Critical evaluation of two models of flow cytometers for the assessment of sperm DNA fragmentation: an appeal for performance verification / 亚洲男科学杂志(英文版)

Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P < 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of >99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 ± 0.062 and 0.746 ± 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)

The effects of curcumin on sperm parameters and nitric oxide production in varicocelized rats / Efectos de la cúrcuma sobre los parámetros espermáticos y producción de óxido nítrico en ratas varicocelizadas

One of the proposed mechanism by which varicocele induces its damage is excessive release of nitric oxide (NO). Several studies have shown the role of NO in poor sperm quality in infertile patients with varicocele. Scientific studies have demonstrated the beneficial effects of curcumin on the sperm parameters. Curcumin as an atoxic antioxidant can reduce production of NO. The aim of this study was to determine the effect of curcumin on NO levels and investigate if curcumin can improve sperm parameters in varicocelized male rats. Thirty male Wistar rats were randomly divided into 5 groups (V1 and V2 (varicocele), T (treatment), Sh (sham) and C was control). In groups V1, V2, T and Sh, the left renal vein was partially ligated to induce varicocele. In groups V1 and V2, sperm parameters and NO level were evaluated 8 and 16 weeks respectively after varicocele induction. Groups T and Sh received 100 mg/kg curcumin and placebo respectively, daily for 8 weeks after 2 months of induced varicocele. Sperm parameters (count, motility, viability and morphology), epididymis and testis weight and also NO concentration were measured. Sperm parameters (count, motility and viability) in groups V1, V2 and Sh were significantly low in comparison with control and treatment groups. The level of NO was significantly increased in serum of rats in groups V1 and V2, whereas group T rat serum in which curcumin was administered, showed decreased NO levels. The values of the epididymis and testis weight had no significant changes (P 0.05) in all groups. Administration of curcumin as a free radical scavenger, can decrease NO level and improve sperm parameters in varicocelized male rats.

Uno de los mecanismos propuestos por el cual los varicoceles inducen daño es la excesiva liberación de óxido nítrico (ON). Varios estudios han demostrado el efecto del ON en la mala calidad del semen en pacientes infértiles con varicocele. Investigaciones han demostrado los efectos beneficiosos de la cúrcuma sobre los parámetros de esperma. La cúrcuma como un antioxidanteatóxico puede reducir la producción de ON. El objetivo de este estudio fue determinar el efecto de la cúrcuma en el nivel de ON e investigar si la cúrcuma puede mejorar los parámetros del semen en ratas macho. Treinta ratas macho Wistar fueron divididas aleatoriamente en 5 grupos (V1y V2 (varicocele), T (tratamiento), Sh (simulado) y C (control)). En los grupos V1, V2, T y Sh, la vena renal izquierda fue parcialmente ligada para inducir varicocele. En los grupos de V1 y V2, los parámetros de semen y nivel de ON se evaluaron a las 8 y 16 semanas respectivamente, después de la inducción de varicocele. Los grupos T y Sh recibieron diariamente 100 mg/kg de cúrcuma y placebo durante 8 semanas, después de 2 meses de inducir el varicocele. Fueron medidos los parámetros del semen (recuento, motilidad, viabilidad y morfología espemática), peso del epidídimo y testículos, junto con la concentración del ON. El recuento, motilidad y viabilidad de los espermatozoides en los grupos V1, V2 y Sh fueron significativamente más bajos en comparación con los grupos C y T. El nivel de ON se incrementó significativamente en el suero de las ratas de los grupos V1 y V2, mientras que el suero del grupo T, en el que se administró cúrcuma, hubo una disminución de los niveles de ON. El peso del epidídimo y testículos tuvieron cambios significativos (P 0,05) en todos los grupos. La administración de cúrcuma como un eliminador de radicales libres, puede disminuir el nivel de ON y mejorar los parámetros espermáticos en ratas macho varicocelizadas.

Fenitrothion induced oxidative stress and morphological alterations of sperm and testes in male sprague-dawley rats

OBJECTIVE: Fenitrothion residue is found primarily in soil, water and food products and can lead to a variety of toxic effects on the immune, hepatobiliary and hematological systems. However, the effects of fenitrothion on the male reproductive system remain unclear. This study aimed to evaluate the effects of fenitrothion on the sperm and testes of male Sprague-Dawley rats. METHODS: A 20 mg/kg dose of fenitrothion was administered orally by gavages for 28 consecutive days. Blood sample was obtained by cardiac puncture and dissection of the testes and cauda epididymis was performed to obtain sperm. The effects of fenitrothion on the body and organ weight, biochemical and oxidative stress, sperm characteristics, histology and ultrastructural changes in the testes were evaluated. RESULTS: Fenitrothion significantly decreased the body weight gain and weight of the epididymis compared with the control group. Fenitrothion also decreased plasma cholinesterase activity compared with the control group. Fenitrothion altered the sperm characteristics, such as sperm concentration, sperm viability and normal sperm morphology, compared with the control group. Oxidative stress markers, such as malondialdehyde, protein carbonyl, total glutathione and glutathione S-transferase, were significantly increased and superoxide dismutase activity was significantly decreased in the fenitrothion-treated group compared with the control group. The histopathological and ultrastructural examination of the testes of the fenitrothion-treated group revealed alterations corresponding with the biochemical changes compared with the control group. CONCLUSION: A 20 mg/kg dose of fenitrothion caused deleterious effects on the sperm and testes of Sprague-Dawley rats.

Sperm morphology and natural biomolecules from marine snail Telescopium telescopium: a phylogenetic perspective / Morfología de los espematozoides y biomoléculas naturales del caracol marino Telescopium telescopium: una perspectiva filogenética

Biochemical analysis of the cytosol fraction isolated from the ovotestis/spermatheca glands of marine mollusc Telescopium telescopium and it's sperm microtubular structure revealed that relatively similar biomolecules like different enzymes, hormones, minerals and structures of the sperm are also exist in humans. Moreover, antiserum of the cytosol fraction was found to cross-react with the human sperm antigen indicated presence of a common sperm surface antigenicity between these two diversified species. These findings might support and / or hypothesize about the origin and diversification of the vertebrate molecules from its ancestral form (s) from the invertebrates, and basic physiological functions of these ancestral biomolecules including some of the cellular structures plausibly remain the same regardless their structural changes even after evolution.

El análisis bioquímico de la fracción aislada del citosol desde las glándulas ovotestes/espermateca del molusco marino Telescopium telescopium y su estructura tubular espermática revelaron biomoléculas relativamente similares como tales como diferentes enzimas, hormonas, minerales y estructuras de los espermatozoides que también existen en los seres humanos. Por otra parte, en el antisuero de la fracción citosólica se encontró una reacción cruzada con los antígenos del esperma humano indicando la presencia de una superficie espermática de antigenicidad común entre estas dos diversificadas especies. Estos hallazgos pueden apoyar y/o hipotetizar sobre el origen y la diversificación de las moléculas de los vertebrados desde su forma (s) ancestral desde los invertebrados, y funciones básicas fisiológicas de estas biomoléculas ancestrales incluyendo algunas de las estructuras celulares siendo plausiblemente las mismas, independientemente de sus cambios estructurales incluso después de la evolución.

Characterization of arylsulphatase A in a 70 kDa protein isolated from goat spermatozoa having Na+, K+-ATPase inhibitory activity.

