The objective of this study was to establish a rapid and efficient assay that would assess acrosomal status and function of the stallion acrosome. Ejaculates from fertile and subfertile stallions were extended to 25x106/mL and divided into aliquots (1mL) treated with no ionophore (control) or 10µM A23187 and incubated at 37ºC for 0, 1, 2, and 3h. Following incubation, samples were fixed with 2% paraformaldehyde for 10 minutes at room temperature; then stored at 4°C in Dulbecco’s Phosphate-buffered saline (DPBS) for 0, 24, and 72 hours (i.e. post-fixation storage). After post-fixation storage samples were then permeabilized with 95% ethanol at -20ºC for 10 minutes. Samples were resuspended in 20% fetal bovine serum in DPBS, labeled with fluorescein isothiocyanate for 10 minutes, and analyzed by flow cytometry. Post-fixation storage produced fewer (P<0.05) acrosome intact (AI) spermatozoa and a higher (P<0.05) fluorescence intensity than respective fresh samples. Regardless of incubation time or treatment, cool-stored samples averaged ~6% lower (P<0.001) AI spermatozoa than the corresponding fresh semen; however, cooled storage did not alter (P>0.2) the overall fluorescence properties as compared to fresh semen (730±8.08 vs. 734±8.01 fluorescence intensity units, respectively). For fertile stallions, the percentage of AI spermatozoa was higher (p<0.01) in control samples than A23187 samples at incubation times 1, 2, and 3h (Control-59, 56, and 51% vs. A23187- 46, 29, and 23%, respectively), but not at Time 0. For subfertile stallions, the percentage of AI spermatozoa was not affected by ionophore treatment (P>0.05) or incubation period (P>0.05). The results suggest that post-fixation storage in DPBS for up to three days is still representative of the acrosomal competence of the original sample. In addition, spermatozoa stored for 24 hours in an Equitainer™ exhibited a small (~6%) but significant decrease in the percentage AI spermatozoa. Storage conditions may therefore, affect acrosomal integrity and contribute to reduced fertility when cooled-semen is used. Subfertile stallions exhibited little response [<11% acrosome reacted (AR)] after 3h of A23187 exposure, while the fertile stallions demonstrated a substantial response (≥ 36% AR) as soon as 1h after ionophore exposure. This assay diagnosed acrosomal dysfunction in stallions with unexplained subfertility.

Abstract Several recent studies have indicated that radiofrequency electromagnetic fields (RFEMF) have an adverse effect on human sperm quality, which could translate to an effect on fertilization potential. The present study evaluated the effect of RF-EMF on spermspecific characteristics in order to assess the fertilizing competence of sperm. Highly motile human spermatozoa, were exposed for one hour to 900 MHz mobile phone radiation at a specific absorption rate (SAR) of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed by computer assisted sperm analysis (CASA). Significant reduction in sperm head area (9.2 ± 0.7 μm2 vs. 18.8 ± 1.4 μm2) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) were reported among exposed sperm compared with unexposed controls. Sperm–zona binding was assessed directly after exposure using the hemizona assay (HZA). The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p<0.05), respectively. This study concludes that while RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilisation potential.

Thesis (MScMedSci)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Introduction: Mondia whitei commonly known as "White Ginger" is a highly acclaimed medicinal plant that is extensively used across Africa. M. whitei is used as treatment for sexual dysfunction and is considered to be an aphrodisiac by traditional medicine practitioners. Yet, scientific evidence to support these claims are minimal and those that are published possess ambiguity. To date, only one study reporting the in vitro effect of the aqueous rhizome extract of M. whitei on human sperm motility is available. Therefore, the aim of the study was to determine the in vitro effects of M. whitei in human sperm functions. Materials and Methods: Roots of Mondia whitei obtained from the tropical Kakamega rain forest, located in the Western Province of Kenya, were cleaned and chopped into smaller segments. These pieces were ovendried at 25℃ for 3 days and milled to form a powdery substance which was infused with hot (about 70℃) distilled water for 1 hour. After cooling and filtration, the extract was frozen at -20℃ and subsequently freeze-dried. The dried extract was then stored at 4℃ in a closed container until experimentation. A total of 60 semen samples were collected: 28 of them represented healthy sperm donors and 32 infertile patients. Among these subjects, oligozoospermic and asthenozoospermic semen samples were identified and analysed separately. Sperm were washed using human tubular fluid medium supplemented with bovine serum albumin (HTF-BSA) and incubated for 1 hour at 37℃ with different concentrations of M. whitei (0.0185, 0.185, 1.85, 18.5 and 185 μg/ml). A sample without M. whitei served as control. Sperm cell motility, vitality, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), capacitation, acrosome reaction and DNA fragmentation were assessed. Results: Total motility and the percentage of sperm with intact MMP showed significant dose-dependent increases in both groups (patient and donor), while, the percentages of progressively motile sperm only revealed significant increases in the patient group. Besides, the percentage of ROS-positive spermatozoa showed significant trend towards higher concentrations in the patient group only. Conversely, a trend towards reduced sperm DNA-fragmentation could be observed in the patient, but not the donor group. Similar tendencies were noted in oligozoospermic and asthenozoospermic, but not for normozoospermic subjects. Yet, sperm vitality, capacitation, acrosome reaction and kinematic parameters were not affected. Conclusions: Phytochemicals present in M. whitei root extract maintains spermatozoa total motility, progressive motility and intact-MMP and DNA integrity. However, at therapeutic concentration (<1.85 μg/ml) it does not trigger sperm intrinsic superoxide production nor increase ROS by causing oxidative stress, that leads to DNA fragmentation. / National Research Foundation (NRF)

In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...

