A novel homozygous frameshift variant in DNAH8 causes multiple morphological abnormalities of the sperm flagella in a consanguineous Pakistani family / 亚洲男科学杂志(英文版)

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

Biallelic mutations in WDR12 are associated with male infertility with tapered-head sperm / 亚洲男科学杂志(英文版)

Teratozoospermia is a rare disease associated with male infertility. Several recurrent genetic mutations have been reported to be associated with abnormal sperm morphology, but the genetic basis of tapered-head sperm is not well understood. In this study, whole-exome sequencing (WES) identified a homozygous WD repeat domain 12 (WDR12; p.Ser162Ala/c.484T>G) variant in an infertile patient with tapered-head spermatozoa from a consanguineous Chinese family. Bioinformatic analysis predicted this mutation to be a pathogenic variant. To verify the effect of this variant, we analyzed WDR12 protein expression in spermatozoa of the patient and a control individual, as well as in the 293T cell line, by Western blot analysis, and found that WDR12 expression was significantly downregulated. To understand the role of normal WDR12, we evaluated its mRNA and protein expression in mice at different ages. We observed that WDR12 expression was increased in pachytene spermatocytes, with intense staining visible in round spermatid nuclei. Based on these results, the data suggest that the rare biallelic pathogenic missense variant (p.Ser162Ala/c.484T>G) in the WDR12 gene is associated with tapered-head spermatozoa. In addition, after intracytoplasmic sperm injection (ICSI), a successful pregnancy was achieved. This finding indicates that infertility associated with this WDR12 homozygous mutation can be overcome by ICSI. The present results may provide novel insights into understanding the molecular mechanisms of male infertility.

Cisplatin-induced azoospermia and testicular damage ameliorated by adipose-derived mesenchymal stem cells

BACKGROUND: The testes are highly susceptible to the adverse effects of chemotherapy and radiation at all stages of life. Exposure to these threats mainly occurs during cancer treatment and as an occupational hazard in radiation centers. The present study investigated the regenerative ability of adipose-derived mesenchymal stem cells (ADMSCs) against the adverse effects of cisplatin on the structure and function of the testes. METHODS: New Zealand white rabbits (N = 15) were divided into three groups of five: a negative control group (no treatment), a cisplatin group (single dose of cisplatin into each testis followed three days later by a PBS injection), and a cisplatin + ADMSCs group (cisplatin injection followed three days later by an ADMSC injection). On day 45 post-treatment, serum testosterone levels were evaluated, and the testes and epididymis were collected for histology, oxidative stress examination, and epididymal sperm analysis. RESULTS: Cisplatin caused damage to the testicular tissue and decreased serum testosterone levels, epididymal sperm counts, and oxidants. An antioxidant imbalance was detected due to increasing malondialdehyde (MDA) and reduced glutathione (GSH) levels in testicular tissue. The ADMSC-treated group displayed a moderate epididymal sperm count, adequate antioxidant protection, suitable hormone levels, and enhanced testicular tissue morphology. CONCLUSIONS: ADMSCs treatment repaired damaged testicular tissue, enhanced biochemical parameters, and modified pathological changes caused by cisplatin.

Sperm-specific protein ACTL7A as a biomarker for fertilization outcomes of assisted reproductive technology / 亚洲男科学杂志(英文版)

Obtaining high-quality embryos is one of the key factors to improve the clinical pregnancy rate of assisted reproductive technologies (ART). So far, the clinical evaluation of embryo quality depends on embryo morphology. However, the clinical pregnancy rate is still low. Therefore, new indicators are needed to further improve the evaluation of embryo quality. Several studies have shown that the decrease of sperm-specific protein actin-like 7A (ACTL7A) leaded to low fertilization rate, poor embryo development, and even infertility. The aim of this study was to study whether the different expression levels of ACTL7A on sperm can be used as a biomarker for predicting embryo quality. In this study, excluding the factors of severe female infertility, a total of 281 sperm samples were collected to compare the ACTL7A expression levels of sperms with high and low effective embryo rates and analyze the correlation between protein levels and in-vitro fertilization (IVF) laboratory outcomes. Our results indicated that the ACTL7A levels were significantly reduced in sperm samples presenting poor embryo quality. Furthermore, the protein levels showed a significant correlation with fertilization outcomes of ART. ACTL7A has the potential to be a biomarker for predicting success rate of fertilization and effective embryo and the possibility of embryo arrest. In conclusion, sperm-specific protein ACTL7A has a strong correlation with IVF laboratory outcomes and plays important roles in fertilization and embryo development.

