Sperm Fractions Research Articles

Sperm Nuclei Analysis and Nuclear Organization of a Fertile Boar-Pig Hybrid by 2D FISH on Both Total and Motile Sperm Fractions.

Simple SummaryMammalian hybrids frequently form in nature between closely related species, and the majority are sterile due to the production of chromosomally unbalanced gametes. In Italy, the widespread wild boar has had negative consequences for free-range pig farming, which is considered the best practice for pig welfare and is a common method of farming most autochthonous pig breeds. This study aimed to analyze the sperm DNA integrity, sperm meiotic segregation and nuclear spatial organization in a boar–pig hybrid, for the first time, to evaluate its fertilizing capacity. The results show that the hybrid presented a high frequency (64%) of motile spermatozoa with a regular chromosome composition and a specific spatial distribution. This study underlines how fertile boar–pig hybrids represent a growing problem for conserving autochthonous pig breeds.A wide range of mammalian hybrids has recently been found by chance or through population-screening programs, but studies about their fertilizing capacity remain scarce and incomplete. Most of them are assumed to be sterile due to meiotic arrest caused by the failure of chromosome pairings. In this study, we evaluated both sperm meiotic segregation, by 2D fluorescent in situ hybridization (FISH) analysis, and sperm quality (Sperm Chromatin Structure Assay) by flow cytometer in a fertile boar–pig hybrid (2n = 37,XY) originating from a Nero Siciliano pig breed (Sus scrofa domesticus) and a wild boar (Sus scrofa ferus). Spermatozoa were also separated by a dual-layer (75–60%) discontinuous Percoll gradient, resulting in two fractions with a significantly better overall quality in the motile sperm fraction. These data were confirmed by FISH analysis also, where the frequencies of spermatozoa with a regular chromosome composition were 27% in total sperm fraction and 64% in motile sperm fraction. We also evaluated the nuclear architecture in all counted spermatozoa, showing a chromatin distribution changing when chromosome abnormalities occur. Our results demonstrate that the chromosome pairing has a minimal effect on the sperm segregation and semen quality of a boar–pig hybrid, making it fertile and harmful for the conservation of autochthonous pig breeds.

The impact of cryopreservation on the morphology of spermatozoa in men with oligoasthenoteratozoospermia

The cryopreservation of ejaculate can reduce the viability, motility, and morphological characteristics of the spermatozoa of infertile men. Oligoasthenoteratozoospermia (OAT) is the most common cause of male subfertility. The aim of this study was to evaluate the morphological characteristics and viability of progressive motile sperm fraction before and after cryopreservation, and to determine whether cryopreservation of progressive motile sperm fraction is effective in eliminating morphologically abnormal sperm in men with OAT. An increased proportion of spermatozoa with normal morphology in fresh progressive motile sperm fraction compared with fresh ejaculate has been observed. After cryopreservation, the motility was 65.5 ± 8.8%; the proportion of spermatozoa with normal morphology increased non-significantly compared with freshly prepared motile sperm fraction (35.6 ± 5.5%). Concurrently, the proportion of cryopreserved spermatozoa with head defects increased significantly by 1.7 times (to 38.4 ± 4.7%) and the proportion of almost all morphologically abnormal sperm cells, particularly spermatozoa with multiple abnormalities, was reduced significantly. These data appear to be a novel finding in the context of patients with OAT. Using such spermatozoa for in vitro fertilization leads to a significant decrease in both a number of embryos at the cleavage stage and the blastocysts formation rate. High-magnification sperm morphology examination and selection, IMSI, post-cryopreservation significantly increased the likelihood of successful oocyte fertilization and subsequent embryo development.

Comparative Analysis of Effectiveness of Clomiphene Citrate and Letrozole Combined with Low Dose Human Menopausal Gonadotropin for Controlled Ovarian Stimulation in Intrauterine Insemination Cycles

