published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

by Will W.M. Lee. / Thesis (M.Phil.) - Chinese University of Hong Kong. / Bibliography: leaves 101-110. / Chapter CHAPTER I --- GENERAL INTRODUCTION / Chapter A --- Spermatogenesis --- p.1 / Chapter B --- Sperm Maturation --- p.2 / Chapter C --- Ejaculation --- p.3 / Chapter D --- Sperms in Fertilization --- p.3 / Chapter E --- Aim of Spermatozoal Motility Studies --- p.7 / Chapter CHAPTER II --- AN ASSAY TO MEASURE THE SPERMATOZOAL PROGRESSIVE MOTION / INTRODUCTION --- p.8 / MATERIALS AND METHODS --- p.15 / Chapter A --- Sample Collection --- p.15 / Chapter B --- Cell Wash --- p.17 / Chapter C --- Media --- p.18 / Chapter D --- Motility Assay Chamber --- p.18 / RESULTS AND DISCUSSIONS --- p.22 / Chapter A --- Sperm Entry --- p.22 / Chapter B --- Sperm Motility --- p.32 / Chapter CHAPTER III --- EFFECT OF VARIOUS GROUPS OF CHEMICALS ON THE MOTILITY OF SPERMATOZOA / INTRODUCTION --- p.48 / MATERIALS AND METHODS --- p.55 / RESULTS AND DISCUSSIONS --- p.57 / Chapter A --- Energy Source --- p.57 / Chapter B --- "Phosphodiesterase Inhibitors and Cyclic 3',5'-Adenosine Monophosphate (cAMP)" --- p.59 / Chapter C --- p-Nitrophenyl Compounds --- p.64 / Chapter D --- Motility of Spermatozoa from Addicted Rat and Effect of Morphine on tozoa In VitroSperma- --- p.68 / Chapter E --- Metallic Ions and EDTA --- p.68 / Chapter CHAPTER IV --- EFFECT OF SEMINAL PLASMA ON SPERM MOTILITY --- p.77 / INTRODUCTION / MATERIALS AND METHODS --- p.79 / RESULTS --- p.82 / DISCUSSIONS --- p.98 / REFERENCES --- p.101 / Chapter APPENDIX I --- Spermatozoa Repellent as a Contraceptive --- p.111 / Chapter APPENDIX II --- Effect of p -Nitrophenylglycerol on Motility of Rat Epididymal Spermatozoa --- p.117

Sperm mobility denotes the net movement of a sperm population. Previous work implicated mitochondrial function as a basis underlying phenotypic variation in this quantitative trait. Our objective was to determine if mitochondrial function was indeed critical to expression of phenotype. Phenotype was assigned to roosters within a random bred population (n=242). A representative subpopulation (n=40) was used to correlate sperm mobility with oxygen consumption (r=0.83). In contrast, sperm mobility was independent of mitochondrial helix length in a sample of males (n=7) representing the range of phenotype observed within the population. Thus, mitochondrial function rather than number appeared to be critical to expression of phenotype. This hypothesis was tested by ultrastructural analysis of sperm midpieces. Males from the lower and upper tails of the distribution were characterized with high and low proportions of sperm containing aberrant mitochondria in 47 and 4% of the cells respectively. When sperm from average males were allowed to segregate into immobile and mobile subpopulations, 40% of immobile sperm contained aberrant mitochondria. In contrast, only 9% of sperm from the same males contained aberrant mitochondria in non-segregated populations. In conclusion, the mitochoridrion is an organelle that may account for phenotypic differences in sperm mobility. / Graduation date: 2002

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Call number: LD2668 .T4 1964 C288 / Master of Science