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.

Efecto del zumo de sandía (citrullus lanatus) en el estrés oxidativo en espermatozoides humanos / Effect of watermelon juice (citrullus lanatus) on oxidative stress in human sperm

INTRODUCCIÓN: Las alteraciones reproductivas de causa masculina relacionadas con el estrés oxidativo son cada día más estudiadas y dan cuenta de causas de infertilidad diagnosticada como idiopáticas. El objetivo del presente trabajo fue evaluar el efecto del zumo de sandía sobre los parámetros seminales convencionales y funcionales in vitro e in vivo. MATERIALES Y MÉTODOS: Cinco muestras de espermatozoides puros fueron incubados con peróxido de hidrógeno (H2O2, 5mM) y 0,45% de extracto de sandía, se determinó la movilidad espermática al tiempo 0, 30 y 60 minutos. En los ensayos in vivo se incluyeron 20 individuos a los cuales se les determinaron los parámetros espermáticos convencionales, funcionales y la capacidad antioxidante del plasma seminal por microscopía, citometría y espectrofotometría en los días 0, 7 y 15 después de iniciar el consumo diario de 16 onzas de zumo de sandía. RESULTADOS: El extracto de sandía protege a los espermatozoides del efecto deletéreo del H2O2 sobre la movilidad espermática. Además, el consumo regular de jugo de sandía disminuye la lipoperoxidación de la membrana espermática, la producción intracelular de especies reactivas del oxígeno, el índice de fragmentación del ADN el día 15 y la capacidad antioxidante el día 7 y 15. CONCLUSIONES: El extracto de sandía genera un efecto protector sobre los espermatozoides humanos in vitro, protegiendo su movilidad del efecto negativo del H2O2. Además, si bien el consumo regular de zumo de sandía no mejora los parámetros seminales convencionales, si mejora algunos parámetros funcionales relacionados con el estrés oxidativo.

OBJETIVE: Male reproductive alterations related to oxidative stress are increasingly studied and account for causes of infertility diagnosed as idiopathic. The aim of this work was to evaluate the effect of watermelon juice on conventional and functional seminal parameters in vitro and in vivo. MATERIALS AND METHODS: Five samples of pure sperm were incubated with hydrogen peroxide (H2O2, 5mM) and 0.45% watermelon extract, sperm motility was determined at time 0, 30 and 60 minutes. In vivo assays, 20 individuals were included. Conventional and functional sperm parameters, and antioxidant capacity of seminal plasma using microscopy, cytometry and spectrophotometry were determined on days 0, 7 and 15 after starting daily consumption of 16 ounces of watermelon juice. RESULTS: Watermelon extract protects sperm cells from the deleterious effect of H2O2 on sperm motility. In addition, regular consumption of watermelon juice decreases sperm membrane lipoperoxidation, intracellular production of reactive oxygen species, DNA fragmentation index on day 15 and antioxidant capacity on day 7 and 15. CONCLUSION: Watermelon extract generates a protective effect on human sperm in vitro, protecting sperm motility from the negative effect of H2O2. In addition, although regular consumption of watermelon juice does not improve conventional seminal parameters, it does improve some functional parameters related to oxidative stress.

Estrés oxidativo: ¿un estado celular defectuoso para la función espermática? / Oxidative stress: a defective cell state for sperm function?

En los organismos vivos, las cantidades de radicales libres y especies reactivas del oxígeno (ROS) son controladas por un complejo sistema de homeostasis, capaz de mantener niveles fisiológicos de ROS necesarios para el funcionamiento y regulación de algunas biomoléculas. Paralelamente, los organismos poseen sistemas bioquímicos de protección contra el estrés oxidativo, que consiste en el desbalance entre la producción de especies químicas altamente reactivas y las defensas antioxidantes de la célula. Dicho estrés contribuye de manera importante a la etiología tanto de la senescencia celular como de algunas enfermedades. En el contexto reproductivo, las células espermáticas pasan por una serie de cambios fisiológicos durante los procesos de maduración, capacitación y fecundados, entre los que se incluyen las modificaciones de las proteínas existentes, reguladas por señales procedentes del entorno espermático, donde las ROS modulan importantes vías bioquímicas, involucradas en procesos fundamentales de la función del espermatozoide y que se pueden alterar en estados de estrés oxidativo. El objetivo de esta revisión de literatura es describir algunos de los procesos que contribuyen al estrés oxidativo y sus implicaciones sobre la funcionalidad espermática.

