Sperm morphophysiology in different sections of the rooster reproductive tract / Morfofisiología espermática en diferentes secciones del tracto reproductivo del gallo

It is important to know the morphological changes that occur in the spermatozoa of rooster during their passage through the reproductive tract, which help to understand what they acquire their fertilization capacity. The morphophysiological changes related to the capacitation and acrosomal reaction processes in the different segments of the rooster reproductive system were analyzed. Samples were obtained from various regions of the rooster reproductive tract and dorso-ventral massage to obtain ejaculates, 25 roosters were used Rhode Island Red with proven fertility, assessments were performed with chlortetracycline and Lectin WGA-FITC to determine the morphophysiological parameters. Sperm motility increases (p<0.05) during the passage of spermatozoa from the testis until they are ejaculated. The parameters of viability and morphology also show differences (p<0.05) in the different segments of the tract. Sperm morphometry shows a spermatic contraction (p<0.05) in the cranial and medial segments of the vas deferens. The acrosomal reaction capacity evaluated with chlortetracycline (CTC) or Wheat germ agglutinin (WGA), was evident increasing the parameters (p<0.05) with the use of the perivitelline layer in the spermatozoa of the reproductive tract and of the ejaculate. Spermatozoa of the reproductive tract of the rooster demonstrate acrosomal reaction capacity without requiring a previous sperm capacitation condition. On the other hand, they do not show parameters of incapacity, which implies that they cannot be stored in any segment of the reproductive tract.

Es importante conocer los cambios morfológicos que se producen en los espermatozoides del gallo durante su paso por el tracto reproductivo y que ayudan a comprender como adquieren su capacidad de fertilización. Se analizaron cambios morfofisiológicos relacionados con los procesos de capacitación y reacción acrosomal de los espermatozoides presentes en los diferentes segmentos del tracto reproductor del gallo. Se obtuvieron espermatozoides de diferentes regiones del tracto reproductor del gallo y de espermatozoides de eyaculado. Se usaron 25 gallos Rhode Island Red con fertilidad probada. Se realizaron evaluaciones básicas, con clortetraciclina (CTC) y lectina Wheat germ agglutinin conjugada con isotiosionato de fluoresceína (WGA-FITC) para determinar los parámetros morfofisiológicos. La motilidad del esperma aumenta (P<0,05) durante el paso de los espermatozoides desde el testículo hasta que son eyaculados. Los parámetros de viabilidad y morfología también muestran diferencias (P <0,05) en los diferentes segmentos del tracto. La morfometría mostró una contracción de los espermatozoides (P<0,05) en los segmentos craneal y medial del conducto deferente. La capacidad de reacción acrosomal evaluada con clortetraciclina CTC o WGAFITC, fue evidente al aumentar los parámetros (P<0,05) con el uso de membrana perivitelina en los espermatozoides del tracto reproductivo y del eyaculado. los espermatozoides del tracto reproductivo del gallo demuestran capacidad de reacción acrosomal sin requerir una condición previa de capacitación espermática. Por otro lado, no muestran parámetros de descapacitación espermática lo que implica que no pueden almacenar en ningún segmento del tracto reproductivo.

Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm / 亚洲男科学杂志(英文版)

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l-1), whereas higher concentrations (>5 μmol l-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.

Novel DPY19L2 variants in globozoospermic patients and the overcoming this male infertility / 亚洲男科学杂志(英文版)

Globozoospermia has been reported to be a rare but severe causation of male infertility, which results from the failure of acrosome biogenesis and sperm head shaping. Variants of dpy-19-like 2 (DPY19L2) are highly related to globozoospermia, but related investigations have been mainly performed in patients from Western countries. Here, we performed a screening of DPY19L2 variants in a cohort of Chinese globozoospermic patients and found that five of nine patients carried DPY19L2 deletions and the other four patients contained novel DPY19L2 point mutations, as revealed by whole-exome sequencing. Patient 3 (P3) contained a heterozygous variant (c.2126+5G>A), P6 contained a homozygous nonsense mutation (c.1720C>T, p.Arg574*), P8 contained compound heterozygous variants (c.1182-1184delATC, p.Leu394_Ser395delinsPhe; c.368A>T, p.His123Arg), and P9 contained a heterozygous variant (c.1182-1184delATCTT, frameshift). We also reported intracytoplasmic sperm injection (ICSI) outcomes in the related patients, finding that ICSI followed by assisted oocyte activation (AOA) with calcium ionophore achieved high rates of live births. In summary, the infertility of these patients results from DPY19L2 dysfunction and can be treated by ICSI together with AOA.

