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.

Specialized in form and function, the sperm cell is a unique microsystem unto itself where in the cytoskeletal processes and structures of the somatic cell often find new purpose and characteristics within the sperm. Unlike other cells in the human body, this unique cell polarizes and transforms itself from a line of germ cells to evolve into a functional, hydrodynamic haploid spermatozoon. The success of fertilization is dependent on this haploid cell and its specially designed vesicular structure, the acrosome, which provides the leading edge of oocyte penetration. To date, there is little insight into the mechanics of how acrosomic vesicles are successfully targeted and transported to the nuclear envelope and tether to its surface. Our laboratory has identified a novel 15 kDa sperm specific histone variant, SubH2Bv, which possesses a distinct and functional nuclear localization signal (NLS) that associates with the acrosomic vesicle. This study provides evidence that SubH2Bv’s bipartite NLS (an NLS with two basic domains linked together by 10-12 amino acid residues) is responsible for directing acrosomic vesicles to the nuclear envelope using the somatic import receptor, karyopherin alpha (Kap α). Based on bipartite NLS-receptor conventions, where karyopherin alpha is known to specifically associate with this NLS-type, SubH2Bv would be the karyophilic cargo and karyopherin alpha would act as part of the underlying transport mechanism. Western blot analysis and immunohistochemistry characterized Kap α as a membrane-associated sperm protein that is co-localized with SubH2Bv around the proacrosomic granules and the acrosomic vesicle during spermiogenesis. Their co-expression and co-localization, as demonstrated by immunolabelling, suggested a potential binding relationship that was confirmed by a His-tag-recombinant SubH2Bv-pull-down assay. The co-developmental acrosomic expression of Kap α and SubH2Bv in haploid cells, combined with the pull-down evidence of their binding affinity, provides a compelling argument that these two proteins work in concert to traffic the acrosomic vesicles to the nucleus. The exclusion of these two otherwise nuclear proteins from the nucleus, and their co-localization to the subacrosomal region in elongating spermatids, also implies a contingent role for SubH2Bv and Kap α in acrosomal docking, that may involve the classical bipartite/Kap α nuclear import pathway. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-09-02 16:11:17.429

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

L'infertilité masculine est une préoccupation croissante dans les sociétés modernes. Parmi les différentes causes d'infertilités masculines, certaines ont une origine génétique. C'est le cas de la globozoospermie de type I, une infertilité sévère caractérisée par la production exclusive des spermatozoïdes ayant une tête ronde et sans acrosome. Cette pathologie présente un phénotype complexe associant à l'absence de l'acrosome, des défauts de compaction nucléaire, des lésions de l'ADN et une déficience d'activation ovocytaire.Récemment notre équipe a montré que le gène DPY19L2 est impliqué dans 60 à 80% des cas de globozoospermie de type I et représente donc la cause majeure de la globozoospermie de type I chez l'homme. La fonction de la protéine étant complètement inconnue au moment de l'identification du gène DPY19L2, nous avons alors réalisée une étude fondamentale sur les mécanismes moléculaires expliquant la pathogénie moléculaire de cette maladie et avons démontré, grâce à l'utilisation du modèle des souris KO pour le gène Dpy19l2, que la protéine est localisée dans la membrane nucléaire interne et est nécessaire à l'ancrage de l'acrosome sur l'enveloppe nucléaire. Dans le but d'identifier d'autres acteurs impliqués dans l'attachement de l'acrosome au noyau spermatique, nous nous sommes intéressés à l'étude de la protéine Sun5 de l'enveloppe nucléaire interne située en regard de l'acrosome qui pouvait être un partenaire de Dpy19l2. On a trouvé que les deux protéines n'étaient pas des partenaires puisque Sun5 était, contrairement à ce qui a été décrit dans la littérature, du côté postérieur opposé à l'acrosome. En deuxième temps, nous nous sommes intéressés à la compréhension des principales causes moléculaires de l'absence de fécondation ou du très faible taux de réussite lors d'ICSI réalisées avec des spermatozoïdes globozoospermiques.Pour ce faire, nous avons recherché la protéine PLC ζ, le principal facteur spermatique activateur de l'ovocyte, dans les spermatozoïdes humains provenant de patients délétés pour le gène DPY19L2 et murins (de souris KO Dpy19l2-/-) et avons montré qu'effectivement cette protéine est absente tant chez les patients que dans le modèle d'étude murin. Nous avons également réalisé une étude comparative des principales étapes de la compaction du génome spermatique entre les souris mâles WT et Dpy19l2-/- et nous avons montré que la compaction du génome des spermatozoïdes globozoospermiques était défectueuse avec un défaut de protamination et une rétention des histones. Ainsi, la déficience en PLC ζ, les défauts de compaction et le taux élevé de fragmentation de l'ADN sont susceptibles d'expliquer l'échec de la fécondation après ICSI, et le développement très réduit des embryons issus des patients déficients pour le gène DPY19L2 ainsi que des souris Dpy19l2-/-. La compréhension de cette physiopathologie devrait permettre la proposition de nouvelles stratégies thérapeutiques permettant d'améliorer le taux du développement embryonnaire en AMP. / Male infertility is a growing concern in modern societies. Among the various causes of male factor infertility, some have a genetic origin. This is the case of type I globozoospermia, a severe infertility characterized by the presence in the ejaculate of a majority of round spermatozoa devoid of acrosome. This pathology presents a complex phenotype associating to the absence of the acrosome, sperm nuclear compaction defects, DNA damage and failure in egg activation.Recently our team identified the DPY19L2 gene as responsible for a large majority of pure globozoospermia cases (>80%) and therefore represents the major cause of type I globozoospermia in humans. The function of the protein was completely unknown at the time of identification of DPY19L2 gene, we then performed a fundamental study of the molecular mechanisms underlying the molecular pathogenesis of the disease and have shown, through the use of knockout mice model for the Dpy19l2 gene, that the protein is localized in the inner nuclear membrane and is necessary for the anchoring of the acrosome to the nuclear envelope. In order to characterize new actors of acrosome attachment, we assessed the importance of Sun5 (also called Spag4l) in acrosome attachment which was previously shown to be expressed in NE of spermatogenic cells, facing the acrosome and that could be a partner of Dpy19l2. Interestingly we demonstrate that Sun5 and Dpy19l2 are not partners and that Sun5, is not as initially reported, facing the acrosome but is in fact excluded from this zone. We were also interested in understanding the main molecular causes of the poor fertilization ability or the very low success rate in ICSI when performed with globozoospermics sperm.Therefore we have searched the PLC ζ protein, the sperm factor thought to induce the Ca2+ oscillations responsible for egg activation, in human spermatozoa from patients with DPY19L2 deleted gene and in murine spermatozoa from Dpy19l2 Knock-Out mice and showed that actually this protein is absent both in patients and in KO mice. We also made a comparative study of the main stages of genome compaction of sperm from both WT and Dpy19l2 KO mice and we showed that Dpy19l2 deficient sperm displays defective genome condensation and DNA alterations. Thus, PLC ζ deficiency, the compaction defects and the high rate of DNA fragmentation may explain the failure of fertilization after ICSI, and the poor development of embryos from patients deficient for the gene DPY19L2 as well as Dpy19l2 KO mice.Understanding the physiopathology of globozoospermia should allow the proposal of new therapeutic strategies that improves the rate of embryonic development in ART.

Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...

Research Doctorate - Doctor of Philosophy (PhD ) / In Australia almost 40% of the carnivorous marsupials, or dasyurids, are threatened. Assisted reproductive techniques (ART), especially genome resource banking, have the potential to contribute to the conservation of these species by reducing the loss of genetic diversity. This project aimed to advance the knowledge of ART in dasyurids by focusing on the long term preservation of male and female gametes and establishing protocols for the production of mature oocytes for use in future ART. These studies used the fat tailed dunnart (Sminthopsis crassicaudata) as a model dasyurid and replicated many of the findings on threatened dasyurids. Dasyurid spermatozoa had a relatively unstable acrosome which lacked acrosomal membrane disulphide stabilisation. There was no evidence that S. crassicaudata spermatozoa were susceptible to high concentrations of cryoprotectants, but spermatozoa frozen with up to 40% glycerol using a rapid freezing protocol were not viable. Nonetheless the morphology and acrosomal integrity of frozen spermatozoa was normal and there was no evidence of DNA damage. The lack of success with cryopreservation is likely to be an artifact of cold shock, which was observed in S. crassicaudata and had not previously been described in any other marsupial. This susceptibility to low temperature can be overcome by slow cooling spermatozoa to 0 ºC at 0.5 ºC minute -1 with up to 20% egg yolk, and it is likely that this finding will result in successful sperm cryopreservation in the near future. Freeze drying spermatozoa represents an additional strategy for long term sperm preservation and freeze dried S. crassicaudata spermatozoa had normal morphology and nuclear integrity. In this study preserved dasyurid spermatozoa were immotile and non-viable but had no nuclear damage, suggesting that fertilisation may be achieved with intracytoplasmic sperm injection (ICSI). As ICSI requires a large number of mature oocytes to be collected, a reliable timed ovarian stimulation protocol was established in S. crassicaudata. This protocol enabled the collection of up to 28 oocytes which were either mature, or able to be cultured to the first polar body stage within 48 hours. Despite the success of induced ovulation, methods for preservation of the female gamete are essential to genome resource banking. This study also described a protocol for the enzymatic dissociation of dasyurid ovarian tissue allowing collection of high quality individual preantral follicles. The oocytes inside these follicles were able to be vitrified without any loss of viability and short term in vitro culture of immature follicles repaired the small amount of vitrification-induced damage to the surrounding granulosa cells. This collection of studies describes progress in genome resource banking for spermatozoa and oocytes from dasyurids and the development of protocols allowing the collection of a large number of oocytes for use in fertilisation experiments. These advances provide a solid and comprehensive framework for continuing the study of dasyurid ART which is timely due to the urgent need for genome resource banking in several threatened dasyurid marsupials.

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.