Living organisms regulate the load of free radicals and reactive oxygen species (ROS) by a complex homeostatic system, capable of maintaining physiological levels of ROS, necessary for the action and regulation of some biomolecules. In parallel, organisms harbor biochemical protection systems against oxidative stress, consisting of an unbalance state between oxygen reactive chemical species and antioxidant defense production; this kind of biochemical stress has been shown to contribute to cellular senescence and the development of different diseases. In the reproductive field, the spermatic cells undergo a serial of physiological changes during the maturation, capacitation and fertilization process. Such changes include the modification of proteins regulated by signals from the sperm environment, where ROS modulate important biochemical pathways involved in fundamental processes of sperm function, and that could be altered under oxidative stress conditions. The objective of this review is to describe some of the processes that contribute to oxidative stress and its implications on sperm functionality.

Efecto de los factores solubles de Staphylococcus aureus, Staphylococcus capitis y Staphylococcus epidermidis sobre la funcionalidad espermática / Effect of soluble factors of Staphylococcus aureus, Staphylococcus capitis and Staphylococcus epidermidis on sperm functionality

ANTECEDENTES: La interacción entre los espermatozoides con algunas especies bacterianas o sus factores solubles influyen en el deterioro de la calidad seminal, alterando la función reproductiva del hombre. OBJETIVO: El objetivo de este trabajo fue determinar el efecto de los factores solubles de Staphylococcus aureus, Staphylococcus capitis y Staphylococcus epidermidis sobre la calidad seminal. MÉTODO: Los factores solubles producto del metabolismo bacteriano de las cepas de S. aureus y S. Capitis sensible a oxacilina y S. aureus y S. Epidermidis resistente a oxacilina se incubaron con las muestras de semen de 20 voluntarios y se cuantificaron los parámetros seminales convencionales y funcionales por microscopía y citometría de flujo, respectivamente. RESULTADOS: Se observó una disminución en la movilidad espermática con los factores solubles de S. aureus, esta disminución fue mayor con la cepa sensible y el efecto negativo sobre la movilidad fue inmediato. Al incubar los espermatozoides con los factores solubles de S. aureus sensible a oxacilina, se afectaron todos los parámetros funcionales excepto la integridad de la cromatina y se observó menor liberación de especies reactivas de oxígeno; con los factores solubles de la cepa de S. aureus resistente a oxacilina se observó una disminución en la lipoperoxidación de membrana y en la expresión de anexina V. CONCLUSIÓN: Este estudio da cuenta del efecto negativo de los factores solubles de la bacteria S. aureus tanto sensible como resistente a oxacilina sobre los parámetros espermáticos convencionales y funcionales, y por ende en su función reproductiva.

BACKGROUND: The interaction between sperm with some bacteria species and their soluble factors are the deterioration of semen quality by altering the reproductive function of man. AIM: The aim of this study was to determine the effect of soluble factors Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus capitis on semen quality. METHODS: The soluble factors product of bacterial metabolism of the strains of S. aureus and S. capitis methicillin sensitive and S. aureus and S. epidermidis resistant to oxacillin, were incubated with semen samples from 20 volunteers. Subsequently, conventional seminal parameters were measured and functional quantified by microscopy and flow cytometry, respectively. RESULTS: A decrease was observed in sperm motility with soluble factors of S. aureus, this decrease was higher with the sensitive strain that with oxacillin resistant strain and the negative effect on motility was immediate. By incubating the sperm with soluble factor from oxacillin-sensitive S. aureus, all functional parameters were affected except the chromatin integrity and reduced release of reactive oxygen species, mean fluorescence intensity in oxacillin resistant S. aureus strain was decrease in membrane lipid peroxidation and annexin V expression. CONCLUSIONS: This study reports the negative effect of soluble factors of bacteria either S. aureus sensitive and resistant to oxacillin, over conventional and functional sperm parameters, and therefore in their reproductive function.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Histone Acetylation Level and Histone Acetyltransferase/Deacetylase Activity in Ejaculated Sperm from Normozoospermic Men

