaf extract at a concentration of 200 μg mL-1 inhibited cell viability compared to the negative control (p< 0.001). The percentage of abnormal cells in leaf extract was shown in 100 and 200 μg mL-1, the conditions at which a higher percentage of morphological irregularities of observed (15% and 16% respectively). The results show that there was cellular detachment in the seminiferous epithelium in the experimental groups treated with methanolic extracts. Sperm death was observed without decreasing the number of sperm. The methanolic extracts of Azadirachta indica have a modulating effect on the spermatogenesis of experimental rats through sperm morphological alterations.

The role of infections and leukocytes in male infertility.

Declining birth rates are one of the problems facing society today. Male counterparts are responsible for about half of the infertility cases, and genitourinary tract infections may play a contributing role in approximately 15% of male infertility cases. Leukocytospermia is an established indicator of infection in the male urogenital tract, although other microorganisms such as bacteria and virus may also be contributors to the etiology of male infertility. The pathophysiology of these infectious agents may be initiated by a local inflammatory reaction resulting in an increase in reactive oxygen species (ROS). This results in testicular injury, thereby affecting sperm morphology, sperm motility, sperm viability and elevation of the seminal leukocyte as a result of the genital tract infection. The infectious and inflammatory changes can result in male infertility. It is proposed that high concentrations of seminal leukocyte and infectious agents may affect sperm function resulting in clumping of motile spermatozoa, decreasing acrosomal functionality and also causing alterations in sperm morphology. However, the literature has poorly clarified the role of infection in male infertility, provoking further debate and research on this topic.

Ultra-structural analysis of human spermatozoa by aperture scanning near-field optical microscopy.

Scanning near-field optical microscopy (SNOM) represents a potential candidate for investigation of ultrastructure in human spermatozoa. It is a noninvasive optical technique that offers two main advantages: minimal sample preparation and simultaneous topographical and optical images acquisition with a spatial resolution beyond the diffraction limit. This enables the combination of surface characterization and information from the inner cellular organization in a single acquisition providing an immediate and comprehensive analysis of the cellular portions. In this work spermatozoa are immobilized on poly-L-lysine coated coverslips, fixed according to a standard protocol and imaged by aperture-SNOM in air. In the SNOM images, all peculiar sperm portions show well-resolved optical features, which exhibit good similarities with the structures revealed in transmission electron microscopy images, as compared with literature data. The optical features of anomalous spermatozoa are clearly different as respect with those observed for healthy ones. This analysis reveals the potentialities of SNOM and opens to its application to high-resolution analysis of sperm morphological alterations, which might be relevant in reproductive medicine.

The emerging risk of exposure to nano(micro)plastics on endocrine disturbance and reproductive toxicity: From a hypothetical scenario to a global public health challenge

Humans are potentially exposed to nano(micro)plastics, however their interaction with tissues and cells in humans remains largely unknown. This premise is particularly notable with nano-sized plastic particulates, a potentially most pernicious form of plastic pollution. In this study, even in a hypothetical scenario in terms of dose (1, 3, 6 and 10 mg/kg-day) and exposure time (five weeks), the potential endocrine disturbances with particular reference to reproductive toxicity of polystyrene nanoplastics (PS NPs, average size = 38.92 nm) was studied in male rats considering biomarkers of semen quality, changes in hormonal milieu and molecular signatures of endocrine disruption. Sperm DNA integrity and its chromatin structure were also analyzed. There observed significant inverse associations between exposure to PS NPs and serum concentrations of testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Tissue and cell impairments were also noticed even at the lowest tested dosage, though the severity of lesions followed a clear dose-response pattern. DNA damage as well as alterations in sperm morphology and viability were evident, again proportionally with the amount of exposure dosage. RT-qPCR data were in accordance with the results of physio-histological alterations and fluorescence imaging, as significant down-regulation of PLZF, DAZL, FSH and LH gene expressions were noticed in the testis of exposed animals, suggesting that exposure interferes with spermatogenesis and also with HPT-axis. However, among those with highest exposure dosage, expressions of FSH and LH were significantly increased. ABP modulation further revealed evidence of a nonlinear dose response. The association between PS NPs exposure and GnRH was indeed in an unexpected positive direction, though these end-points were less sensitive at higher doses. Although additional evidence is warranted, the present work provides new insights into the possible risks of plastic nanoparticles exposure in humans, especially since the problem is growing and will persist for a long time.

The influence of benign prostatic hyperplasia on sperm morphological features and sperm DNA integrity in dogs.

Benign prostatic hyperplasia (BPH) has a high incidence in older intact dogs. Due to the increased prostatic oxidative stress and hormonal imbalance of BPH, sperm damage can arise, such as sperm morphological alterations and DNA fragmentation. This study aimed to compare the reproductive potential of healthy dogs and those affected by benign prostatic hyperplasia. Ten dogs were assigned to two experimental groups: dogs without BPH (control; n=5) and dogs diagnosed with BPH (n=5), based on clinical signs and ultrasonographic findings. Three semen collections were performed from each dog within one month and analysed using computer-assisted sperm analysis (CASA) and functional tests. Control group showed higher percentage of sperm DNA integrity (95±1.8%) compared to the BPH group (79.2±6.4%). On the other hand, the percentage of minor sperm defects, amplitude of lateral sperm head displacement of the spermatozoa and medium sperm mitochondrial activity were higher in the BPH group. In conclusion, BPH decreases sperm DNA integrity, increases mitochondrial activity, as well as modifies sperm movement pattern. Therefore, a careful sperm analysis of aged dogs with BPH is required before a reproductive programme can be established for such patients.

Sperm quality and fertility in rats after prenatal exposure to low doses of TCDD: A three-generation study.

Exposure to Tetrachlorodibenzo-p-dioxin (TCDD) in male rats promotes, decreased sperm concentration, alterations in motility and in sperm transit time. We evaluated the effect transgenerational of in utero exposure to low doses TCDD in the sperm quality. Pregnant rats (F0) were exposed to 0.1; 0.5 and 1.0μg of TCDD, on gestational day 15, coincides with the end of most organogenesis in the fetus. Adult male offspring (F1, F2 and F3 generation) were investigated for fertility after artificial insemination in utero. After collection of the uterus and ovaries, the numbers of corpora lutea and implants were determined. TCDD provoked alterations in sperm morphology and diminution in serum testosterone levels and sperm transit time in the cauda epididymis. The fertility significantly decreased in all the generations, at least at one dose. In conclusion, TCDD exposure decreases rat sperm quality and fertility in adult male offspring and this effects persist into the next generation.

Sperm Morphology Assessment in Captive Neotropical Primates.

The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO-2010) standards and Sperm Deformity Index (SDI) criteria, objective computer-assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p<0.01) observed among primate species in values obtained from WHO-2010, SDI, CASMA and SSD sperm analysis methods. In addition, multiple significant positive and negative correlations were observed between the sperm morphological traits (SDI, Sperm Deformity Index Head Defects, Sperm Deformity Index Midpiece Defects, Sperm Deformity Index Tail Defects, Normal Sperm, Head Defects, Midpiece Defects and Tail Defects) and the sperm morphometric parameters (SSD, Area (A), Perimeter (P), Length (L), Width (W), Ellipticity, Elongation and Rugosity) (p≤0.046). In conclusion, our findings using different evaluation methods indicate that pronounced sperm morphological variation exists among these four neotropical primate species. Because of the strong relationship observed among morphological and morphometric parameters, these results suggest that application of objective analysis methods could substantially improve the reliability of comparative studies and help to establish valid normative sperm values for neotropical primates.

Assessment of the effect of prolonged forced swimming on CD-1 mice sperm morphology with and without antioxidant supplementation

As physical exercise has been shown to negatively affect sperm morphology, this study was undertaken to assess the effect of a 3-min forced swimming protocol during 50 days, with and without administration of antioxidants [N-acetylcysteine (NAC) and trans-resveratrol], on sperm morphology in CD-1 mice. Forty-four 13-week-old CD-1 mice were randomly allocated to four different groups: mice not submitted to exercise, control group (CG), mice submitted to swimming without administration of antioxidants (EX), mice submitted to swimming that received trans-resveratrol supplementation [exercise group (EX)+Resv] and mice submitted to swimming exercise that received NAC supplementation (EX+NAC). The EX showed 30.5% of spermatozoa with normal morphology, showing significant differences with regard to the CG, which showed 58.5%. The groups receiving antioxidant supplements showed significantly higher percentages of spermatozoa with normal morphology in comparison with the EX group (EX+Resv: 64.1%, EX+NAC: 48.2%). The imposed model of forced swimming caused alterations in sperm morphology. The antioxidants employed seem to be suitable antioxidants for avoiding exercise-associated sperm morphology anomalies in prolonged forced swimming exercise. Trans-resveratrol has proven to be more efficient for this purpose.