Objective: To compare the efficacy of clomiphene citrate and letrozole in combination with low dose human menopausal gonadotropin for controlled ovarian stimulation in intrauterine insemination (IUI) cycles.Methods: During January-2018 to December-2019 for intending 496 IUI cycles, controlled ovarian stimulation was performed with either clomiphene or letrozole combined with human menopausal gonadotropin (hMG), in two arms: subjects in one arm (Group A) were with clomiphene and hMG in 222 cycles; those in the second arm (Group B) were with letrozole and hMG in 274 cycles. Pregnancy rate and clinical pregnancy rate of both groups were considered as the primary outcomes.Results: Patient characteristics like female age, indications for IUI, type of IUI, endometrial thickness and total motile fraction (TMF) of spermatozoa of male partners were seen similar in both groups. The letrozole-hMG group (Group B) had significantly higher numbers of cycles with single dominant follicle (P=0.01) and human chorionic gonadotropin (hCG) was more frequently used as the ovulation trigger (P=0.03). Pregnancy rate (18.5% vs. 15.3%, P=0.35) and clinical pregnancy rate (18.5% vs. 15.3%, P=0.35) were similar in groups A and B, respectively.Conclusion: Clomiphene citrate and letrozole combined with low dose human menopausal gonadotropin were equally effective for controlled ovarian stimulation in IUI cycles.

Supplementation with Proline Improves Haemato-Biochemical and Reproductive Indicators in Male Rabbits Affected by Environmental Heat-Stress.

Simple SummaryThe exposure of rabbits, such as other mammals, to environmental heat stress drastically affects homeostasis and reproductive function. The current trial indicates that a dietary supplementation with 50–100 mg/kg DM of proline improves redox status, blood metabolites, and reproductive traits of rabbit bucks.Twenty-four adult rabbit bucks (n = 6 per treatment) were fed a basal diet supplemented with 0 (control), 50, 100, and 150 mg proline/kg dry matter (DM) diet for 12 weeks to determine possible usefulness for alleviating the negative impact of environmental heat stress on redox status, haemato-biochaemical attributes and semen quality. There were significant dose–response effects, with increments in levels of dietary proline (LDP) quadratically improving red blood cell counts (p = 0.017), rectal temperature (p = 0.009), and respiratory rate (p < 0.001). Increasing LDP cubically affected superoxide dismutase activity in blood plasma (p = 0.012) and total antioxidant capacity in both blood and seminal plasma (p < 0.001 and p = 0.006, respectively). The optimal response was observed at 30 and 80 mg proline/kg DM for blood and seminal plasma, respectively. With regards to homeostasis indexes, increments in LDP cubically modified blood plasma concentrations of total protein (p = 0.002) and albumin (p < 0.001), with an optimal response found at 70 mg proline/kg DM. A linear relationship (p = 0.005) was also observed between LDP and blood plasma glucose concentrations, with the optimal response being found at 100 mg proline/kg DM. Increasing LDP also showed positive effects on reproductive traits, with quadratic increases in blood plasma testosterone and cortisol concentrations (p < 0.001; optimal responses at 50 and 60 mg proline/kg DM, respectively), a positive linear relationship with in libido, ejaculate volume, sperm concentration and total sperm count (p < 0.001 for all; optimal responses observed at 100 mg proline/kg DM) and a quadratic increase in total functional sperm fraction (p < 0.001; optimal response at 70 mg proline mg/kg DM). Hence, the optimal positive effects of dietary proline supplementation on redox status, blood metabolites, and reproductive traits of rabbit bucks may be achieved at 50–100 mg/kg DM.

Zika RNA and Flavivirus-Like Antigens in the Sperm Cells of Symptomatic and Asymptomatic Subjects.

Zika virus (ZIKV) RNA has been found to remain in human semen for up to one year after infection, but the presence of Flavivirus antigens in the different compartments of semen has been largely unexplored. Following the introduction of ZIKV in Nicaragua (2016), a prospective study of patients with clinical symptoms consistent with ZIKV was conducted in León to investigate virus shedding in different fluids. ZIKV infection was confirmed in 16 male subjects (≥18 years of age) by RT-qPCR in either blood, saliva or urine. Of these, three provided semen samples at 7, 14, 21, 28, 60 and 180 days postsymptom onset (DPSO) for Flavivirus antigens and RNA studies. These cases were compared with 19 asymptomatic controls. Flavivirus antigens were examined by immunofluorescence (IF) using the 4G2 Mabs, and confocal microscopy was used to explore fluorescence patterns. The three (100%) symptomatic subjects and 3 (16%) of the 19 asymptomatic subjects had Flavivirus antigens and viral RNA in the spermatozoa fraction. The percentage of IF Flavivirus-positive spermatozoa cells ranged from 1.9% to 25% in specimens from symptomatic subjects, as compared with 0.8% to 3.8% in specimens from asymptomatic controls. A marked IF-pattern in the cytoplasmic droplets and tail of the spermatozoa was observed. The sperm concentrations (45 × 106/mL vs. 63.5 × 106/mL, p = 0.041) and the total motility percentage (54% vs. 75%, p = 0.009) were significantly lower in specimens from ZIKV-positive than in those of ZIKV-negative. In conclusion, this study demonstrated the presence of Flavivirus antigens and RNA within a time frame of 28 DPSO in sperm cells of symptomatic and asymptomatic subjects during the ZIKV epidemic. These findings have implications for public health, in terms of nonarthropod-born, silent transmission facilitated by sperm cells and potential transmission from asymptomatic males to pregnant women, with consequences to the fetus.