This study was conducted to investigate the effect of platelet activating factor (PAF) on the acrosome reaction and fertilizing capacity of spermatozoa, and development of the resulting embryos in the rabbit. Rabbit spermatozoa were exposed to PAF, Iyso-PAF, or high ionic strength medium (HIS) prior to subzonal sperm injection (SUZI) into 326 mature oocytes, or morphological assessment of the acrosome reaction. The rates of fertilization and blastocyst formation were compared among the three treatment groups. Acrosome reaction was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining and electron microscopy. PAF-treated spermatozoa fertilized the oocytes at a significantly higher rate (56.1%) than did lyso-PAF-(36.8%, P< 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa. The embryos produced by PAF-treated spermatozoa showed significantly higher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%, P < 0.050) or HIS-(8.8%, P< 0.05) treated spermatozoa. FITC-PSA staining demonstrated a significantly higher incidence of acrosome reaction in PAF-treated spermatozoa (45.8%) than in Iyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01) treated spermatozoa. Acrosome reaction of PAF-treated spermatozoa was also confirmed by electron microscopy. PAF treatment of spermatozoa enhances fertilizing capacity for SUZI possibly by augmenting the acrosome reaction. Enhanced embryonic development was also found in the oocytes fertilized by SUZI of PAF-treated spermatozoa.

ABSTRACT REGULATION OF THE ACROSOME REACTION BY THE TRANSMEMBRANE ADENYLYL CYCLASE SEPTEMBER 2012 DOUGLAS HADDAD, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST M.Sc., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Pablo Visconti Capacitation prepares mammalian sperm to undergo a process known as the acrosome reaction, which enables them to penetrate the zona pellucida. The standard method of measuring the acrosome reaction has been over the years the staining of the acrosome and visual counting using light or fluorescence microscopy. In this study we explored the intracellular signaling that results in the acrosome reaction using a novel method. This method employs the use of transgenic mice that contain green fluorescent protein, GFP, in the sperm acrosome. The quantity of sperm either containing or lacking GFP is precisely and rapidly quantified with flowcytometry. Currently little is known about the signaling processes that lead to the acrosome reaction. It has been proposed that this reaction is regulated by the cAMP activated guanine nucleotide exchange factor, Epac. In human sperm, stimulation of this pathway leads to an increase in acrosomal exocytosis. Furthermore, previous studies from our laboratory indicated that Gαs is present in the mouse sperm anterior head. These results suggest that in mouse sperm, the cAMP pathway leading to the acrosome reaction could be stimulated by Gαs associated with a transmembrane adenylyl cyclase. In this study we first validated the ability of known reaction inducers to increase the rate of the acrosome exocytosis in mouse sperm. Progesterone, solubilized zona pellucida and calcium ionophore A23187 all showed to be very effective at inducing the acrosome reaction in mouse sperm. We then investigated the role of the cAMP pathway using a battery of cAMP agonists and antagonists. We observed that stimulation of the cAMP dependent pathway through the transmembrane adenylyl cyclases, using forskolin, inhibit the increase in acrosome reaction induced by soluble zona pellucida, progesterone and the calcium ionophore A23187. This inhibitory effect was observed only when forskolin was used before the start of capacitation. Consistent with this observation, addition of cAMP analogues including an Epac specific cAMP analogue 8-pCPT-2’-O-Me-cAMP can also inhibit the increase in acrosome reaction by progesterone. This inhibition was seen only when the pathway was stimulated from the beginning of capacitation. Altogether, these data suggest that transmembrane cyclases are involved in the regulation of mouse sperm acrosome reaction.

Dynamics of acrosome reaction during intra-specific sperm competition in rodents Sperm acrosome integrity is disturbed in promiscuous species field mice (Apodemus) and more than half of the spermatozoa undergoing spontaneous acrosome reaction (AR) before binding to the zona pellucida. In Muridae it is documented a generally high rate of spontaneous AR, and the percentage increases in promiscuous species up to 60 % in 60 min capacitation in vitro. The acrosome integrity positively corellates with presence of CD46 protein which absence in wood mouse is fenotypicaly same as in CD46 knock-out mouse leading to accelerated spontaneous AR. It is necessary to clarify whether for mouse sperm it is essential the primary binding of intact sperm to zona pellucida of the egg or whether it is preferred secondary sperm binding after spontaneous AR. In this context, the question is whether there is a relocalization of the key fusion protein IZUMO in sperm during spontaneous AR. IZUMO relocalization was monitored by immunofluorescence at specific times of capacitation in vitro during spontaneous and induced AR. IZUMO relocalization as closely connected to actin cytoskeleton, and β1 integrins. Dynamics and localization of β1 integrin during spontaneous and induced AR was also detected by immunofluorescence. Our results...

