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. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Fundamentals of forensic deoxyribonucleic acid (DNA) typing for sexual assault samples require the successful application of a differential extraction. Gynecological swabs containing vaginal epithelial cells and sperm cells are commonly encountered in forensic casework. A priority in sexual assault casework is the identity of the male contributor and in order to identify the male contributor, separation of the vaginal epithelial cells and sperm cells must be achieved. Two considerations when separating the different cell-types from a substrate are cellular elution and purity of the DNA fractions. Maximizing DNA yield is directly proportional to the number of cells eluted off of the swab and the extraction method utilized. The trypsin-ZyGEM extraction method has shown results of increased DNA recovery on liquid mixture samples compared with the standard differential extraction, which uses proteinase K and dithiothreitol (DTT). The trypsin-ZyGEM differential extraction protocol calls for the use of proteases EA1, incorporated in the forensicGEM extraction kit, and trypsin, a serine proteinase that has been discovered to effectively digest the DNA bound protamines in sperm cells. The trypsin-ZyGEM protocol follows a similar preferential lysis procedure to the standard differential extraction; however, everything is incorporated into one tube, therefore, minimizing loss of DNA from transfer techniques in the trypsin-ZyGEM protocol. Initially, epithelial cells are lysed using the forensicGEM enzyme and removed from solution after centrifugation. Samples are subsequently treated with trypsin, digesting sperm cells. The resultant solution contains the sperm cell DNA and other cell components. The standard differential extraction commonly uses a Qiagen silica membrane column to purify DNA away from cellular proteins and other contaminants, ensuring successful downstream DNA testing with the polymerase chain reaction (PCR). However, treatment with the trypsin-ZyGEM method is followed by a second incubation with forensicGEM after sperm cells are lysed with trypsin. Extracted samples can be quantified and followed through to DNA typing. Expanding previous studies and optimizing conditions of the dual enzyme approach were explored in this study for semen samples on cotton swabs. When comparing DNA yields of extracted samples, tris-ethylenediaminetetraacetic acid (TE) buffer recovered more cells from the cotton swabs than phosphate buffered saline (PBS) buffer and Buffer ATL, used in the Qiagen QIAamp® DNA Investigator Kit. Relative to DNA recovery of the standard Qiagen differential protocol, the trypsin-ZyGEM method on the cotton swab samples appeared subpar and could be attributed to ineffective cellular elution. Additionally, carryover DNA into the non-sperm cell fraction was exhibited. Procedures with Accumax, a cell detachment solution, were implemented in an attempt to elute more cells. The resulting DNA yields were significantly lower in the presence of Accumax. Contrastingly, incorporating trypsin directly into the elution TE buffer exhibited significant increases in DNA recovery. The direct lysis of sperm cells on the swabs, rather than the two-phase method of eluting the cells from the swab and subsequently extracting the DNA, proved to be much more effective. The swab remains from the elution and subsequent trypsin-ZyGEM method were re-extracted using the direct lysis method. Whilst comparing DNA yields, it was discovered that approximately 90% of the cellular DNA was retained on the swab after the two-phase extraction method was performed. Experiments utilizing direct lysis with the trypsin-ZyGEM method, the standard Qiagen differential protocol, and the trypsin lysis followed by Qiagen extraction were performed on swab samples comprised of different semen concentrations resulting DNA yields and short tandem repeat (STR) DNA profiles were compared. Results obtained indicated that the trypsin-ZyGEM method provided substantially larger DNA recovery yields and provided full STR profiles to a target mass of 0.0625 ng and average peak height ratios above 60%. Successful implementation of this extraction procedure will require further studies with the addition of epithelial cells on the swab samples to mimic vaginal swab mixture samples. These samples will help to determine purity of the DNA fractions and effects of epithelial cells on the trypsin-ZyGEM protocol. / 2017-11-03T00:00:00Z

The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...

The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...

To identify factors important to the efficiency of artificial insemination in cattle, four studies were conducted. In the first study, the addition of cream to the inseminate was used in an attempt to increase accessory sperm number. On d 6 after insemination, 60 embryos were evaluated. The addition of cream to the inseminate had no effect on accessory sperm number. In the second study, cryopreserved semen of a marked bull (spermatozoa exhibiting a semi-flattened anterior head) was matched with semen from an unmarked bull (conventional sperm head shape) to determine competitively the effect of a deep uterine insemination on accessory sperm number. Forty embryos were recovered 6 d after insemination and the ratio of accessory sperm observed was different: 62:38 for unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 for unmarked semen in the uterine horn and marked semen in the uterine body (P < .05). In the third study, superovulated cows were utilized to determine the effect of artificial insemination time on fertilization status and accessory sperm number. Cows were inseminated once at 0 h (n=10), 12 h (n=10), or 24 h (n=10) after the first standing event. On d 6 after insemination, 529 embryos(ova) were recovered. Fertilization rates were 29% (0 h); 60% (12 h); and 81% (24 h)(P < .01). Percentages of embryos with accessory sperm were: 5 (0 h); 8 (12 h); and 41(24 h) (P < .01). In the fourth study, three experiments utilizing superovulated cows were conducted to provide a basis for distinguishing unfertilized ova from very early embryonic death. In Exp. 1, recovered d 6 unfertilized ova were classified morphologically as either: 1) typical, 2) satellite, or 3) fragmented. In Exp. 2, recovered d 6 unfertilized ova from the third study were classified morphologically, and typical ova were fixed. In Exp. 3, ultrastructural features of preovulatory, tubal-stage, and typical d 6 unfertilized ova were investigated. Preovulatory ova revealed normal ultrastructure; tubal-stage ova exhibited evidence of degeneration; typical d 6 ova were degenerated and contained no discernable organelles. The first three studies support the use of accessory sperm evaluation as an alternative measure of fertility. The final study provides a basis from which future embryologists may distinguish fertilization failure from very early embryonic death. / Ph. D.