Sperm selection of cryopreserved milt of catfish (Rhamdia quelen) by density gradient centrifugation with AllGrad® 90%

Density gradient centrifugation is a technique used to wash or separate samples of cryopreserved milt, mainly in humans and bovines allowing, for example, reducing the concentration of cryoprotectants or choosing the best portion of sperm. The proposed method seeks to reduce the presence of cryoprotectant in the cryopreserved milt of the Rhamdia qhelen and to obtain a fraction of better quality sperm. Gradient centrifugation was formed from 90% AllGrad® and different centrifugation times and forces were compared. The separated sperm presented a low increase in motility and decreased head damage and presence of gout, however, it was better compared to the non-separated samples. The speed of 1000 × g for 10 min, 4 °C, allowed 22.25 ± 4.64% of normal spermatozoa, that is, 9.25% more than the non-centrifuged milt (p = 0.0013).•The centrifugation method allows a fraction of spermatozoa morphologically less affected by cryopreservation.•Density gradient centrifugation with AllGrad® 90% is proposed as a tool of easy adaptation and application for the separation of cryopreserved sperm of R. quelen.•Density gradient centrifugation method at 1000 × g for 10 min allows obtaining a better fraction of normal sperm.

Physiological response, testicular function, and health indices of rabbit males fed diets containing phytochemicals extract under heat stress conditions.

Objective:The present study aimed to evaluate the impact of dietary turmeric or garlic extract on physiological responses, hematological parameters, antioxidants status, testicular functions, and health indices of heat-stressed rabbit males.Materials and Methods:Seventy bucks were distributed into seven experimental groups (ten/group). A commercial diet (CD) was fed to rabbit males in the control group, while males in G2, G3, and G4 were fed CD with turmeric at 30, 60, and 90 mg/kg, respectively. Males in G5, G6, and G7 were fed CD with garlic extract at 50, 75, and 100 mg/kg, respectively, for 8 weeks.Results:The results showed that turmeric or garlic extract levels decreased ear temperature, respiration rate, germ cell apoptotic number, total cholesterol, triglycerides, malondialdehyde level, libido, and sperm of dead and abnormal tail (p < 0.05). The hemoglobin and red and white blood cells, platelets, total antioxidants capacity, mass motility, mitochondrial potential, normal, acrosome reacted, normality, and a total functional fraction of spermatozoa and relative of epididymal weight were increased (p < 0.05). The increased packed cell volume percentage and initial semen fructose were significant only when 100 mg per kg garlic extract was supplemented.Conclusion:Phytochemicals extract supplementation can be an effective dietary supplementation to eliminate heat stress and improve health, oxidative capacity, and testicular functions of rabbit males.