O objetivo deste estudo foi avaliar os efeitos de três diluidores diferentes na criopreservação do sêmen bovino sobre a motilidade, hiperativação espermática, reação acrossomal, capacitação e peroxidação lipídica das membranas espermáticas. Foram realizadas seis colheitas de sêmen em intervalos quinzenais de dez touros zebuínos da raça Nelore. O sêmen in natura foi avaliado, dividido em três tratamentos: uma fração foi diluída em meio a base de gema de ovo (Botu-Bov® - diluidor 1), a segunda fração diluída em meio a base de lecitina de soja (Botu-Bov® - diluidor 2) e a terceira fração em meio AndroMed® (diluidor 3) também a base de lecitina de soja. Logo após as amostras foram submetidas à congelação. As avaliações do sêmen após descongelação consistiram na análise computadorizada das características de motilidade e nas análises pela citometria de fluxo quanto à reação acrossomal (PI/FITC-PSA/H33342); peroxidação lipídica (PI/C11-BODIPY581/591/H33342) e capacitação espermática através da estabilidade da membrana plasmática (Merocianina 540/Yo-Pro1/H33342). As variáveis foram submetidas à análise de variância e ao teste LSD para comparação das médias, ao nível de 5% de significância. O diluidor a base de gema de ovo preservou melhor a motilidade espermática, a população de células com integridade de membrana plasmática e acrossomo não reagido no sêmen bovino pós-descongelação que os diluidores a base de lecitina de soja. Os espermatozóides criopreservados com o diluidor a base de gema de ovo apresentou menor sub população de células hiperativas e com membrana plasmática desestabilizada quando comparados com os diluidores a base de lecitina de soja e o diluidor a base de gema de ovo possibilitou uma diminuição da peroxidação lipídica das membranas espermáticas / The objective of this experiment was evaluate the effects of three diferent extenders in bovine semen cryopreservation about motility, sperm hiperactivation, acrosome reaction, capacitation, and sperm membrane lipid peroxidation. It was made six collection of semen samples each 15 days of 10 Nelore bulls. Semen samples in natura was verify and divided in three treatments: The first one was extended in egg yolk base (Botu-Bov® Extender 1), second one was extended in soy lecithin base (Botu-Bov® Extender 2) and the third was extended in AndroMed® soy lecithin base too. After that, semen samples were submitted to freeze process. Semen evaluations after thawing were made with computer assisted sperm analysis (CASA) and flow citometry for acrosome reaction (PI/FITC-PSA/H33342), lipid peroxidation (PI/C11-BODIPY581/591/H33342) and sperm capacitation by plasma membrane stability (Merocianina 540/Yo-Pro1/H33342). Data obtained from experimental proceeding were submitted to variance analysis and LSD test to compare means with 5% of significance. Egg yolk base extender preserved better sperm motility, cell population with plasma membrane integrity and non-reacted acrosome in bovine semen after thawing than soy lecithin extenders. Cryopreserved sperm with egg yolk base extender displayed less subpopulation of hiperactivated cells, less destabilized plasma membrane and permitted a decrease of lipid peroxidation of membranes when it was compared with soy lecithin extender

Orientador: Fabiana Ferreira Souza / Resumo: As proteínas dos fluido e células do sitema reprodutor são constantes objetos de estudo, a fim de elucidar eventos fisiológicos e buscar biomarcadores das funções reprodutivas, facilitando a escolha de reprodutores. Em vista disso, este estudo objetivou caracterizar as proteínas dos espermatozoides do ejaculado e da cauda do epidídimo, do plasma seminal e do fluido epididimário de touros. Foram utilizados 10 touros adultos da raça Brangus. O sêmen foi colhido por eletroejaculação e, posteriormente os machos foram orquiectomizados para a colheita dos espermatozoides e fluido do epidídimo. As células do ejaculado e da cauda do epidídimo foram analisadas subjetivamente após a colheita, e o plasma seminal e fluido do epidídimo foram separados por centrifugação. Então, as amostras foram preparadas para a espectrometria de massas (ESI-QTof MS/MS), com um pool de cada grupo. A concentração de proteínas totais não diferiu entre os grupos. Foram encontradas 67 e 66 proteínas nos espermatozoides do ejaculado e da cauda do epidídimo, e 20 e 16 no plasma seminal e líquido epididimário, respectivamente. Além disso, 52 proteínas foram comuns entre os espermatozoides obtidos do ejaculado e epidídimo, e 9 entre o plasma seminal e fluido epididimário. Atividade catalítica foi a principal função molecular nas células espermáticas; já no plasma seminal foi de ligação e no fluido epididimário, atividade catalítica. As proteínas que se destacaram foram: 14-3-3 protein zeta/delta, A-kinase anchor ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre