This study aims to elucidate the process and mechanism of sperm penetration through eggs of Penaeus monodon. Sperm penetration of the vitelline envelopes (VEs) of P. monodon eggs were observed with the scanning electron microscope. The characteristics of sperm proteases in the sperm extracts from seminal receptacles of females were analysized. In P. monodon, mating and sperm transfer to the thelycum of female occur soon after maturity moult. Females store the sperm in the seminal receptacles. At spawning, they release stored sperm and eggs simultaneously into the water column. The outermost investment of a newly spawned egg is the VE. Sperm bind to the VE via the tip of their anterior spike. They rapidly undergo the acrosome reaction, which composes of depolymerization of the spike and exocytosis of the acrosome vesicle, pass through the VE and become bound to the egg oolemma. The isolated sperm suspended in artificial seawater were disrupted by sonication on ice. The supernatants after microcentrifuged were collected as sperm extracts. Sperm extracts were analyzed by gelatin SDS-PAGE. Sperm extract from sperm isolated from seminal receptacles of females showed clear bands of protease activity, whereas sperm extract from vas deferens and spermatophore of males did not. This results indicated that sperm of P. monodon do proceed capacitation in the seminal receptacles, and obtain sperm protease activity after capacitation. Using fluorescent peptidyl-MCA as sperm protease substrates, high trypsin-like and aminopeptidase-like activities were observed in sperm extracts. The sperm protease activity was inhibited by trypsin inhibitors aprotinin, p-aminobenzamidine (PAB), soybean trypsin inhibitor (SBTI), N-£\-p-tosyl-L-lysinechloromethyl ketone (TLCK); but was not inhibited by chymotrypsin inhibitor N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and metalloprotease inhibitor 1,10-phenanthroline (1,10-P). The results indicated that sperm undergo the acrosome reaction and release sperm proteases including trypsin-like protease, which has been implicated in facilitating sperm passage through vitelline envelope. Sperm proteases were highly active in the weak base environment, exhibiting maximum activity at pH 8.0. The protease activities were enhanced by addition of calcium chloride and magnesium chloride in the incubation medium.

Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep[TM], or Cushion Fluid[TM]. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 149-174).

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Neutrophils ordinarily infiltrate the female reproductive tract subsequent to mating or artificial insemination, resulting in reduced fertility. Recently, it was demonstrated that equine neutrophil extracellular traps (NETs) entangled sperm in these DNA-rich structures, interfering with their normal transport through the female reproductive tract. Seminal plasma (SP) or proteinaceous extracts from SP inhibited sperm-neutrophil binding and specifically degraded sperm-activated NETs, without suppressing bactericidal activity of neutrophils. Fertility-associated antigen (FAA), a 31 kDa naturally occurring heparin-binding protein (HBP) produced by the accessory sex glands, has been shown to bind to sperm and potentiate heparin-induced capacitation. FAA shares 87% identity with DNase I-like family members, and contains two internal DNase-I-like peptide motifs. The purpose of this study was to determine if a recombinant form of FAA displayed capacitating effects associated with the native protein and to determine whether rFAA displayed DNase activity similar to SP or SP protein extracts to inhibit sperm-neutrophil binding.

Sperm senescence, a decline in sperm quality caused by male ageing and by sperm ageing before or after copulation, may have fitness costs manifested as infertility or lowered genetic quality of offspring. This thesis tested the distinct evolutionary roles of sperm senescence using a laboratory-adapted population of Drosophila melanogaster. We developed a practical approach to avoid confounding male age with sperm age by standardizing pre-copulatory sperm age and mating history in young and old male age groups. Applying this approach, we documented sperm senescence in D. melanogaster and discussed its potential evolutionary importance. First, ageing males declined in fitness as evidenced by the reduction in fertilization potential of their ejaculates but not by decreased offspring fitness (the ability that a fly can survive to adulthood, successfully mate and produce viable offspring). This suggests a decline in the quality or quantity of seminal fluid or spermatozoa, with no decline in the genetic quality of sperm that actually fertilized ova. Second, post-copulatory sperm senescence has significant negative impacts on offspring fitness, indicating degraded genetic integrity of the spermatozoa stored in females. In both cases, male ageing and sperm ageing had similar fitness impact on male and female offspring, different from what has been suggested by previous work. In addition, We demonstrated that female fecundity, fertility, and length of the fertile period after a single mating were positively associated with the concentration of yeast in their food, and were negatively associated with the duration of yeast restriction in their diet, which suggested that sperm storage is affected by the nutritional status of the females. By revealing the significance of sperm senescence on male and female fertilization success and the fitness of the next generation, this thesis sheds light on a number of evolutionary and applied issues, and provokes new questions for future research on sperm senescence. / Thesis (Ph.D, Biology) -- Queen's University, 2014-03-07 10:38:12.879

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.

An unusually high level of inter- and intraspecific variability in spermatozoa has been well documented. However, recent evidence indicates that the level of variation within spermatozoa differs markedly across taxa. In particular, it appears that the variability in spermatozoa tends to decrease across species as the risk of sperm competition increases. In this thesis, I present a model that explains how variability in spermatozoa may arise due to errors made during the sperm production process. In doing so, I also provide an explanation for why variability in sperm traits tends to decrease as the level of sperm competition experienced by males of a given species increases. The model presented in this study provides a novel perspective on spermatozoa and their production. While many sperm traits are thought to be selected upon, I suggest that variability in spermatozoa may also be the result of evolutionary forces such as sperm competition. Variability in spermatozoa, then, can be adaptive and can represent an optimal reproductive strategy. / Thesis (Master, Biology) -- Queen's University, 2009-09-25 21:53:23.172