A protein having inhibitory effect on Na+, K+-ATPase as well as showing arylsulphatase A activity (ASA) was isolated from the cytosolic fraction of goat spermatozoa and characterized biochemically. The molecular mass of the protein was found to be 70 kDa (P70) on 10% SDS-PAGE after 35% ammonium sulphate precipitation, followed by hydroxyapatite column chromatographic separation. The isoelectric point (pI) of the protein was found to be 4.9. The sequencing results of first ten N-terminal amino acid residues of protein showed 100%, 90%, and 80% homology with N-terminal 18-27 amino acid residues of mice, pig and human testicular ASA, respectively. The optimum pH, temperature and incubation time for maximum ASA activity of the protein was 5.5, 37°C and 30 min respectively. The ASA activity of protein and AS from a commercial source was studied with respect to the sensitivity to different metal ions, vanadate, carbonyl compounds and ascorbate. Inhibition of AS activity of P70 by silver nitrate suggested that it was related to ASA. Comparable effects of different polyunsaturated fatty acids (eicosapentaenoic and docosahexaenoic acids) and purified anti P70-antibody on P70 and AS from commercial source were observed. The findings suggested that protein was novel in nature, having both regulatory and catalytic functions and showed similarities with the ASA reported from different sources.

Human sperm centrin levels & outcome of intracytoplasmic sperm injection (ICSI)--a pilot study.

BACKGROUND & OBJECTIVE: The objective of the present study was to compare the levels of sperm centrin a centrosomal protein that influences cell migration, in normal fertile donors and in oligoasthenozoospermic males (count 5 million/ml and motility <40%, grade c+d) undergoing intracytoplasmic sperm injection (ICSI) and to correlate with the outcome of ICSI. METHODS: The prospective study carried out at Inkus IVF Centre, Mumbai, India, during (January-December 2003). It included 20 normal fertile donor males (group I) and 20 oligoasthnozoospermic (OA) males (group II). Group II was further divided in II a and II b according to the centrin levels. Centrin levels were measured by using enzyme linked immunosorbent assay (ELISA) in both groups. All participants underwent an ICSI procedure and the levels of centrin and outcome of ICSI were correlated. RESULTS: Centrin levels were significantly lower (P<0.001) in group II (0.39) as compared with group I (1.34). With centrin levels <0.45 optical density (OD) (group II a) the pregnancy rate was further reduced, with only 2 pregnancies (out of 14) both of which, ended in abortion. Cases in group II showed levels of centrin much lower than in the fertile group. Further lowered centrin levels were associated with lowered pregnancy rates in OA males, but statistically was not significant. INTERPRETATION & CONCLUSION: The study revealed that lower centrin levels in OA males resulted in lower pregnancy percentage in this group after ICSI. Disturbances in centrosomal protein could be one of the possible causes of ICSI failure.

Effect of some ions on sperm activation in Brycon henni(Eigenmann 1913)

Spermatozoa in Characid fish remain immobile in seminal plasma and are activated when freed into water where the ionic balance seems to be the main factor starting the activation process. This process was the target of the present study with emphasis on the activation of motility and on motility maintenance over time. The effect of isosmotic solutions was analyzed taking into account the possible combinations of the following ions, Ca2+, K+, Mg2+ and Na+ as well as the effect of channel blocking agents. The parameters measured were cells with motility (per cent), duration of motility (s), plasma membrane potential, and the effect of channel blockers on activation time and on motility. There was an increased motility when the semen was incubated in solutions containing K+ (p<0.05) compared with the control (CaNaMgK solution); the longest duration of motility was attained when the incubation was performed in solutions containing Na+ and Mg2+ (p<0.05). All solutions induced a change in membrane potential detected after 15 s of activation. Blocking K+, Ca2+ and Na+ channels did not alter motility but decreased the activation time (p<0.05). Potassium induced activation at all concentrations up to 105 mM, but motility was drastically decreased at concentrations higher than 140 mM (p<0.05). The conclusion is that interaction of the ionic environment with the cell membrane leads to changes in membrane potential and intracellular signalling that trigger sperm motility in Brycon henni.