Does conventional freezing affect sperm DNA fragmentation? / 대한생식의학회지

OBJECTIVE: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. METHODS: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. RESULTS: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. CONCLUSION: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

Iodixanol supplementation during sperm cryopreservation improves protamine level and reduces reactive oxygen species of canine sperm

The objective of this study was to analyze the protective effects of iodixanol on dog spermatozoa during cryopreservation. The optimal concentration of iodixanol, 1.5%, was determined using fresh spermatozoa and was applied in the following experiments. The 1.5% iodixanol group showed significantly increased sperm motility from that in the control (p < 0.05). Lower mitochondrial reactive oxygen species (ROS) modulator (ROMO1) and pro-apoptotic gene (BAX) expressions, together with higher expressions of protamine-2 (PRM2), protamine-3 (PRM3), anti-apoptotic B-cell lymphoma-2 (BCL2), and sperm acrosome associated-3 (SPACA3) genes were detected in the iodixanol-treated group. In addition, decreased protamine deficiency and cryocapacitation were observed in the treatment group. Our results show that supplementation with 1.5% iodixanol is ideal for reducing production of ROS and preventing detrimental effects during the canine sperm cryopreservation process, effects manifested as increased motility and reduced cryocapacitation in frozen-thawed spermatozoa.

Microscopic sperm head damage and abnormalities as heat stress indicators in Australian Merino rams (Ovis aries) in Northern Patagonia, Argentina / Danos e anormalidades microscópicas das cabeças espermáticas como indicadores de estresse por calor em carneiros Merino Australiano (Ovis aries) da Patagônia Norte, Argentina

In Northern Patagonia, the mating season starts on March 15th, when rams are submitted to summer temperatures. Exposure of rams to heat stress increases the prevalence of microscopic damage to spermatozoa, morphological abnormalities, and reductions in fertility. This study assesses the adaptive capabilities of six unshorn and six shorn Australian Merino rams, half of which were treated in a heat chamber for eight hours for five days, gradually reaching a temperature of up to 40 °C. Microscopic damage, abnormalities and ultramicroscopic alterations of the plasma membrane and the acrosome of sperm head were analysed. There were significant differences in the percentage of tailless spermatozoa and proximal cytoplasmic droplets between post-treatment periods. Temperature primarily affected the shorn rams and the sperm heads during spermiogenesis. Submicroscopic alterations were observed when the plasma membrane was present in the anterior segment. These alterations can be intact, waved, or dilated. When the plasma membrane was absent, the acrosome might be intact, dilated, and waved. In addition, the outer acrosomal membrane may completely lose its contents or have a nude nucleus. The plasma membrane assumes a waved shape as a result of the effect of temperature on the epididymis. According to this study, the tailless head, proximal cytoplasmic droplets, and the ultramicroscopic categories studied were robust indicators of semen heat stress. After ten weeks, the sperm head recovered its normal shape. Unshorn rams are better adapted to summer heat stress than shorn ones. Microscopy and transmission electron microscopy alterations have been shown to be excellent indicators of thermal stress in Australian Merino rams and may be useful tools to help sheep farmers choose when to begin the mating season, which will vary depending on the environmental conditions of the summer.(AU)