No description available.

After ejaculation, mammalian spermatozoa must undergo a series of complex and poorly understood cellular events known as “capacitation” in order to be able to fertilize an oocyte. Among these, biochemical changes such as an increase in tyrosine phosphorylation of some sperm proteins have been correlated with the sperm capacity to fertilize an egg and found to be regulated by a cAMP dependent pathway. The influx of ions such as Ca2+ and HCO3- induce the activation of a soluble adenylyl cyclase (SACY) increasing the cAMP levels within the cell that leads to the activation of a protein kinase A (PKA), and a subsequent increase in protein tyrosine phosphorylation. This modification in sperm proteins seems to be essential for induction of a change in the motility pattern known as hyperactivation that enables the sperm to penetrate the zona pellucida of the oocyte and initiate fertilization. Since PKA is a serine/threonine kinase, it is not clear how it mediates protein tyrosine phosphorylation during sperm capacitation. Based on the finding that in somatic cells PKA activates c-Src, it has been proposed that the Src family of protein kinases (SFK) are the intermediate players involved in tyrosine phosphorylation induced by PKA activity. In order to better understand the molecular mechanisms involved in stallion sperm capacitation, the objectives of our study were: (1) To analyze PKA activity during stallion sperm capacitation (2) To evaluate the involvement of the Src family of protein kinases (SFK) on stallion sperm phosphorylation events associated with capacitation. Standard In Vitro Fertilization (IVF) has not been reproducibly successful in the horse. Recent data indicate that good fertilization rates may be achieved after treatment of sperm with procaine to induce hyperactivation. Our objectives were also to determine if drugs used in other species as well as procaine induce hyperactivation in stallion sperm and to evaluate biochemical changes such as protein tyrosine phophorylation.

The Testis Specific Serine Kinases (Tssks) are a family of proteins that show testis and sperm-specific expression. Members of this family are most conserved among mammals, however there are homologs in vertebrates like birds and amphibians, chordates, and other invertebrates like insects and cnidarians. This specific expression suggests that these kinases are highly regulated. Analysis of murine and human Tssk1, Tssk2, and Tssk6 sequences show that these genes are comprised of one exon each, suggesting they are retrotransposons. The expression of these genes shows their importance, since many retrotransposons are silenced due to the foreign nature of the DNA, and knock-out mouse models have shown that these kinases are required for fertility. Understanding the properties of these kinases not only expands our scientific knowledge, but also lends itself to understanding fertility issues in men as well as being a contraceptive target. We looked at an epigenetic regulation factor, DNA methylation at CpG dinucleotides, to see if this caused the testis-specific gene expression we saw. Tssk2 and preliminary results from Tssk1 showed that there is no differential methylation at CpG dinucleotides or between tissues. Preliminary results for Tssk6 did show one site that may be differentially methylated, thus the tissue specific expression. We then started looking further into biochemically characterizing TSSK1 and TSSK2 to determine functionally relevant sites and new substrates. Understanding how these kinases function in sperm is relevant in our understanding in the fertility field and poses new targets for developing contraceptives.

Prostaglandin F2a (PGF2a), oxytocin and gonadotropin releasing hormone (GnRH) have been used in bulls, rams, boars, stallions or rodents to increase sperm numbers in the ejaculate. Improving sperm quantity in the canine ejaculate would benefit all assisted reproductive techniques used in this species. The purpose of the present study was to evaluate the effects of PGF2a, oxytocin and GnRH on canine ejaculate characteristics. Eight, mature, medium size (25-30 kg), mixed breed dogs were randomly assigned to one of four treatment groups (N=2 dogs each); each group received one treatment per week for four weeks. Treatments were assigned based on a Latin Square design. A two-week training period was used to acclimate the dogs to manual semen collection. Treatments were 0.1 mg/kg PGF2a 15 minutes prior to collection, 2.5 units/dog oxytocin 10 minutes prior to collection, 50 mg/dog GnRH 60 minutes prior to collection, or 1.0 ml of saline 30 minutes prior to collection. An evaluator that was blinded to treatment analyzed ejaculate characteristics. Samples were evaluated for semen volume, concentration of spermatozoa per milliliter, motility, morphology, total sperm number and total morphologically normal motile sperm number (TNMS). In addition, a subjective ease of collection score was assigned following each collection (Scale 1-9, 1 being easiest to manually ejaculate). Semen concentration, motility and morphology were not different between treatments. Semen volume was greater for dogs treated with PGF2a or oxytocin compared to saline. Total sperm number and TNMS were greater when dogs were treated with PGF2a compared to oxytocin, GnRH and saline (p<0.05). The subjective ease of collection score was lower for dogs receiving PGF2a compared to GnRH or saline (p<0.05). In summary, administration of PGF2a or oxytocin prior to semen collection increased semen volume and PGF2a increased total sperm number in the ejaculate of the dog. It did not appear that treatment with GnRH had an effect on semen parameters evaluated in this study. / Master of Science

No description available.