Estrogens and male reproduction: a new concept

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them estrogens are the end products obtained from the irreversible transformation of androgens by a microsomal enzymatic complex named aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rats, the aromatase activity is mainly located in Sertoli cells of immature rats and then in Leydig cells of adult rats. We have demonstrated that germ cells represent an important source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes compared to gonocytes or round spermatids; conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank voles, bears, and monkeys express aromatase. In humans, we have shown the presence of a biologically active aromatase and of estrogen receptors (alpha and ß) in ejaculated spermatozoa and in immature germ cells in addition to Leydig cells. Moreover, we have demonstrated that the amount of P450arom transcripts is 30 percent lower in immotile than in motile spermatozoa. Alterations of spermatogenesis in terms of number and motility of spermatozoa have been described in men genetically deficient in aromatase. These last observations, together with our data showing a significant decrease of aromatase in immotile spermatozoa, suggest that aromatase could be involved in the acquisition of sperm motility. Thus, taking into account the widespread localization of aromatase and estrogen receptors in testicular cells, it is obvious that, besides gonadotrophins and androgens, estrogens produced locally should be considered to be physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis.

Purification of protein from a crude mixture through SDS-PAGE transfer method.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer method was used for purification and enrichment of the protein from crude sample. Coomassie bluc/ZnSO4 stained protein band(s) containing intact polyacrylamide gel were loaded on to another polyacrylamide gel either alone or as pooled gel bands. Two/three bands were combined together and arranged tightly over one another, sealed with stacking gel and ran in another gel, which was quite useful for enrichment and purification of a particular protein from a complex mixture. Recovery of protein by gel transfer method was found to be 70% in case of ZnSO4 staining, whereas around 30% recovery was possible, following Coomassie blue staining. The method described here for purification of protein(s) from a complex mixture, following gel transfer procedure could be useful for further characterization of the desired protein.

Paternity after vasectomy with two previous semen analyses without spermatozoa

CONTEXT: The risk of paternity after vasectomy is rare but still exists. Overall failure to achieve sterility after vasectomy occurs in 0.2 to 5.3 percent of patients due to technical failure or recanalization. The objective of this report was to describe a rare but notable case of proven paternity in which the semen analyses had not given evidence of spermatozoa. CASE REPORT: A 44-year-old vasectomized man whose semen analyses had shown azoospermia became a father four years after sterilization. Blood sample DNA analysis on the child and husband proved biological paternity. Vasectomy may fail in the long run even without spermatozoa in semen analysis. The patient must be aware of this possibility.

CONTEXTO: O risco de paternidade após vasectomia é raro, mas existente. Falha em atingir esterilidade após vasectomia ocorre em 0.2 por cento a 5.3 por cento dos pacientes devido à falha técnica ou recanalização. O objetivo é descrever um caso raro, mas importante, de paternidade comprovada cujo espermograma mostrava ausência de espermatozóides. RELATO DE CASO: Um homem de 44 anos vasectomizado, cujo espermograma evidenciou azoospermia, tornou-se pai após quatro anos da esterilização. Análise sanguínea por DNA da criança e do marido provou paternidade biológica. Vasectomia pode falhar a longo prazo até com azoospermia no espermograma. O paciente deve estar informado dessa possibilidade.

Evaluación del sistema antioxidante en el semen normal / Evaluation of antioxidant system in normal semen

Background. Reactive oxygen species (ROS) formation have the ability to alter reversibly or irreversibly the cellular function in humans. It has been proposed that the ROS alters the biochemistry and the physiology of the sperm. On the other hand, the antioxidative mechanisms could protect the sperms from the damage produced by free radicals. Aim. To determine the normal values for superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) and nitric oxide (NOx) in the seminal liquid of healthy humans. Procedures. Semen samples from 45 healthy men (22 to 47 years of age) were studied. The samples were obtained by masturbation and were collected in conical sterile tubes. Once centrifuged at 4 °C they were divided in aliquots to measure the concentration of SOD, GPx, MDA, and NOx. The analysis of the samples was realized in conformity with biochemical widely accepted methods. Results. The concentrations of SOD and MDA both in the seminal liquid and in the spermatozoids were similar, SOD 0.43 ± 0.09 U/mg prot. in the seminal liquid and 0.45 ± 0.07 U/ mg prot. in spermatozoids, and MDA 0.33 ± 0.07 nmoles/mg prot. and 0.37 ± 0.10 nmoles/mg prot. in the seminal liquid and spermatozoids respectively. With regard to GPx it increased almost 13 times more in the spermatozoids (2547.77 ± 48.59 U/mg prot.) than in the seminal liquid (197.54 ± 25.21 U/mg prot.). The NOx also increased lightly in the spermatozoids (4.45 ± 0.43 /imol) when compared with the seminal liquid (3.91 ± 0.16 /imol). Conclusions. The measurement of the antioxidative and oxidative agents could serve to evaluate human infertility in those cases where the result of the spematobioscopy appears normal.