411 Sperm movement, storage, and release from the oviduct

Abstract After semen deposition, a fraction of sperm is transported through the female reproductive tract to the lower oviduct, the isthmus, where sperm are retained to form a reservoir. Some of these sperm are released to move to the upper oviduct, the site of fertilization. For sperm to make this journey, they must overcome challenges including possible phagocytosis, high fluid viscosity, and peristaltic contractions of the tract. The study of sperm transport is complex because so few sperm reach the site of fertilization. We have focused our studies on how sperm are retained in the reservoir, how storage prolongs sperm lifespan, and how sperm are released to fertilize oocytes. Sperm storage is particularly important in situations in which ovulation is not tightly synchronized with semen deposition. This occurs in domestic animals, but is especially notable in some species of bats, birds, and insects. Using porcine sperm and an array of 400 common glycans, we identified two specific glycan motifs found in all structures that bound sperm, a Lewis X trisaccharide and a branched 6-sialylated oligosaccharide. Tandem MS profiling indicated that both motifs were abundant in the asparagine-linked glycans of the oviduct epithelium and several larger oligosaccharides were identified that contained both motifs. When these motifs were immobilized, each could retain sperm, suppress Ca2+ influx and lengthen sperm lifespan. Secretions from the cumulus-oocyte complex (COC) and progesterone released sperm from immobilized oviduct glycans, suggesting that COCs can themselves signal sperm release. Progesterone-induced release required CatSper channels, sperm hyperactivation and was dependent on sperm protein degradation. These studies support a model in which sperm are retained in the isthmus by specific glycans on the epithelium, which extends sperm lifespan until COCs produce releasing factors that promote sperm liberation from the isthmus and movement to the ampulla to fertilize the COCs.

Induction of vacuolization in spermatozoa from men with oligoasthenoteratozoospermia after cryopreservation with glycerol and polyvinylpyrrolidone

Background. The two–step method using penetrating cryoprotectant glycerol is a routine for cryopreservation of spermatozoa from men with normal spermatogenesis. However, the use of this freezing method in the case of oligoasthenoteratozoospermia (OAT) leads to additional cells damage and there is a need to search for new effective cryopreservation method. The sperm vacuolization inherent in male gametes plays a negative role, reducing the fertilizing ability of cells. Objective. The aim of the study was to assess the degree of vacuolization induced in human OAT sperm after cryopreservation by a two–stage method using glycerol and polyvinylpyrrolidone (PVP), molecular mass 360000. Methods. The isolated sperm fraction was divided into 3 groups: group I – native spermatozoa, II – spermatozoa cryopreserved by the two–stage method with 10% glycerol, group III – spermatozoa cryopreserved by the two–stage method with 10% PVP. Results. About 5% of spermatozoa contained vacuoles in group I. The number of spermatozoa with one small vacuole increased to (19.68 ± 2.27)% in group II. And in group III the number of cells without vacuoles was comparable with native spermatozoa: 95.59 ± 1.59 and (96.09 ± 2.02)%, respectively. Conclusion. Thus, the use of a penetrating cryoprotectant of glycerol at 10% concentration for two–stage method cryopreservation of human spermatozoa initiates the vacuoles formation while the use of non–penetrating PVP can prevent vacuolization.

DNA fragmentation of sperm: a radical examination of the contribution of oxidative stress and age in 16 945 semen samples.

What is the relationship between sperm DNA fragmentation and oxidative stress (OS) with increasing male age? Sperm DNA fragmentation increases with age and is likely related to both defective spermatogenesis and increasing OS levels. Sperm quality declines with age. The presence of DNA damage in a high fraction of spermatozoa from a raw semen sample is associated with lower male fertility in natural conception and intrauterine insemination. A retrospective cohort study of 16 945 semen samples analysed at a single reference laboratory between January 2010 and December 2018. All males were undergoing an infertility evaluation. The cohort was divided into seven age categories: <30, 30-34, 35-39, 40-44, 45-49, 50 to <54 and ≥55 years. The mean age was 37.6 years (SD 6.8). Sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) were calculated using flow cytometry. OS levels were measured using the oxidative stress adducts (OSA) test, by spectrophotometry. ANOVA with weighted polynomial contrast analysis was used to evaluate trends for DFI, OSA and HDS values across age categories. Mean DFI significantly increased across all age groups (Ptrend < 0.001). OSA was lowest in patients <30 years old (mean 3.6, SD 1.0) and also increased as age increased (Ptrend < 0.001). There was a statistically significant difference between age groups for each of the three parameters (P < 0.001). There was a significant linear trend for DFI, OSA and HDS across the seven age categories (P < 0.001). Among patients with high DFI, there was a decreasing age-dependent trend in the patients observed with high OSA (P < 0.001). This is a retrospective study. All males included in the study were undergoing a work-up for infertility and may not be representative of a fertile population. Additional patient demographics and clinical data were not available. DNA and/or oxidative damage in sperm may be just as important to understand as the chromosomal aberrations that are carried in the oocyte. Further studies are needed to evaluate the effect of advancing paternal age on the male genome and, ultimately, on the health of the offspring. No funding was obtained for this study. V.D. is an employee of Reprosource/Quest Diagnostics. D.S. reports he was a Scientific Advisor to Cooper Surgical. N/A.