Na Patagônia Norte, os ovinos têm sua estação de acasalamento iniciada em 15 de março, portanto, ficam sujeitos às temperaturas do verão. A exposição de carneiros a estresse térmico aumenta a prevalência de danos microscópicos e anomalias morfológicas nos espermatozoides, que implica uma redução na fertilidade. Este trabalho avaliou a capacidade adaptativa de carneiros Merino Australiano com lã (N = 6) e tosquiados (N = 6): metade ficou ao ar livre e outra metade foi mantida em uma câmara climática por oito horas, durante cinco dias, chegando gradualmente a uma temperatura máxima de 40 °C. Foram analisados danos microscópicos, anormalidades e alterações ultramicroscópicas da membrana plasmática e do acrossoma da cabeça dos espermatozoides. Os resultados microscópicos confirmaram a existência de diferença significativa na porcentagem de espermatozoides sem cauda e com gota citoplasmática proximal, entre os ejaculados pós-tratamento. A temperatura afetou os carneiros tosquiados, principalmente a cabeça de seus espermatozoides, durante a espermatogênese. Alterações submicroscópicas foram observados na membrana plasmática quando ela estava presente no segmento anterior: quando não intacta, ficava ondulada ou dilatada. Quando a membrana plasmática estava ausente, o acrossoma podia se apresentar ondulado ou dilatado. Além disso, sob efeito do calor, a membrana acrossomal externa pode perder completamente seu conteúdo ou apresentar núcleo desnudo. A membrana plasmática assume uma forma ondulada pelo efeito da temperatura no epidídimo. Depois de dez semanas, a cabeça dos espermatozoides recuperou sua forma normal. Como demonstrado neste estudo, a cabeça sem cauda, as gotas citoplasmáticas proximais e as categorias ultramicroscópicas estudadas são indicadores do efeito do estresse térmico no sêmen, e os carneiros com maior cobertura de lã se adaptam melhor ao estresse por calor. Alterações de microscopia e de microscopia eletrônica de transmissão têm se mostrado excelentes indicadores de estresse por calor em carneiros Merino Australiano e podem ser ferramentas úteis para ajudar criadores de ovelhas a escolher quando começar a época de acasalamento, o que irá variar de acordo com as condições ambientais do verão.(AU)

The role of human lysozyme-like protein 4 in fertilization and its enzymatic properties / 中华男科学杂志

Objective@#To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties.@*METHODS@#The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods.@*RESULTS@#Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity.@*CONCLUSIONS@#LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.

Resveratrol protects human sperm against cryopreservation-induced injury / 中华男科学杂志

<p><b>Objective</b>To investigate the effects of resveratrol in the cryopreservation medium on the quality and function of post-thaw sperm.</p><p><b>METHODS</b>Semen samples were obtained from 50 normozoospermic and 50 oligoasthenozoospermic men, liquefied and then cryopreserved in the glycerol-egg yolk-citrate (GEYC) medium with or without 30 μmol/L resveratrol. Sperm motility, viability and acrosome reaction (AR) were examined before and after thawing. Sperm lipid peroxidation and the level of reactive oxygen species (ROS) were measured using commercial malondialdehyde (MDA) and the ROS assay kit. Sperm mitochondrial membrane potential (MMP) and DNA damage were determined by Rhodamine 123 staining and TUNEL.</p><p><b>RESULTS</b>The percentage of progressively motile sperm (PMS), total sperm motility, sperm viability, MMP and AR were significantly decreased (P <0.05) while the levels of sperm ROS, MDA and DNA fragmentation index (DFI) remarkably increased in both the normozoospermia and oligoasthenozoospermia groups after cryopreservation as compared with those in the fresh ejaculate (P <0.05). In comparison with the non-resveratrol control, the post-thaw sperm cryopreserved with 30 μmol/L resveratrol showed markedly higher PMS (［32.7 ± 4.8］ vs ［43.1 ± 6.3］ %, P <0.05), total motility (［44.8 ± 6.9］ vs ［56.9 ± 7.4］ %, P <0.05), viability (［52.3 ± 6.1］ vs ［67.5 ± 5.6］ %, P <0.05), MMP (［56.5 ± 7.0］ vs ［63.4 ± 7.5］ %, P <0.05) and AR (［16.6 ± 3.8］ vs ［26.3 ± 4.7］ %, P <0.05) but lower ROS, MDA and DFI (all P <0.05) in the normozoospermia group, and so did the post-thaw sperm in the oligoasthenozoospermia group, with a particularly lower DFI (［28.5 ± 4.8］ vs ［36.3 ± 5.7］%, P <0.01).</p><p><b>CONCLUSIONS</b>Resveratrol in the cryopreservation medium can improve the quality and function of post-thaw human sperm by reducing cryopreservation-induced sperm injury and the level of ROS.</p>