Homogeneous Microscopic Abnormalities in Sperm Morphology and Immotility as A Cause of Male Infertility

Objective To study the identification of the cause of specific sperm abnormalities. Methods Two adult men with specific alterations in sperm morphology causing 100% immobility were included in this study. The study of sperm used: transmission electron microscopy (both patients); apoptotic markers, DNA fragmentation test and fluorescence in-situ hybridization (patient 1) and immunocytochemistry study of sperm flagellum using anti-β tubulin antibodies and ciliary activity test (patient 2). Results Increased DNA fragmentation (52.6%) and apoptosis biomarkers were detected in patient 1, and loss of the central pair of microtubules in patient 2 (‘9+0’ axoneme); the nasal ciliary activity was normal. Conclusion Results suggest an apoptotic origin of the abnormalities in the sperm from patient 1 and dysplasia of the fibrous sheath in patient 2.

Deltamethrin-induced Alterations in Sperm Morphology and Spermatogenesis Impairment in Adult Sprague-Dawley Rats

The aim of this investigation was to assess Deltamethrin-induced alterations in sperm quality and testicular toxicity. Sixty adult male Sprague-Dawley rats were assigned Four Groups (A-D). A acted as control and received deionised distilled water, B-D were exposed orally to 6.25, 12.5 and 25 mg/kg/day of Deltamethrin Emulsifiable concentrate for 35 days. The results showed significant (p<0.05) reduction in bodyweight at 25 mg/kg/day with no effect on vital organs weights. Accesory sex organ weights declined significantly (p<0.05). A concentration-dependent reduction (p<0.05) in the testes weight (2.81±0.52 to 2.56±0.22 g) epididymal weight (2.36±0.12 to 2.18±0.22 g) occured. Histopathological evaluation of the testes showed degenerative changes and impairment of spermatogenesis accompanied by cellular losses at 12.5 and 25 mg/kg/day. The most affected spermatogenic elements were the primary spermatocytes. Post testicular toxicity was confirmed from epididymal sperm quality with reduction in motility and viability, increased percentage dead sperm cells and percentage abnormality in the sperm morphology including retention of cytoplasmic droplet and sperm head abnormality. There was elevation in the concentration of Aspartate aminotransferase which was positively and significantly (p<0.01) correlated with Deltamethrin concentration (r = 0.9060), R 2 = 0.8209, Alanine aminotransferase (ALT) r = 0.9015, R 2 = 0.8127 and Alkaline phosphatase (ALP) r = 0.9099, R 2 = 0.828. It was concluded that Deltamethrin has the potential to impair fertility and general reproductive health by inhibiting spermatogenesis and thus, limiting sperm production potential. Periodic evaluation of reproductive health especially in males constantly exposed to Deltamethrin due to their occupation is recommended.

A TIME-COURSE CHARACTERIZATION OF MALE REPRODUCTIVE TOXICITY IN RATS TREATED WITH METHYL METHANESULPHONATE (MMS)

Methyl methanesulphonate (MMS), a potent alkylating agent and testicular toxicant, was orally administered to rats for 5 days at doses of 20, 30, and 40 mg/kg. During the recovery period of 5 weeks, males were evaluated for multiple endpoints such as organ weights, fertility, and sperm parameters. The 5-week recovery periods are designated as follows: Day 1 (1 day after final treatment); Week 1, Week 2, Week 3, Week 4, and Week 5 (first, second, third, fourth, and fifth week after final treatment). A clear time-course of dominant lethals was observed. The peak severities of the dominant lethals were observed in Week 2. It was judged that the most sensitive cellular targets for the dominant lethals are late spermatids. Sperm examination revealed a clear time-course and dose-dependent changes in the frequency of sperm morphological abnormalities. The peak severities of the sperm morphological alterations in cauda epididymis were observed in Week 4. Sensitive cellular stages for the induction of sperm morphological abnormalities were judged to be late spermatocytes and early spermatids. The most frequently observed type of morphologically abnormal spermatozoa was tailless sperm, followed by no-hook head sperm. Although the initial cause for both sperm morphological alterations and dominant lethals was suggested to be genetic insult to the germ cells, there were no obvious relationships observed between these two findings.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection.

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

Attainment of spermatogenesis in Swedish cross-bred boars.

Twenty peripubertal Swedish cross-bred boars were used to study the testicular differentiation and the maturation of spermatozoa (epididymal) between 100 and 180 days of age. In all animals the sex cords were small and solid at 100 days of age. Spermatogenesis started at approximately 115 days of age and was completed, i.e. the cellular organization of the seminiferous tubules indicated a sexually mature testis with normal spermatogenesis, at 180 days of age. By 125 days of age only a few spermatozoa were found in the cauda epididymidis Furthermore, a high frequency of sperm morphological alterations were seen. The sperm concentration in the cauda increased with increasing age, and simultaneously the frequency of abnormal spermatozoa decreased. However, not all boars had a 'mature' semen picture in the cauda epididymis at 180 days of age.

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm.

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.

Protein factor of fertility in men in conjugal couples with reproductive disorders

Humans are potentially exposed to nano(micro)plastics, however their interaction with tissues and cells in humans remains largely unknown. This premise is particularly notable with nano-sized plastic particulates, a potentially most pernicious form of plastic pollution. In this study, even in a hypothetical scenario in terms of dose (1, 3, 6 and 10 mg/kg-day) and exposure time (five weeks), the potential endocrine disturbances with particular reference to reproductive toxicity of polystyrene nanoplastics (PS NPs, average size = 38.92 nm) was studied in male rats considering biomarkers of semen quality, changes in hormonal milieu and molecular signatures of endocrine disruption. Sperm DNA integrity and its chromatin structure were also analyzed. There observed significant inverse associations between exposure to PS NPs and serum concentrations of testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Tissue and cell impairments were also noticed even at the lowest tested dosage, though the severity of lesions followed a clear dose-response pattern. DNA damage as well as alterations in sperm morphology and viability were evident, again proportionally with the amount of exposure dosage. RT-qPCR data were in accordance with the results of physio-histological alterations and fluorescence imaging, as significant down-regulation of PLZF, DAZL, FSH and LH gene expressions were noticed in the testis of exposed animals, suggesting that exposure interferes with spermatogenesis and also with HPT-axis. However, among those with highest exposure dosage, expressions of FSH and LH were significantly increased. ABP modulation further revealed evidence of a nonlinear dose response. The association between PS NPs exposure and GnRH was indeed in an unexpected positive direction, though these end-points were less sensitive at higher doses. Although additional evidence is warranted, the present work provides new insights into the possible risks of plastic nanoparticles exposure in humans, especially since the problem is growing and will persist for a long time.

Reproductive tract lesions resulting from subchronic administration (63 days) of tri- o-cresyl phosphate in male rats

An initial dose-range pilot study where animals were gavaged with between 100 and 1600 mg tri- o-cresyl phosphate (TOCP)/kg/day for 14 days resulted in decreased epididymal sperm density and disruption of the seminiferous epithelium in 100% of treated animals. A subchronic 63-day study (reflecting the 49-day length of the rat seminiferous epithelium cycle plus the 14-day transit time of spermatids through the epididymis was initiated. Dose-dependent (10 to 100 mg TOCP/kg/day) decreases in cauda epididymal sperm motility and density, testicular enzyme activities, and alterations in sperm morphology were observed. Concurrent pair-fed controls (matched to the highest dose group, 100 mg TOCP/kg/day) indicated that weight loss resulting from TOCP administration had minimal contributory effects to the testicular toxicity seen. Plasma α-tocopherol acetate (vitamin E) and testosterone concentrations were unaffected. Tri- p-cresyl phosphate (TPCP), the nonneurotoxic structural analog of TOCP, produced no toxic effects, demonstrating the necessity of the ortho-cresol moiety for induction of damage. A minimum effective (threshold) dose for observable testicular toxicity was determined to be 10–25 mg TOCP/kg in this study. These data suggest that TOCP interferes with spermatogenic processes and sperm motility directly and not via an androgenic mechanism or decreased vitamin E availability.