Antecedentes. Las especies reactivas del oxígeno (ERO), tienen la capacidad de alterar reversible o irreversiblemente la función celular. Se ha propuesto que las ERO modifican la bioquímica y la fisiología del espermatozoide. Por otro lado, los mecanismos antioxidativos pudieran proteger a los espermatozoides del daño producido por las ERO. Objetivo. Determinar los valores normales para el superóxido dismutasa (SOD), glutatión peroxidasa (GPx), malondialdehído (MDA) y óxido nítrico (NOx) en el líquido seminal y espermatozoides de humanos sanos. Procedimientos. Se estudiaron 45 muestras de semen de sujetos aparentemente sanos. Las muestras se obtuvieron por masturbación y se colectaron en tubos estériles. Una vez centrifugadas, se fraccionaron en alícuotas para medir la concentración de SOD, GPx, MDA y NOx. El análisis de las muestras se realizó conforme a métodos bioquímicos ampliamente aceptados. Resultados. Las concentraciones de SOD y MDA en el líquido seminal como en los espermatozoides fueron similares (SOD 0.43 ± 0.09 en semen y 0.45 ± .07 U/mg prot. en espermatozoides, y MDA 0.33 ± .07 y 0.37 ± 0.10 nmoles/mg prot. en líquido seminal y espermatozoides, respectivamente. Con respecto a la GPx, está aumentada casi 13 veces más en los espermatozoides (2547.77 ± 48.59 U/mg prot.) que en el líquido seminal (197.54 ± 25.21 U/mg prot.), el NOx también se incrementa ligeramente en los espermatozoides (4.45 ± 0.43 µmol) cuando se compara con el líquido seminal (3.91 ± 0.16 µmol). Conclusiones. La medición de los antioxidantes y oxidantes pudieran servir para evaluar la infertilidad humana en aquellos casos donde los resultados de la espermatobioscopia aparezcan como normales.

Isolating sperm cells and preparing DNA from mixture sample / 法医学杂志

OBJECTIVE@#To establish a method of preparing and analyzing DNA of sperm cells from sample mixtures.@*METHODS@#Sperm cells were captured by micromanipulation method, DNA template was prepared from these sperm cells, and then amplified by multiple displacement amplification (MDA).@*RESULTS@#High-yield of highly integrated DNA was produced by MDA from very small amount of DNA template of 20 sperm cells. The DNA could be amplified at least 30,000-fold, and the length of most the DNA segments were greater than 15 kb, providing sufficient amount of short tandem repeat (STR) products for DNA genotyping.@*CONCLUSION@#The micromanipulation method is efficient to isolate sperm cells from sample mixtures and multiple displacement amplification can provide sufficient amount of DNA products for STR genotyping. The method seems to be feasible in detecting DNA from rape cases.

Efecto de la pluviosidad y el brillo solar sobre la producción y características del semen en el pez Brycon henni (Pisces: Characidae) / Effect of pluviosity and sun shine on sperm production and seminal characteristics of the fish Brycon henni (Pisces: Characidae)

In Colombia the fish Brycon henni is a protected endemic species. It inhabits water bodies in coffee producing areas (700-1900 ma.s.l.; 4º35’56’’ N -74º04’51’’ W; 18-28°C). Insufficient knowledge of its basic biology and behavior prevent the commercial culture of this promising fish. We studied the production and sperm physiology of captive males. Along a year 20 samples were taken from each of 10 males. The sample was obtained by abdominal cefalo-caudal massage and transported to the laboratory at 4°C. Except for September and October (maximum rainfall),sperm was always obtained in at least 50% of the males. Color, osmolality and pH were similar in all the samples. Volume, concentration, viability, motility and activation time were variable: sunshine had a positive effect on volume (Spearman p<0.05) and on sperm concentration (Spearman p<0.05) while pluviosity had a negative effect on volume and viability. The proportion of ions (Na,K,Mg,Ca)was constant along the year; Na being 10-fold higher than K and 100-fold higher than Mg and Ca; however the absolute concentration of all ions was slightly higher in April and in July (with no apparent relation with the other variables analyzed).

El pez Brycon henni es una especie endémica protegida por la legislación colombiana, que habita cuerpos de agua de zonas cafeteras (700-1900 m. s. n. m),comprendidas entre los 4º35’56’’ N y 74º04’51’’ W, con temperaturas que oscilan entre los 18 y los 28ºC. A pesar de las características promisorias de esta especie, su reproducción en cautiverio a nivel comercial no ha sido posible por falta de conocimientos básicos de su biología y comportamiento. El objetivo del presente trabajo fue caracterizar la producción y la fisiología espermática de machos en cautiverio. A lo largo de un año se tomaron 20 muestras de cada uno de 10 ejemplares. El semen se obtuvo mediante masaje abdominal cráneo-caudal y se transportó a 4ºC para su análisis en el laboratorio. Con excepción de septiembre y octubre que fueron los meses más lluviosos, siempre se obtuvo semen de al menos el 50% de los animales. El color, la osmolalidad y el pH fueron similares en todas las muestras a lo largo del año. El volumen, la concentración, la viabilidad, la movilidad y el tiempo de activación fueron variables: El efecto del brillo solar fue positivo sobre el volumen (Spearman p<0.05) y sobre la concentración espermática (Spearman p<0.05), mientras que la pluviosidad tuvo un efecto negativo sobre el volumen y la viabilidad. La proporción entre los iones (Na, K, Mg, Ca) se mantuvo constante durante todo el año: el sodio fue el mayor, seguido por el potasio, y por el magnesio y el calcio, 10 y 100 veces menos, respectivamente; sin embargo la concentración absoluta de estos iones aumentó durante los meses de abril y julio sin una relación aparente con las otras variables analizadas.

Characters that differ between diploid and haploid honey bee (Apis mellifera) drones

Diploid males have long been considered a curiosity contradictory to the haplo-diploid mode of sex determination in the Hymenoptera. In Apis mellifera, 'false' diploid male larvae are eliminated by worker cannibalism immediately after hatching. A 'cannibalism substance' produced by diploid drone larvae to induce worker-assisted suicide has been hypothesized, but it has never been detected. Diploid drones are only removed some hours after hatching. Older larvae are evidently not regarded as 'false males' and instead are regularly nursed by the brood-attending worker bees. As the pheromonal cues presumably are located on the surface of newly hatched bee larvae, we extracted the cuticular secretions and analyzed their chemical composition by gas chromatograph-mass spectrometry (GC-MS) analyses. Larvae were sexed and then reared in vitro for up to three days. The GC-MS pattern that was obtained, with alkanes as the major compounds, was compared between diploid and haploid drone larvae. We also examined some physical parameters of adult drones. There was no difference between diploid and haploid males in their weight at the day of emergence. The diploid adult drones had fewer wing hooks and smaller testes. The sperm DNA content was 0.30 and 0.15 pg per nucleus, giving an exact 2:1 ratio for the gametocytes of diploid and haploid drones, respectively. Vitellogenin was found in the hemolymph of both types of imaginal drones at 5 to 6 days, with a significantly lower titer in the diploids.

Protein detection in spermatids and spermatozoa of the butterfly Euptoieta hegesia (Lepidoptera)

This study was undertaken to detect protein components in both sperm types of the butterfly Euptoieta hegesia. These spermatozoa possess complex extracellular structures for which the composition and functional significance are still unclear. In the apyrene sperm head, the proteic cap presented an external ring and an internal dense content; basic proteins were detected only in external portions. In the tail, the paracrystalline core of mitochondrial derivatives and the axoneme are rich in proteins. The extratesticular spermatozoa are covered by a proteic coat, which presented two distinct layers. In eupyrene spermatozoa, acrosome and nucleus were negatively stained, probably because of their high compaction. In the tail, there is no paracrystalline core and the axoneme presented a very specific reaction for basic proteins. The lacinate and reticular appendages are composed of cylindrical sub-units and presented a light reaction to E-PTA and a strong reaction to tannic acid. A complex proteic coat also covers the extratesticular spermatozoa. We found similarities between both extratesticular coats, indicating a possible common origin. Both spermatozoon types are rich in proteins, especially the eupyrene appendages and the extratesticular coats. We believe that both coats are related to the sperm maturation and capacitation processes.

A sort of meliorative method for testing DNA from sperm smear / 法医学杂志

OBJECTIVE@#To establish an effective method for testing DNA from the sperm smear.@*METHODS@#We did exploratory research for ninety-one cases of the sperm smear which were accumulated by our laboratory during working hours. Chelex extraction method was used to extract DNA. The digested solution of sperm was purified and concentrated by using Silicon bead. STR loci were typed after PCR amplification by Profile Plus kit.@*RESULTS@#Although there were a few sperm cells on the slide, it is easy to obtain good DNA typing result from it because the amalgamative method we used was high effective for DNA extraction.@*CONCLUSION@#The DNA analysis of sperm smear could offer satisfactory typing results which would be useful in case.

Cigarette smoking and spermatozoa I. histochemical differences between spermatozoa of cigarette smokers and non-smokers

Thirty semen specimens were taken from non-smokers and another thirty specimens from heavy smokers. Sperms of both groups were examined histochemically for some enzymes concerned with energy production [adenosine triphosphatase, lactate dehydrogenase and succinic dehydrogenase] and penetrating power [non-specific esterase and acrosin]. The histochemical reactions for these enzymes were lower in the sperms of the smokers than those of non-smokers revealing more aspects of the detrimental effects of cigarette smoking on spermatozoa

Surface protein changes in goat spermatozoa during capacitation.

Polypeptides of goat sperm surface before and after capacitation were examined by radiolabelling and immunologically using polyclonal antisera. Radioiodination revealed five protein bands having mol wt of 14.8, 72.4, 81, 100 and 128 kDa in uncapacitated ejaculated spermatozoa and only three bands of 23.4, 27 and 72.4 KDa in capacitated spermatozoa. The protein band with mol wt 72.4 kDa was only feebly iodinated in uncapacitated sperm surface but in capacitated spermatozoa it was heavily labelled. Western blot analysis of detergent-extracted proteins using gamma-globulin fraction of antisera raised against purified goat sperm plasma membrane revealed six antigens (17.8, 29.1, 33.4, 45.6, 85.1, 123.2 kDa) in uncapacitated spermatozoa, four (26, 32.1, 40.1, 45.6 kDa) in capacitated spermatozoa and only one (45.6 kDa) in acrosome-reacted spermatozoa. High mol wt proteins were more numerous on the surface of uncapacitated spermatozoa while the capacitated spermatozoa had relatively low mol wt proteins. An apparent effect of capacitation is the metabolism and reorganisation of proteins on goat sperm surface. Polypeptides on capacitated sperm surface revealed through radiolabelling and polyclonal antisera may have a likely receptor(s) role in the recognition and binding to homologous zona pellucida during fertilization.