ЯКІСТЬ СПЕРМОПРОДУКЦІЇ У КНУРІВ-ПЛІДНИКІВ ЗАЛЕЖНО ВІД ТИПІВ ВИЩОЇ НЕРВОВОЇ ДІЯЛЬНІСТІ

Досліджено особливості впливу типів вищої нервової діяльності на якість спермопродукції у кнурів-плідників. Використовуючи рухово-харчову методику, було протиповано дорослих кнурів-плідників великої білої породи віком 24-36 місяців за основними типами вищої нервової діяльності. Було сформовано чотири групи по 3 голови в кожній. До I-ї групи було віднесено тварин сильного врівноваженого жвавого (рухливого); II групи – сильного врівноваженого спокійного (інертного); III групи – сильного неврівноваженого (нестримного); IV групи – слабкого типів вищої нервової діяльності. Сперму отримували від кнурів-плідників мануально із врахуванням загального часу еякуляції, розділяючи еякулят на 4 фракції – F1 - перша, F2 – друга, F3 – третя, F4 – четверта. Виявлено, що швидкість статевих рефлексів у кнурів-плідників залежить від типу вищої нервової діяльності. Так, час еякуляції у особин сильного нестримного, слабкого та сильного інертного типів є вірогідно меншим (р&lt;0,001), порівняно із тваринами сильного врівноваженого жвавого типів вищої нервової діяльності. Встановлено, що максимальною масою еякулятів характеризуються кнури-плідники сильного врівноваженого жвавого типу, а мінімальною – слабкого типу (р&lt;0,001). Найбільш насиченими сперміями були еякуляти у тварин сильного врівноваженого живого та сильного неврівноваженого нестримного, найменш – сильного врівноваженого спокійного і слабкого типів вищої нервової діяльності. У другій фракції сперми кнурів-плідників рухливість сперміїв є найбільшою. Максимальною функціональною активністю цих гамет характеризувались тварини сильного врівноваженого живого типу, а мінімальною – слабкого (р&lt;0,001). Спермії четвертої фракції мали найнижчу рухливість, особливо у тварин сильного врівноваженого жвавого типу. Спермії другої фракції сперми характеризуються найвищою життєздатністю, а найменшою - четвертої. У третій фракції сперми спермії більш швидко втрачають функціональну активність, особливо у тварин сильного врівноваженого жвавого і сильного врівноваженого спокійного типів вищої нервової діяльності, відповідно на 22,8% та 17%.

The Impact of Obesity on Various Semen Parameters and Sex Hormones in Iranian Men with Infertility: A Cross-Sectional Study.

ObjectiveThe aim of this study was to examine the correlations between body mass index (BMI) and waist circumference (WC) and semen parameters (semen volume, sperm count, motility, and morphology) and sex hormones in Iranian men with infertility.Materials and MethodsIn this cross-sectional study, a total of 119 male patients who had lived as a partner in an infertile couple for at least 1 year, after regular unprotected sexual intercourse in their married life were investigated. BMI and WC were assessed, and a morning blood sample was taken assessing serum levels of testosterone (T), sex hormone-binding globulin (SHBG), prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2) and leptin. Semen-analysis parameters were also measured.ResultsBased on BMI and WC, the sperm count, total motility and progressive sperm were significantly lower in overweight and obese infertile males compared to that in normal weight infertile males and those with WC<102 cm. In addition, the fraction of sperm with abnormal morphology was significantly higher in infertile men with WC>102 cm compared to that in those with WC<102 cm. Moreover serum levels of LH, FSH, and leptin were significantly higher in overweight and obese infertile males compared to that in normal weight infertile males and those with WC<102 cm. Moreover serum level of E2 was significantly higher in obese infertile males compared to the normal weight infertile males and in those with WC>102 cm compared to the WC<102 cm. Furthermore, serum level of T was significantly lower in obese infertile males compared to the overweight infertile males and in those with WC>102 cm compared to the WC<102 cm. The mean of T/E2 ratio also was significantly lower in obese infertile males vs overweight and normal weight infertile males and in those with WC>102 cm compared to the WC<102 cm.ConclusionWe concluded that overweight and obesity in infertile men compared to those with normal weight may worsen the infertility situation.

Sperm antioxidant system in ocellate river stingray Potamotrygon motoro at transition from seminal vesicle to cloaca.

The importance of reactive oxygen species and the antioxidant system in sperm biology has been recognized for different bony fishes but nothing is known in this regard for chondrichthyans. For the first time for cartilaginous fishes, the enzymatic antioxidant system was shown herein to be present in both fractions of sperm (spermatozoa and seminal fluid) collected from two different places (seminal vesicle and cloaca). In internally fertilizing freshwater ocellate river stingray, Potamotrygon motoro, the activity of superoxide dismutase and glutathione peroxidase was not changed upon sperm transition from the seminal vesicle to the cloaca. The activity of catalase was significantly increased for both sperm fractions at transition from the seminal vesicle to the cloaca (1.6 times for spermatozoa and 1.9 times for seminal fluid). The role of the sperm antioxidant system for different aspects of internal fertilization is discussed. The presented results are the initiatory step in uncovering the biochemical events of internal reproduction in Chondrichthyes.

Analysis of meiotic segregation by triple-color fish on both total and motile sperm fractions in a t(1p;18) river buffalo bull.

Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among them, translocations present a specific meiotic segregation pattern able to give a higher percentage of unbalanced gametes that can induce fertility problems. In this study, the meiotic segregation patterns of 1p, 1q and 18 Bubalus bubalis chromosomes were analyzed in both total sperm fraction and motile sperm fraction of a t(1p;18) carrier and a control bulls by triple-color FISH analysis with a pool of specific BAC probes. The frequencies of each total sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 39%, 20%, 1% and 38%, respectively. On the other hand, the frequencies of each motile sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 93%, 5%, 0% and 2%, respectively. The frequencies of normal sperms in the carrier were 27% and 69% in total sperm fraction and motile sperm fraction, respectively. The frequencies detected in motile sperm fraction were also validated by comparison with bull’s progeny. To our knowledge, this is the first report on the meiotic segregation patterns in motile sperm fractions of B. bubalis bull carrying a chromosomal translocation. These data suggest that translocation has a very limited effect on aneuploidy in the gametes, and therefore, on the reproductive abilities of the bull.

Insemination with Man’s Sperm: Results of Fertility Recovery in Married Couples with Male Infertility

The paper analized the results of reproductive function restoration in 140 married couples with male infertility, which passed the program of insemination with man’s sperm in 2013–2015 at the Institute of Reproductive Medicine (Kyiv). It was found that the number of pregnancies and childbirths after 3 attemps (420 cycles) with an interval of 3–5 months was reached in 17,0±1,8% and 15,0±1,8% cases, respectively. The presence of a linear relationship between the fact of fertility recovery and factors such as the woman’s age, duration of non-set of natural pregnancy, and the response of the ovulatory function to stimulation was confirmed. The influence of the spermogram indicators on the result is objectified: the type of pathospermia, the concentration of sperm, which is inversely dependent on the presence of predictors and determinants that burden the general condition of the man, as well as the percentage of actively moving sperm fractions.

Changes in the Cellular Distribution of Tyrosine Phosphorylation and Its Relationship with the Acrosomal Exocytosis and Plasma Membrane Integrity during In Vitro Capacitation of Frozen/Thawed Bull Spermatozoa.

During sperm capacitation, intracellular signaling leads to protein tyrosine phosphorylation (PTP) of multiple cellular structures. However, the connection of this molecular signaling to the physiology of capacitated spermatozoa is not completely understood. This is the case of the short lifespan of capacitated spermatozoa and their increased susceptibility to initiate acrosomal exocytosis (AE) during incubation. Herein, by employing frozen/thawed bull spermatozoa, we aimed to study the relationship between PTP with AE and with plasma membrane integrity (PMI) at the cellular level. For this, we employed double staining following immunofluorescence for PTP combined with fluorescence probes for the acrosome (PNA-FITC) and PMI (LIVE/DEAD Fixable Dead Cell Stain Kit). Our results revealed that the presence of PTP at sperm head was less abundant in the sperm fraction that triggered the AE after 3 h of incubation under capacitating conditions, or by its induction with calcium ionophore, compared to the unreacted fraction. Furthermore, PTP at the equatorial region of the head (PTP-EQ) was enriched in the fraction showing damaged membrane while induction of AE with calcium ionophore did not alter the PMI and its relation to PTP-EQ. These results suggest that spontaneous AE and induced AE trigger similar cellular events regarding PTP and the spermatozoa showing PTP-EQ are more prone to suffer plasma membrane damage.

MP44-14 SORTING OF SPERM WITH CHROMASELECT DOES NOT SKEW THE RATIO OF X TO Y CHROMOSOME BEARING SPERM

You have accessJournal of UrologyInfertility: Therapy I (MP44)1 Apr 2020MP44-14 SORTING OF SPERM WITH CHROMASELECT DOES NOT SKEW THE RATIO OF X TO Y CHROMOSOME BEARING SPERM Darius Paduch*, Anna Mielnik, and Russ Hayden Darius Paduch*Darius Paduch* More articles by this author , Anna MielnikAnna Mielnik More articles by this author , and Russ HaydenRuss Hayden More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000899.014AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: ChromaSelect is a highly sensitive and specific method of identification and isolation of intact and live human sperm from ejaculate, epididymal or testis specimens with a low concentration (up to 100 sperm /ml). As Y ch is much smaller than X, it is theoretically possible that Y or X bearing sperm will show different fluorescence intensity and thus could result in a skewed selection of one of the sexes in sorted sperm.The aim of our study was to evaluate the ratio of Chr X and Y bearing sperm in intact, live sperm and dead/apoptotic sperm fraction after ChromaSelect identification and sorting. METHODS: Ejaculated human sperm were resuspended to a concentration of 10x106/ml in MHM-C medium, stained using ChromaSelect protocol, and then sorted on SONY-FX500 fluorescent activated cell sorting (FACS). Three groups of samples were prepared: CONTROL – unsorted but stained sperm, LIVE- after sorting intact sperm, and DEAD: after sorting apoptotic and dead sperm based on specific gates. Sperm from each group were placed on microscope slides and number of sperm bearing X, Y, and Chr 8 was counted in at least 500 sperm from each group using FISH with Vysis CEP X (DXZ1) Spectrum Green, CEP Y (DYZ3) Spectrum Orange, and Vysis CEP 8 (D8Z2) Spectrum Aqua DNA. Sperm specific decondensation protocol for FISH was developed by us. The automatic FISH protocol for peripheral WBCs was not adequate for the hybridization of DNA probes in sperm. Only sperm with one signal from Chr 8 were used in the analysis. Four channels epifluorescent microscope was used to acquire images that were then processed using the Bioimaging station. Z-stage deconvolution was used to assure a single signal from each probe was counted. DAPI was used for counterstaining. Experiments were performed in triplicates. JIM 14 was used for statistical analysis. RESULTS: The rate of aneuploidy in any of the specimens was less than 0.5% and was negligible. Single signal for Chr 8 and either X or Y was observed in each counted sperm. The mean ratio of X:Y sperm for CONTROL was 0.99 (49.7% of X positive sperm), for LIVE was 0.99 (49.72 % of sperm had Chr X) and for DEAD was 1.03 (50.68% sperm had Chr X). The total number of sperm counted was 829 in CONTROL, 720 in LIVE, and 916 in DEAD groups. There was no statistically significant difference in the number of X or Y chromosomes bearing sperm between any of the groups. (Chi-square) CONCLUSIONS: Sorting of human sperm using the ChromaSelect method does not skew the ratio of X or Y carrying sperm hence it will not result in any change in the sex ratio in children born using sorted sperm. Source of Funding: NIH grant 1R03HD094046 – 01 to DAP, Irena and Howard Laks and Robert Dow Foundation © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e661-e661 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Darius Paduch* More articles by this author Anna Mielnik More articles by this author Russ Hayden More articles by this author Expand All Advertisement PDF downloadLoading ...

Human papillomavirus in spermatozoa is efficiently removed by washing: a suitable approach for assisted reproduction.