PURPOSE: The aim of this work was to evaluate nuclear histone acetylation level and total histone acetyltransferase (HAT) and deacetylase (HDAC) activity in ejaculated sperm and their relevance to conventional sperm parameters. MATERIALS AND METHODS: Thirty-three normozoospermic men were included in this study. Semen samples were processed by swim-up and then immunostained by six acetylation antibodies (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac). Our preliminary study verified the expression of HAT/HDAC1 in mature human sperm. From vitrified-warmed sperm samples, total HAT/HDAC activity was measured by commercially available kits. Nuclear DNA integrity was also measured by TUNEL assay. RESULTS: The levels of six acetylation marks were not related with conventional sperm parameters including sperm DNA fragmentation index (DFI) as well as HAT/HDAC activity. However, sperm DFI was positively correlated with HAT activity (r=0.038 after adjustment, p<0.02). HAT activity showed a negative relationship with HDAC activity (r=-0.51, p<0.01). Strict morphology was negatively correlated with acetylation enzyme index (=HAT activity/HDAC activity) (r=-0.53, p<0.01). CONCLUSION: Our works demonstrated a significant relationship of acetylation-associated enzyme activity and strict morphology or sperm DFI.

Simultaneous deletion of floxed genes mediated by CaMKIIalpha-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goalpha

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIalpha) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIalpha-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice generated by crossing CaMKIIalpha-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIalpha-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Delta)) without inheriting the Cre transgene. The Gnao(Delta/Delta) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIalpha-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIalpha-Cre mice as breeding pairs.

Fatty acid profiles of ram's sperm after removing some fatty acid sources from the diets and persistency of fatty acids in sperm

Background: Mammalian spermatozoa are characterized by a high proportion of polyunsaturated fatty acids [PUFAs], but reliable data concerning dietary effects on fatty acid [FA] profile in ram's sperm and the persistency of FA in the ration to the FA in sperm has not been reported. Therefore, the aim of this study was to determine the stability of saturated and unsaturated FAs in ram's sperm despite removing FA sources from their diet

Materials and Methods: Nine Kalkoohi rams were used in a completely randomized design and they were assigned to 3 groups. The treatments were diet supplemented [35 g/d/ram] by C16:0 [RP-10[registered]], C18: 2 [Sunflower oil; SO] and n-3 [Fish oil; FO] with Vitamin E. Fifteen weeks after the start of the supplemented diet, rams were offered a basal diet without any supplementary FA source for 35 days when the sperm's FA ratio was determined. The data were analyzed by ANOVA [Analysis of variance] using the General Linear Model [GLM] procedure of SAS Institute

Results: Thirty five days after removing the fat supplement from the diet, major FA in sperm consisted of: C14:0, C16:0, C18:0, C18:1 cis, C18:2 cis and C22:6 n-3 docosahexaenoic acid [DHA]. The percentage of C14:0 [p=0.8] and C18:1 cis [P=0.4] were similar among all the treatments. Interestingly, 35 days after the removal of fatty acid source, the percentage of C22:6 was highest in the FO treated group

Conclusion: The different sperm FA profile among various groups suggests that dietary FA had significant direct or indirect impacts on sperm FA profile after 35 days which might lead to physical and chemical changes in sperm characteristics

Cigarette smoking impairs sperm bioenergetics

Objective: The growing consensus on the negative impact of cigarette smoking on fertility prompted us to compare the rate of sperm respiration in smokers and non-smokers. Materials and methods: Semen samples from 20 smokers and 58 non-smokers consulting at the andrology laboratory for fertility evaluation were used. Smoking was defined as consumption of at least a half a pack per day. A phosphorescence analyzer that measures O2 concentration in sperm suspensions as function of time was used to determine the rate of respiration. In a sealed vial, the rate of sperm respiration (k) was defined as -d[O2]/dt; where [O2] was obtained from the phosphorescence decay rate of a palladium phosphor. [O2] in solutions containing sperm and glucose declined linearly with time, showing the kinetics of O2 consumption was zero-order. Inhibition of O2 consumption by cyanide confirmed the oxidations that occurred in the sperm mitochondrial respiratory chain. Results: There were no differences (p > 0.28) between smokers and non-smokers for ejaculate volume, motility, concentration, normal morphology, viability and hypo-osmotic swelling test. The rate (mean ± SD, in µM O2/min/108 sperm) of sperm mitochondrial O2 consumption in the smokers was 0.96 ± 0.58 and in the non-smokers 1.39 ± 0.67 (p = 0.004). Conclusions: The rate of sperm respiration was significantly lower in smokers. This negative impact of cigarette smoking on sperm aerobic metabolism may, in part, explain the lower rate of fertility in smokers.