Differentiation of human male germ cells from Wharton's jelly-derived mesenchymal stem cells / 대한생식의학회지

OBJECTIVE: Recapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium. METHODS: Human WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at 36℃ for 3 more weeks. RESULTS: The gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted. CONCLUSION: Human WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.

Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation / Inibidor do ativador do plasminogênio 1 e antipaína preservam a integridade do acrossoma de espermatozoides bovinos durante a criopreservação

Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.(AU)

A criopreservação é parcialmente prejudicial à fertilidade do sêmen de bovinos e induz mudanças semelhantes à capacitação em espermatozoides. O plasma seminal contém serina-proteases e inibidores de serina-proteases que estão envolvidos na fertilização de mamíferos, e os inibidores podem ser aplicados para evitar uma capacitação espermática induzida pelo frio. Analisaram-se os efeitos de diferentes concentrações de dois inibidores de serina-proteases, inibidor do ativador do plasminogênio 1 - PAI-1 (70ƞg, 140ƞg e 210ƞg) e antipaína (10µg, 50µg e 100µg) na suplementação ao diluidor de criopreservação de sêmen bovino. Trinta e seis ejaculados de quatro bovinos Curraleiro Pé-Duro foram usados para criopreservação. Os efeitos dos inibidores sobre os parâmetros dos espermatozoides (cinética espermática - CASA, integridade acrossomal, integridade da membrana plasmática, potencial de membrana mitocondrial, defeitos espermáticos e taxa de reação acrossomal) foram avaliados no sêmen pós-descongelamento. A criopreservação de espermatozoides com antipaína diminuiu os parâmetros cinéticos pós-descongelamento de MP, VSL, LIN, SRT e a porcentagem de espermatozoides hiperativados, PAI-1 (210ƞg) diminuiu VSL e LIN. Antipaína e PAI-1 não tiveram efeitos nos parâmetros de integridade da membrana plasmática, no potencial de membrana mitocondrial e nos defeitos espermáticos. Espermatozoides criopreservados na presença de antipaína e PAI-1 (70 e 140ƞg) preservaram a integridade acrossomal, assim como foram capazes de completar a reação acrossômica in vitro. Em conclusão, os inibidores de serina-proteases, antipaína e PAI-1 (70 e 140ƞg) são capazes de preservar a integridade acrossomal de espermatozoides criopreservados de bovinos.(AU)

Preparation of a polyclonal antibody against human LYZL4 and its expression in the testis / 中华男科学杂志

Objective@#To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis.@*METHODS@#The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry.@*RESULTS@#rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids.@*CONCLUSIONS@#An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.

Efecto de la hiperviscosidad seminal sobre la integridad acrosómica y la movilidad espermática antes y después de la criopreservación. / Effect of seminal hyperviscosity on acrosomal integrity and sperm motility before and after cryopreservation