Is it possible, by sperm-washing spermatozoa from clinically HPV-positive men, to obtain spermatozoa free of human papillomavirus (HPV) to be employed in assisted reproduction? This was an observational study performed on HPV-positive men. Freshly ejaculated semen was collected and readily processed by gradient separation followed by swim-up from the washed pellet. The resulting fractions were seminal plasma, cell pellet, round cells, non-motile spermatozoa and motile spermatozoa. All fractions were then tested for the presence of HPV DNA. Of the 15 clinically HPV-positive subjects, 67% were positive in at least one of the seminal fractions. If any postivity was detected, the plasma was always HPV positive. No consistent pattern was observed throughout different samples in the cell pellet, round cell and non-motile spermatozoa fractions. However, after the sperm-wash procedure, the fraction of motile spermatozoa was never found to be HPV-positive. The sperm-washing technique, which was previously successfully used to remove human immunodeficiency virus, can efficiently remove HPV from spermatozoa. However, the present study was conducted on a small population so a larger follow-up study is recommended. HPV screening should be performed in sperm samples and, upon HPV positivity, sperm-washing should be considered before assisted reproduction techniques are used.

Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa.

Defective mammalian spermatozoa are marked on their surface by proteolytic chaperone ubiquitin. To identify potential ubiquitinated substrates in the defective spermatozoa, we resolved bull sperm protein extracts on a two-dimensional gel and isolated a 64-65-kDa spot (p64) corresponding to one of the major ubiquitin-immunoreactive bands observed in the one-dimensional Western blots. Immune serum raised against this protein recognized a prominent, possibly glycosylated band/spot in the range of 55-68kDa, consistent with the original spot used for immunization. Internal sequences obtained by Edman degradation of this spot matched the sequence of arylsulfatase A (ARSA), the sperm acrosomal enzyme thought to be important for fertility. By immunofluorescence, a prominent signal was detected on the acrosomal surface (boar and bull) and on the sperm tail principal piece (bull). A second immune serum raised against a synthetic peptide corresponding to an immunogenic internal sequence (GTGKSPRRTL) of the porcine ARSA also labeled sperm acrosome and principal piece. Both sera showed diminished immunoreactivity in the defective bull spermatozoa co-labeled with ananti-ubiquitin antibody. Western blotting and image-based flow cytometry (IBFC) confirmed a reduced ARSA immunoreactivity in the immotile sperm fraction rich in ubiquitinated spermatozoa. Larger than expected ARSA-immunoreactive bands were found in sperm protein extracts immunoprecipitated with anti-ubiquitin antibodies and affinity purified with matrix-bound, recombinant ubiquitin-binding UBA domain. These bands did not show the typical pattern of ARSA glycosylation but overlapped with bands preferentially binding the Lens culinaris agglutinin (LCA) lectin. By both epifluorescence microscopy and IBFC, the LCA binding was increased in the ubiquitinated spermatozoa with diminished ARSA immunoreactivity. ARSA was also found in the epididymal fluid suggesting that in addition to intrinsic ARSA expression in the testis, epididymal spermatozoa take up ARSA on their surface during the epididymal passage. We conclude that sperm surface ARSA is one of the ubiquitinated sperm surface glycoproteins in defective bull spermatozoa. Defective sperm surface thus differs from normal sperm surface by increased ubiquitination, reduced ARSA binding, and altered glycosylation.

Leptin and reproductive dysfunction in obese men.

Infertility is somewhat more prevalent in men who are obese. They are also reported to have low sperm concentration, higher fraction of spermatozoa that look morphologically abnormal, higher DNA fragmentation index and evidence of oxidative stress. The precise cause for this remains uncertain. Leptin levels in serum and percentage body fat correlate positively, and obese men therefore usually have elevated serum leptin levels. Although leptin is important for normal reproductive function, but when present in excess, leptin could seriously affect reproductive function in men. Reports on the findings of sperm parameters in obese men, particularly those who are subfertile or infertile, seem to be similar to those reported from studies on normal-weight rats treated with leptin. Collectively, the observations reported in human and experimental animal studies point to leptin as a possible link between infertility and obesity. Herein, we review some findings on sperm function in obese subfertile or infertile men and those from animal studies following leptin treatment, and discuss the possible link between leptin and reproductive dysfunction in obese men. The large amounts of leptin secreted by the adipose tissue and its higher circulating levels could indeed be responsible for the higher prevalence of infertility in obese men.