Análise computacional da compactação da cromatina de espermatozoides de galo / Computational analysis of chromatin condensation of rooster spermatozoa

Testaram-se variantes metodológicas utilizando azul de toluidina (AT), até se estabelecer um protocolo confiável para a avaliação computacional da compactação da cromatina em espermatozoides de galo. Para tal, foram utilizados sêmen de 10 galos com 35 semanas de idade e sêmen de 10 galos com 60 semanas de idade. O melhor método foi o de hidrólise com ácido clorídrico 1N por 10 minutos, coloração em cubeta com AT 0,025 por cento, pH 4,0, por 20 minutos, desidratação em álcool, diafanização em xilol e montagem com bálsamo do Canadá. Todas as amostras de sêmen foram submetidas a este protocolo e posteriormente avaliadas por análise de imagem computacional, em que foram feitas mensurações da área, comprimento, largura, perímetro, homogeneidade da compactação da cromatina dentro de cada cabeça e intensidade de compactação da cromatina. Os espermatozoides de galos velhos apresentaram mais alterações na cromatina que os de galos jovens. Os galos jovens apresentaram cabeça dos espermatozoides maior que os galos mais velhos. A análise computacional da compactação da cromatina mostrou-se um método menos subjetivo e mais preciso que a avaliação visual das cabeças dos espermatozoides.

The methodological variants using toluidina blue (AT) to establish a trustworthy protocol for the computational analysis of chromatin condensation of rooster spermatozoa were studied. Twenty semen samples were used: ten from 35-week-old roosters and ten from 60-week-old roosters. Different methods of denaturation and staining were tested. The best method was hydrolysis with 1N HCl for 10 minutes, staining in bucket with 0.025 percent AT, pH 4.0, for 20 minutes, dehydration in alcohol, clearing in xylol, and mounted with Canada balsam. All the semen samples were submitted to this protocol and later evaluated by computational image analysis. Area, length, width, perimeter, and chromatin compaction homogeneity of head spermatozoa were measured. The sperm of older roosters presented more chromatin changes than the ones of younger ones. The spermatozoa of younger roosters presented bigger heads than the ones of older roosters. The computational analysis of chromatin compaction showed to be less subjective and more precise than the visual evaluation for identification of chromatin alterations of rooster spermatozoa.

Binding characteristics of sperm with recombinant human zona pellucida glycoprotein-3 coated beads.

Background & objectives: An inability or decreased ability of spermatozoa to bind to the zona pellucida (ZP), an extracellular glycoproteinaceous matrix surrounding egg, is one of the plausible causes of idiopathic infertility. It will be clinically useful to distinguish this condition from other causes of infertility. An assay system, investigating binding of human sperm with ZP glycoprotein may prove useful in this regard. We attempted to develop a simple assay system to analyse the binding of capacitated human spermatozoa to human zona pellucida glycoprotein-3 (ZP3) using baculovirus-expressed recombinant human ZP3 coated beads. Methods: Recombinant baculovirus-expressed ZP3 was purified, labelled with biotin and coated on streptavidin sepharose beads. An in vitro assay system was optimized to study binding of capacitated human sperm to ZP3 coated beads. Results: A higher percentage of baculovirus-expressed recombinant human ZP3 coated beads showed significant (P<0.05) binding of capacitated human sperm as compared to beads coated with fetuin. An inhibition in the binding of sperm to ZP3 coated beads was observed in presence of cold recombinant human ZP3. Further, prior incubation of ZP3 coated beads with monoclonal antibodies (MAbs) against ZP3 but not against ZP2 resulted in the decrease in number of sperm bound to bead. Interpretation & conclusion: An in vitro assay system to study the binding of human sperm to ZP3- primary sperm receptor was established, which may be useful to determine the functional competence of spermatozoa.