Thesis (MMed)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In all Assisted Reproductive Technologies (ART) procedures the semen sample is handled, processed, prepared and manipulated before use in the fertilization process. During these incubation times, the sperm cells are exposed to factors that may inflict damage to the sperm structure and DNA integrity, impair its functional abilities and subsequently lead to fertilization failure and poor ART outcome. Two of the very basic, but important factors that may have an impact on the sperm quality are time and temperature exposure. The primary objective of this study was to prospectively determine the effect of different incubation times and temperatures on motility and the DNA profile of the spermatozoa. Non-processed (n=36) and processed (n=33) semen samples were incubated for different time intervals (before: 20, 40, 60 minutes; after: 30, 60, 90 minutes) and at different temperatures (room temperature [RT] and 37°C). After incubation, sperm parameters were assessed, the CMA3 assay was applied to determine chromatin maturity and compaction and the TUNEL assay to assess the level of DNA fragmentation. The results showed that in the non-processed group, incubation led to a time-dependent, significant decline in the motility. The highest motility was seen at 20 minutes (37°C) and motility declined in a time-dependent manner. Incubation time and temperature did not affect the CMA3 and TUNEL values. Incubation of the processed sample led to a significant time-dependent decrease in the motility; 90 minutes (RT) had the lowest motility. The CMA3 and TUNEL values between the different incubation groups did not differ significantly. The secondary objective was to retrospectively investigate the effect of sperm incubation time after preparation on ART outcome. A total of 901 patient ART cycles (January 2010- December 2012) were included. Fertilization rates, embryo quality and pregnancy rates were examined. The results showed that the sperm incubation time before insemination between in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) differed significantly and the incubation time had a significant negative effect on the fertilization rates in IVF, but not in ICSI. Longer incubation times led to an unexpected significant improvement in the quality of day 2 embryos and were significantly associated with pregnancy failure in IVF and ICSI. These combined findings suggest that non-processed semen samples can be incubated at RT or 37°C, but for no longer than 40 minutes and, for IVF, processed samples should not be incubated for longer than 60 minutes at RT or 37°C. The ICSI sample should not be incubated for more than 60 minutes although longer incubation times do not seem to influence the results for IVF. It can therefore also be concluded that sperm incubation time before insemination should be closely monitored, especially in IVF cycles. / AFRIKAANSE OPSOMMING: In kunsmatige voortplantingstegnieke (ART) word die semen-monster geprosesseer, voorberei en gemanipuleer voordat dit vir die bevrugtingsproses gebruik word. Terwyl die monster geïnkubeer word, word die spermselle blootgestel aan verskeie faktore wat die struktuur van die sperm, die DNS integriteit en die sperm se funksionele vermoë negatief kan beïnvloed. Dit kan lei tot swak bevrugting, embriokwaliteit en swangerskapsyfers. Twee basiese, maar belangrike, faktore wat die spermkwaliteit negatief kan beinvloed is die duur van inkubasie en die temperatuur waarby die spermselle geïnkubeer word. Die primêre doel van die huidige studie was om prospektief te ondersoek wat die effek van verskillende inkubasietye en temperature op die motiliteit en DNA profiel van die sperm het. Monsters is voor en na spermvoorbereiding vir verskillende tydsintervalle (voor: 20, 40, 60 minute; na: 30, 60, 90 minute) en verskillende temperature (kamertemperatuur [KT] en 37°C) geïnkubeer. Na elke inkubasie is ’n spermanalise, ’n CMA3- en ’n TUNEL toets gedoen. Die CMA3 toets bepaal die chromatienmaturiteit en -kompaksie en die TUNEL toets vir die vlak van DNS fragmentasie. Die resultate het getoon dat daar in die voor voorbereiding groep ’n beduidende verskil in motiliteit tussen die verskillende inkubasiegroepe was. Die hoogste motiliteit is in die 20 minute/37°C groep gevind. Die motiliteit het oor tyd afgeneem. Die tyd en temperatuur van inkubasie het nie ’n beduidende effek op die CMA3 en TUNEL uitslae gehad nie. Inkubasie nadat die semen voorberei was het weereens tot ’n beduidende verskil in motilieit tussen die groepe gelei. Die laagste motiliteit is waargeneem by 90 minute/KT. Geen beduidende verskil is tussen die inkubasiegroepe vir CMA3 en TUNEL gevind nie. Die sekondêre doel van die studie was om retrospektief te ondersoek wat die effek van sperminkubasietyd na spermvoorbereiding op die bevrugting, embriokwaliteit en swangerskapsyfers is. 901 pasiëntsiklusse is in die studie ingesluit (Januarie 2010 tot Desember 2012). Die resultate het aangedui dat die inkubasietye van die intrasitoplasmatiese inspuiting (ICSI) en in vitro bevrugting (IVB) beduidend van mekaar verskil het. Langer inkubasietye het ’n beduidende negatiewe effek op die bevrugtinguitslae van IVB siklusse gehad, maar geen effek op ICSI siklusse gehad nie. Langer inkubasietye het ook tot ’n onverwagte verhoging in die kwaliteit van dag 2 embrios gelei en was verder beduidend geassosieer met negatiewe swangerskapuitkoms. Hierdie gesamentlike bevindinge dui aan dat semenmonsters voor voorbereiding by KT of 37°C geïnkubeer kan word, maar nie vir langer as 40 minute nie. Na semenvoorbereiding, behoort die IVB semenmonster vir nie langer as 60 minute voor inseminasie geïnkubeer te word nie (KT of 37°C). Die ICSI semenmonster moet verkieslik binne 60 minute na voorbereiding gebruik word, maar dit wil voorkom asof die tyd hier nie so ’n groot rol speel nie. Daar kan verder afgelei word dat sperminkubasietye voor die gebruik vir inseminasie baie goed gemonitor moet word – veral in IVB siklusse.

published_or_final_version / abstract / Medical Sciences / Master / Master of Medical Sciences

The goal of this research was to examine the role of the novel membrane progestin receptor alpha (mPR[alpha]) in the stimulation of sperm hypermotility by the progestin 17,20[beta],21-trihydroxy-4-pregnen-3-one (20[beta]-S) in two teleosts; the Atlantic croaker (Micropogonias undulatus) and the southern flounder (Platylicthys lethigstomata). In croaker, the expression, localization and hormonal regulation of mPR[alpha] in testis and sperm were investigated, as were the intracellular signaling pathways activated by 20[beta]-S and mPR[alpha] to induce croaker sperm hypermotility. In flounder, stimulation of sperm hypermotility by 20[beta]-S and binding of this steroid to flounder sperm membranes were examined. Finally, expression of mPR[alpha] was investigated in flounder testes and the expression and localization of this receptor in flounder testis and sperm was examined. In croaker sperm, mPR[alpha] was expressed on the plasma membrane and localized to the midpiece. Expression of mPR[alpha] was also shown to be associated with high sperm motility and regulated by gonadotropin. The signaling pathways activated by 20[beta]-S in croaker sperm were shown to involve activation of olfactory G-proteins (Golf). Subsequent activation of membrane adenylyl cyclases was also demonstrated and shown to be necessary for 20[beta]-S-stimulated cAMP production and 20[beta]-S-induction of sperm hypermotility. Furthermore, co-immunoprecipitation studies show mPR[alpha] and Golf physically associate with one another, establishing mPR[alpha] as the mediator of 20[beta]-S actions in croaker sperm. Finally, evidence was obtained for progestin-stimulation of sperm hypermotility and the presence of mPR[alpha] on sperm membranes in another marine teleost species belonging to a different family, the southern flounder. In addition, mPR[alpha] was shown to be expressed on flounder sperm membranes and also localized to the sperm midpiece. Results from the following studies support the hypothesis that mPR[alpha] is the mediator of 20[beta]S-stimulated sperm hypermotility in croaker and is a likely intermediary in southern flounder. Furthermore, these data provide a plausible mechanism by which 20[beta]-S and mPR[alpha] stimulate croaker sperm hypermotility. In addition, these results provide the first evidence of hormonal activation of Golf proteins for any species. Finally, mPR[alpha]-mediated mechanisms to increase sperm motility are suggested to be evolutionarily conserved in teleosts since they also likely exist in a non-sciaenid species, the southern flounder.

No description available.

The aim of this study was to test whether or not the equilibration time of two different cryodiluents influences the post thaw qualities of epididymal African buffalo (Syncerus caffer') sperm. Diluents and equilibration times were compared by assessing the post thaw spermatozoal motility, longevity and the acrosomal integrity. African buffaloes belong to Africa's "Big Five" and are, therefore, popular animals amongst game farmers, hunters and tourists. They are also asymptomatic carriers of foot-and-mouth-disease (FMD) and considered to be a wildlife reservoir for this plague. Other diseases, that are carried and can be transmitted from the African buffalo (Syncerus caffer') to livestock include tuberculosis, brucellosis and theileriosis or corridor disease (CD). Therefore, the transportation of African buffaloes is highly regulated. Disease-free buffalo populations are currently derived from a small genetic 8 pool and are smaller in their trophy size than the free-ranging animals from the diseased areas of the Kruger National Park (KNP) and the Hluhluwe/Umfolozi National Park. Hence there is a special interest in bringing new genetic material into the disease-free populations. Epididymal sperm from 11 mature African buffalo bulls was collected, diluted with two different semen extenders (TriiadylTM [Tris egg yolk extender] and AndroMed® [synthetic extender, i.e. fully defined medium]) and frozen. Pre-freezing equilibration times of 2 and 9 hours were tested. Total and progressive motilities, longevities and acrosomal integrity were measured and compared. Results show that there were no differences in post-thaw sperm quality when equilibration times between 2 and 9 hr were used. The use of the egg yolk containing extender (TriiadlyTM) resulted in higher percentage of post-thaw motilities than the use of the synthetic AndroMed®. Because a high percentage of progressive motile spermatozoa is one of the prerequisites for successful AI it must be concluded that TriladylTM is superior to AndroMed®. As I believe the advantages of higher motility to be bigger than the hygiene risks of a yolk containing extender I conclude that epididymal buffalo sperm should rather be frozen with TriiadylTM than with AndroMed®. / Dissertation (MSc (Production Animal Science))--University of Pretoria, 2003. / Production Animal Studies / unrestricted