La criopreservación del semen es una herramienta útil en la reproducción asistida, la cual puede tener impacto en las características espermáticas durante el congela miento y el descongelamiento. El objetivo de este estudio fue valorar la integridad del acroso ma y la movilidad de los espermatozoides criopreservados y descongelados provenientes de muestras hiperviscosas y no viscosas. Se realizó el espermograma, la integridad del acrosoma, el espermocultivo y los niveles de los marcadores de glándulas accesorias en 60 muestras de semen. Cada alícuota de semen fue inmersa en un crioprotector comercial para congelar a -196°C. Transcurridos 30 días, éstas fueron descongeladas y en el sedimento celular espermá ticesuspendido se evaluó la movilidad y la integridad acrosómica, disminuyendo significa tivamente la movilidad progresiva (p<0,05), la vitalidad espermática (p<0,005) y la integridad acrosómica (p<0,05); dicho descenso fue más evidente en las muestras hiperviscosas. La viscosidad del semen fresco se relacionó inversamente con la movilidad y la integridad del acrosoma antes y después del congelamiento (p<0,05). En veinte muestras de semen se iden tificó la presencia de microorganismos y de anticuerpos IgA anti C. trachomatis , de las cuales quince muestras en la reproducción hiperviscosas. El aumento de la viscosidad seminal y los niveles de ácido cítrico están asociados con disfunción prostática, baja movilidad espermática y reacción prematura del acrosoma, lo que puede reducir la capacidad fecundante de un esper matozoide. La etiología de la hiperviscosidad sigue siendo compleja; sin embargo, para pre servar la movilidad y la integridad del acrosoma, previamente deben investigarse sus causas en las muestras seminales que van a ser sometidas a la criopreservación.

Semen cryopreservation is a useful tool in assisted reproduction, which may have impact on sperm characteristics during freezing and thawing. The aim of this study was to assess the integrity of the acrosome and motility of cryopreserved and thawed spermatozoos in hyperviscous and no viscous samples. In semen samples spermiogram, glandular markers, acrosome integrity, culture and the levels markers accessory glands were measured. Each ali quot of semen was immersed in cryoprotectant and maintained in a commercial freezer at -196 ° C. After 30 days, these were thawed and in the cell pellet resuspended, spermatic motility and acrosomal integrity were evaluated. In thawed samples, there were significant decreases in progressive motility (p <0.05), vitality (p <0.005) and acrosome integrity (p <0.05) with respect to fresh sperm, this decline was most evident in hyperviscous samples. The viscosity of fresh semen was inversely related to motility and acrosome integrity before and after freezing (p <0.05). Twenty semen samples showed the presence of microorganisms and C. trachomatis IgA antibodies, of which fifteen showed hyperviscosity. Biochemical analysis demonstrated that semen samples with low levels of citric acid had less acrosomal integrity both before and after freezing (p <0.05). The viscoelasticity and citric acid levels are associated with prostate dys function, low sperm motility and premature acrosome reaction, which can reduce the fertilizing capacity of sperm. The etiology of hyperviscosity remains complex; however, to preserve mo tility and acrosome integrity, its causes must be investigated previously in the seminal samples to be subjected to cryopreservation.

Combined effect of trolox and EDTA on frozen-thawed sperm quality

The freezing and thawing process not only is associated with serious damage to sperm such as damage to the plasma membrane and the acrosomal membrane but also changes the membrane permeability to some ions including calcium. Also, the generation of oxygen free radicals is increased during the freezing-thawing process. The purpose of this study was to evaluate of the effects of Trolox as an antioxidant and edetic acid [EDTA] as a calcium chelator on frozen-thawed [FT] sperm and compare these effects with those on fresh sperm. This study was done on these men of 25 healthy men, who referred to Shiraz Infertility Centerbetween 2012 and 2013. Normal samples were transferred to the ReproductivePhysiology Laboratory, Department of Physiology, Shiraz University of Medical Sciences, Shiraz. The samples were divided into two groups randomly: fresh and FT sperm groups. Each group was divided into five subgroups: control group, the solvent group [0.1%dimethyl sulfoxide [DMSO]], Trolox group [200microM], EDTA group [1 .ImM], and Trolox+EDTA group. The percentages of motility, viability, and acrosome-reacted sperm were tested. The percentages of motility and viability in the FT sperm were lower than those in the fresh sperm. The progressive motility of the FT sperm was improved nonsignificantly with Trolox+EDTA. However, the effect of Trolox+EDTA on the progressive motility of the FT sperm was much more than that on the fresh sperm. The fewest acrosome-reacted sperm were observed in the EDTA-containing FT sperm. Antioxidant supplementation or omission of extracellular calcium may partly improve motility and also reduce acrosomal damage in FT sperm

Sperm acrosome formation-associated genes in mice: Advances in studies / 中华男科学杂志

Spermiogenesis is a complex process of differentiation and morphologic alteration, in which sperm acrosome formation is an important stage. Acrosome is an essential component of the sperm head, which develops in four distinct phases: Golgi, cap, acro- somal, and maturation, each supported by precise and orderly regulation of various genes. The regulatory genes which act on Golgi ap- paratus include GOPC, Hrb, SPATA16, PICK1, and CK2α', those involved in the cap phase are Fads2, syntaxin 2, Kdm3a, and UBR7, and participating in acrosomal and maturation phases are KIFC1, Rnf19a, and DPY19L2. The abnormalities of these genes may affect male fertility by influencing the connection of the nuclear dense lamina and acroplaxome with the nuclear membrane and then the fusion and transportation of vesicles. This review focuses on the genes involved in different phases of acrosome formation.

Values of the sperm deformity index, acrosome abnormity rate, and sperm DNA fragmentation index of optimized sperm in predicting IVF fertilization failure / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the values of the sperm deformity index (SDI), acrosome abnormity rate (AAR), and DNA fragmentation index (DFI) of optimized sperm in the prediction of fertilization failure (fertilization rate < 25%) in conventional in vitro fertilization (IVF).</p><p><b>METHODS</b>We selected 695 cycles of conventional IVF for pure oviductal infertility in this study, including 603 cycles of normal fertilization and 92 cycles of fertilization failure. On the day of oocyte retrieval, we examined sperm morphology, acrosome morphology, and DNA fragmentation using the Diff-Quik, PSA-FITC and SCD methods. We established the joint predictor (JP) by logistic equation and analyzed the values of different parameters in predicting fertilization failure with the receiver operating characteristic (ROC) curve.</p><p><b>RESULTS</b>The fertilization rate was negatively correlated with SDI (r = - 0.07; P = 0.03), AAR (r = -0.49; P < 0.01), and DFI (r = -0. 21; P < 0.01). The SDI, AAR, and DFI in the normal fertilization group were 1.24 ± 0.20, (7.75 ± 2.28)%, and (7.87 ± 3.15)%, and those in the fertilization failure group were 1.42 ± 0.15, (12.02 ± 3.06)%, and (13.32 ± 4.13)%, respectively, all with statistically significant differences between the two groups (P < 0.05). SDI, AAR, and DFI were all risk factors of fertilization failure ( OR = 2.68, 14.11, and 3.85; P = 0.01, < 0.01, and < 0.01). The areas under the ROC curves for SDI, AAR, DFI, and JP were 0.651 ± 0.033, 0.895 ± 0.019, 0.789 ± 0.022, and 0.915 ± 0.017, respectively. According to the Youden index, the optimal cut-off values of SDI, AAR, and DFI obtained for the prediction of fertilization failure were approximately 1.45, 10%, and 12%.</p><p><b>CONCLUSION</b>The SDI, AAR and DFI of optimized sperm are closely associated with the fertilization rate, and all have the value for predicting fertilization failure in IVF. The AAR is more valuable than the other single predictors, but JP is more effective than the AAR.</p>

Impact of sperm DNA and acrosome integrity and acrosome reaction rate on outcomes of rescue intracytoplasmic sperm injection / 南方医科大学学报

<p><b>UNLABELLED</b>Objective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI).</p><p><b>METHODS</b>This retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed.</p><p><b>RESULTS</b>No significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively.</p><p><b>CONCLUSION</b>Rescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.</p>

Expressions of chemokine CXCL12 and its receptor CXCR4 in human sperm / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the expression patterns of the chemokine CXCL12 and its receptor CXCR4 in human sperm.</p><p><b>METHODS</b>We collected semen samples from 10 fertile men, performed density gradient centrifugation, and then determined the expressions of both CXCL12 and CXCR4 in the sperm by RT-PCR and immunofluorescence staining.</p><p><b>RESULTS</b>RT-PCR revealed the mRNA expressions of CXCL12 (0.641 +/- 0.180) and CXCR4 (0.464 +/- 0.100) in the sperm. However, only CXCR4 rather than CXCL12 was expressed at the protein level, and the positive staining for CXCR4 was observed mainly in the posterior part of the acrosome.</p><p><b>CONCLUSION</b>CXCL12 and CXCR4 are involved as important molecules in regulating the function of human sperm.</p>

Structural modification of the black bengal buck (Capra hircus) acrosome during post-testicular maturation of spermatozoa / Modificación estructural del acrosoma de la cabra black bengal (Capra hircus) durante la maduración de los espermatozoides

The present study was conducted to measure various biometric parameters of intact/normal acrosomes (AC) collected respectively from caput, corpus and cauda epididymis and vas deferens of Black Bengal buck. Giemsa stained acrosomes were measured after camera lucida drawings. Observations revealed dimensional characters of the acrosomal cap diminished gradually and significantly (p<0.01, p<0.05) during spermatozoa maturation phases in the different regions of the excurrent duct. Shape and size of the AC were also found to be influenced significantly (p<0.01, p<0.05) by the age and body weight of the animals. The structural modification along with decrease in the morphology of the AC reflected one of the maturational indexes of the male gametes in Black Bengal buck.

El presente estudio se realizó para medir diversos parámetros biométricos del acrosoma (AC) intacto/normal recogido desde la cabeza, cuerpo y cola del epidídimo y vas deferens de la Cabra Black Bengal. Los AC teñidos con Giemsa fueron medidos después de la captura con cámara lúcida. Las observaciones revelaron caracteres dimensionales del capuchón acrosomal que disminuyeron gradualmente y de manera significativa (p <0,01, p <0,05) durante fases de maduración espermatática en las diferentes regiones del conducto. La forma y tamaño del AC también fueron influenciados de manera significativa (p <0,01, p <0,05) por la edad y el peso corporal de los animales. La modificación estructural junto con los cambios morfológicos del AC refleja uno de los índices de maduración de los gametos masculinos de la Cabra Black Bengal.

Selección espermática en semen congelado/descongelado de equino: evaluación de las membranas plasmática, acrosomal y potencial de membrana mitocondrial / Sperm selection in frozen/thawed semen of equine: evaluation of plasma, acrosome membranes and mitochondrial membrane potential

Los procedimientos de criopreservación inducen cambios morfofuncionales en los espermatozoides. Es importante post descongelación espermática utilizar procedimientos de selección que permitan recuperar espermatozoides altamente funcionales. El objetivo del presente estudio fue comparar la eficiencia del Swim-up y Equipure® en la selección de espermatozoides funcionales en semen descongelado de equino. Semen de 4 potros reproductores Criollos Chilenos (A, B, C y D), fueron descongelados separadamente y procesados (n=15) por: I.- Swim-up (SU) y II.- Equipure® (EQ). Post descongelación se determinó por citometría de flujo la viabilidad e integridad de membrana plasmática (SYBR-14/PI), potencial de membrana mitocondrial (YDm; JC-1), integridad de la membrana acrosomal (FITC-PSA/PI). La motilidad progresiva (%) en dos animales fue más alta (P<0,05) por SU comparado con EQ: A (55,7±5,8% v/s 38,17±3,7%) y C (37,5±7% vs. 32±2,1%, respectivamente). La integridad de la membrana plasmática (%), tres animales presentaron diferencias (P<0,05), siendo más alta por SU en dos animales comparado con EQ (A: 54,3±1,7 vs. 36,7±1,9, C: 36,1±5,7 vs. 29,4±4,8 y D: 34,4±9,4 vs. 52,7±5,2; respectivamente), solamente un animal fue superior EQ. En el YDm (%), diferencias significativas (P<0,05) fueron detectadas en los cuatro animales, siendo más altos en SU comparado con EQ (A: 69,1±8,6% vs. 47,4±3,3%, B: 59,34±12,3% vs. 24,8±1,5%, C: 54,9±12,3% vs. 43,2±3,1% y D: 53,1±17,6% vs. 37,5±5,7%; respectivamente). Los resultados obtenidos en el presente estudio demostraron que los métodos de selección espermática Swim-up y Equipure® permiten recuperar espermatozoides de diferente calidad funcional en semen congelado-descongelado de equino, presentándose diferencias individuales entre los animales con respecto a los métodos. Se observó una tendencia del método Swim-up en seleccionar espermatozoides de equino descongelados con mayor calidad funcional comparado con Equipure®.

Freeze-thaw procedures induce structural and functional changes in sperm. It is important to use post thaw sperm selection procedures that can retrieve highly functional sperm. The aim of this study was to compare the efficiency of the Swim-up and Equipure® in the selection of functional sperm of thawed equine semen. Semen of four Chilean Criollo reproductive stallions (A, B , C and D) were frozen and thawed using a standard protocol and processed separately (n = 15) : I. Swim-up (SU) and II. Equipure® (EQ). Post sperm selection,was determined by flow cytometry. Viability and plasma membrane integrity (SYRB-14/PI), mitochondrial membrane potential (YDm, JC -1), acrosome membrane integrity (FITC-PSA/PI). Progressive motility (%) was higher (P<0.05) in two animals per SU compared with EQ, A (55.7±5.8% vs. 38.17±3.7%) and C (37.5±7.0% vs. 32±2.1%, respectively). The viability and integrity of the plasma membrane (%), three animals showed differences (P<0.05), being higher for SU in two animals compared with EQ (A: 54.3±1.7 vs. 36.7±1.9, C: 36.1±5.7 vs. 29.4±4.8 and D: 34.4±9.4 vs. 52.7±5.2, respectively), only one animal was higher EQ. In YDm (%), significant differences were detected (P<0.05) in all four animals, being higher in SU compared with EQ (A: 69.1±8.6% vs. 47.4±3.3% B: 59.34±12.3% vs. 24.8±1.5%, C: 54.9±12.3% vs. 43.2±3.1% and D: 53.1±17.6% vs. 37.5±5.7%, respectively). The results obtained in this study showed that sperm selection methods Swim-up and Equipure® can retrieve different functional sperm quality in frozen-thawed equine semen, and that individual differences were registered among animals with respect to methods. In the Swim-up method a tendency for selecting higher functional quality in thawed equine sperm was observed when compared to Equipure®.

Quality characteristics of human spermatozoa with hyaluronic acid receptors / 中华男科学杂志

<p><b>OBJECTIVE</b>To analyze the quality characteristics of human spermatozoa with hyaluronic acid (HA) receptors and search for a new indicator for the assessment of sperm quality.</p><p><b>METHODS</b>Using sperm-HA binding assay with HA-coated slides, we determined the binding rate of motile sperm with HA receptors and analyzed its correlation with routine semen parameters, sperm membrane function, sperm fertilizing function and diminished/arrested sperm maturation.</p><p><b>RESULTS</b>The motile sperm with HA binding sites in the acrosomal region showed significantly higher acrosomal integrity ([95.4 +/- 3.9]%) and mitochondrial membrane potential (MMP) ([97.8 +/- 2.1]%) than those in the initial semen ([68.8 +/- 6.2]% and [72.8 +/- 7.4]%) (P < 0.01). The sperm-HA binding scores were correlated mildly with many routine semen parameters (r = 0.195-0.268, P < 0.05), positively with the acrosome reaction level after ionophore challenge (r = 0.666, P < 0.01) and normal sperm morphology (r = 0.417, P < 0.01), and negatively with sperm nucleoprotein immaturation (r = -0.266, P < 0.01), DNA fragmentation (r = -0. 308, P < 0.01) and excessive residual cytoplasm (r = -0.218, P < 0.05).</p><p><b>CONCLUSION</b>Sperm with HA receptors in the acrosomal region exhibit significant advantages in plasma membrane structure, fertilizing potential and maturation. The sperm-HA binding assay, which is based on a relationship between sperm receptors for zona pellucida and HA, is likely to become a new independent indicator for assessing the multiple qualities of spermatozoa.</p>