Morte celular programada e peroxidação lipídica no espermatozoide humano / Programed cell death and lipid peroxidation in human spermatozoa

O principal marcador bioquímico da apoptose precoce em células somáticas é a translocação da fosfatidilserina para o folheto externo da membrana plasmática e fragmentação do DNA. Essas características têm sido também descritas em espermatozoides e são observadas com maior frequência nos ejaculados de homens inférteis. O espermatozoide humano tem uma alta concentração de ácidos graxos poliinsaturados em sua membrama e pouca proteção adequada com antioxidante. Ácidos graxos poli-insaturados são necessários para eventos de fusão da membrana associados à fertilização. No entanto, a presença deles acarreta uma vulnerabilidade para os danos peroxidativos. Sob várias condições de tensão oxidativa, o início da cascata da peroxidação lipídica resulta em perda da fluidez e prejuízo da função espermática. No campo da reprodução humana, principalmente nos aspectos referentes à infertilidade masculina, conhecer os mecanismos da apoptose e da peroxidação lipídica é essencial. Na abordagem da infertilidade masculina, a avaliação da translocação da fosfatidilserina (através da aderência à anexia V como marcador da apoptose) e a aferição da peroxidação lipídica têm como objetivo tentar identificar os casos com mau prognóstico na criopreservação/descongelamento. A identificação de tais casos poderia evitar desgastes psicológicos e econômicos nesses pacientes, bem como a indicação de uma outra abordagem para o seu acompanhamento.

Plasma membrane translocation of phosphatidylserine and DNA fragmentation are considered the main biochemical markers of early apoptosis in somatic cells. These characteristics have been also described in sperm cells and are commonly observed in ejaculated infertile men. The membrane of human spermatozoa contains a high concentration of polyunsaturated fatty acids and inadequate antioxidative protection. Although polyunsaturated fatty acids are required in membrane events associated with potential fertilization, their appearance may increase the vulnerability to peroxidative damage. Under oxidative stress, lipid peroxidation results in motility and sperm function damage. In the field of human reproduction, mainly in the area of male infertility, it is essential to know the apoptosis mechanism and the effects of lipid peroxidation. In the study of male infertility, the evaluation of plasma membrane phosphatidylserine translocation (assessed by annexin V binding as a apoptosis marker) and spontaneous lipid peroxidation aim to identify the bad prognostic cases for those that are submitted to cryopreservation process stress. This would prevent psychological and economic losses in these patients and establish another evaluation method for their infertility.

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.

Oxidative stress & male infertility.

The male factor is considered a major contributory factor to infertility. Apart from the conventional causes for male infertility such as varicocoele, cryptorchidism, infections, obstructive lesions, cystic fibrosis, trauma, and tumours, a new and important cause has been identified: oxidative stress. Oxidative stress is a result of the imbalance between reactive oxygen species (ROS) and antioxidants in the body. It is a powerful mechanism that can lead to sperm damage, deformity and eventually, male infertility. This review discusses the physiological need for ROS and their role in normal sperm function. It also highlights the mechanism of production and the pathophysiology of ROS in relation to the male reproductive system and enumerate the benefits of incorporating antioxidants in clinical and experimental settings.

Oxidative stress and sperm mitochondrial DNA mutation in idiopathic oligoasthenozoospermic men.

Physiological function of reactive oxygen species (ROS) has been known since a long, but recently toxic effects of ROS on spermatozoa have gained much importance in male infertility. Mitochondrial DNA (mtDNA) is believed to be both source and target of ROS. mtDNA unlike nuclear DNA is not compactly packed and hence more susceptible to oxidative stress (OS) than nuclear DNA. In the present study, the role of OS in mitochondrial genome changes was studied in men with idiopathic infertility. The study included 33 infertile oligo-asthenozoospermic (OA) men and 30 fertile controls. Semen analyses were performed and OS was measured by estimating the level of malondialdehye (MDA) in the seminal plasma and ROS in the sperm. Sperm mtDNA was sequenced by standard PCR-DNA sequencing protocol for ATPase and nicotinamide adenine dinucleotide dehydrogenase (ND) groups of genes. Sperm count and progressive motility were found to be significantly lower in infertile group than the fertile controls. Semen MDA and ROS levels of infertile group were significantly higher (p<0.0001), when compared to the control group. However, catalase and glutathione peroxidase (GPx) levels were significantly lower in infertile group, compared to controls, but no significant difference in superoxide dismutase (SOD) activity was observed between control and cases. This might be due to higher expression of SOD alone in order to overcome OS in the semen. mtDNA analysis showed significant and high frequency of nucleotide changes in the ATPase (6 and 8), ND (2, 3, 4 and 5) genes of infertile cases compared to the controls. Hence excess ROS and low antioxidant levels in the semen might cause mtDNA mutations and vice versa in OA men that might impair the fertilizing capacity of spermatozoa. Thus, it is important to understand the etiology of mitochondrial genome mutations in idiopathic OA cases for better diagnostic and prognostic value in infertility treatment/assisted reproductive technique