Masters Degrees (Medical Physiology)

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Browsing Masters Degrees (Medical Physiology) by browse.metadata.advisor "Du Plessis, Stefan"

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    Chronic stress and semen parameters
    (Stellenbosch : Stellenbosch University, 2020-03) Van der Merwe, Esmari; Du Plessis, Stefan; Basson, E.; Stellenbosch University. Faculty of Health and Medical Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    ENGLISH ABSTRACT: It has been well documented that stress has adverse effects on the body and can lead to various health issues. Stress has been investigated as a cause for unexplained infertility in both men and women. Semen quality is a key indicator of male reproductive health. Numerous studies have been done on the effect of stress on semen parameters and an association between chronic psychological stress and poor semen parameters have been reported. Managing psychological stress can help to improve the health of an individual. In order to address the problem it is therefore important to determine if an individual experience high levels of stress. This can be established through psychological questionnaires and various biomarkers, such as the screening test for time urgency perfectionism (TUP) and alpha-amylase in saliva. In general, more or less 84% of couples are estimated to conceive naturally within a year. The remaining 16% of couples are affected by infertility. Within this group, it is estimated that male reproductive factors are the sole cause of one-third of cases and a contributing factor in another 20% of cases. Management of chronic stress in female patients has shown improved IVF rate of 67% or higher. However, as of yet no study has been performed on males to correlate the levels of TUP-stress, alpha-amylase to semen parameters as well as other seminal stress markers such as DNA fragmentation and oxidative stress (ROS). This study compared TUP-categories (Low, Moderate, High) with respect to semen parameters, alpha-amylase levels, age and BMI and investigated if increased alpha-amylase levels correlate with semen parameters, age and BMI. The experiments were performed at Medfem Fertility Clinic in Bryanston Johannesburg and the Division of Medical Physiology in the Department of Biomedical Sciences at Stellenbosch University. A total of 62 male patients of Medfem Fertility Clinic adhering to the basic requirements enrolled in the study. Results showed no significant difference between age, semen parameters and alpha-amylase between TUP categories. Men in the High TUP category had a significant higher BMI compared to those in the Low and Moderate categories. No significant correlation was found between alpha-amylase, age, BMI and semen parameters. This study was unsuccessful in proving a significant relationship between the TUP categories, age and semen parameters. The High TUP category did show a significantly higher BMI compared to the Low and Moderate TUP groups. This finding confirms that there is a link between psychological stress and elevated BMI. Although there was no significant difference between the TUP categories with regards to sORP values, the Moderate and High categories were both higher than the normal value for sORP in semen. This implies that chronic stress leads to elevated levels of oxidative stress in semen. No relationship was found between TUP categories and alpha-amylase levels. Although both are used to detect chronic stress, the TUP questionnaire is used to detect personality types who are prone to chronic stress, whilst salivary alpha-amylase is a biomarker for chronic stress and functions in a completely different way. It is possible that whilst both can be used to detect chronic stress it is not advised to attempt to establish a relationship between the two as the mechanisms of both are very different.
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    A comparison of motility and head morphology of sperm using different semen processing methods and three different staining techniques
    (Stellenbosch : University of Stellenbosch, 2010-12) McAlister, Debra Ann; Du Plessis, Stefan; Van der Horst, Gerhard; Maree, Liana; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
    ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility.
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    The identification of the Dopamine Transporter (DAT) and the effects of dopamine on human sperm parameters
    (Stellenbosch : Stellenbosch University, 2021-03) Ferguson, Lisa Marie; Du Plessis, Stefan; Qulu, Lihle; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Dept. of Biomedical Sciences: Medical Physiology.
    Thesis (MMed)--Stellenbosch University, 2021.
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    Investigating the effects of nicotine on the male reproductive system
    (Stellenbosch : Stellenbosch University, 2013-12) Maartens, Pieter Johann; Du Plessis, Stefan; Windvogel, Shantal; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Division Medical Physiology.
    ENGLISH ABSTRACT: Much has been documented about the detrimental effects of adverse lifestyle factor exposure on the body. Exposure to factors, such as cigarette smoke, have proved to not only be a burden on global health and economy, but have also led to growing concerns about effects on systemic functions such as reproduction. The aim of the present study was to determine the effects of in utero and in vitro nicotine exposure on spermatozoal function and the antioxidant enzyme activity and lipid peroxidation (LPO) status of the male reproductive system. A better understanding of this process is necessary to combat the respective burdens of smoking and male infertility and for the prospective development of treatment strategies. Two experimental models were employed: Wistar rats were exposed to nicotine in utero while human and rat spermatozoa were exposed to nicotine in vitro. In utero studies were achieved by selecting healthy pregnant rats and treating them with 1 mg/kg-bodyweight/day nicotine or 1 ml/kg-bodyweight/day 0.85% physiologic saline throughout gestation and lactation. Male rat pups were selected and sacrificed at each of the following age groups (n=6): 42 days, 84 days and 168 days old. The pups were only exposed to the treatment/saline via placental uptake or lactation. Biochemical analyses of the tissue comprised of measurement of LPO and antioxidant enzyme activity. Results indicated a significant association of maternal nicotine exposure to decreased levels of primary antioxidant enzymes in rat testes. Of particular note was the observation that the treatment group, of which each of the respective antioxidant enzyme levels were significantly less than the control group, was the oldest (d168) rat group. In vitro studies were achieved by collecting sperm samples from healthy human donors (n=12), healthy rats (n=6) and obese rats (n=6). Samples were washed and exposed to different concentrations of high levels of nicotine (Control, 0.1mM, 1mM, 5mM, 10mM) in vitro. Semen parameters such as motility, viability and acrosome reaction were monitored at different time points (30min, 60min, 120min, 180min). Results revealed increasing in vitro nicotine concentrations were associated with decreased viability and acrosomal status of human spermatozoa and decreased progressive motility and viability of rat spermatozoa. Obesity was also associated with decreases in progressive motility and viability of rat spermatozoa. These results indicate that the acute in vitro exposure of spermatozoa to high levels of nicotine could adversely affect semen quality and may be an additive factor to the impediment of male fertility. In utero results reveal maternal nicotine exposure adversely affects male fertility in later life and seems to elicit more detrimental effects on the reproductive system than that of direct nicotine exposure to spermatozoa. Obesity also inhibits parameters of male fertility and these effects are exacerbated by nicotine exposure. The authors believe these adverse effects on the reproductive system to be related to an increased activation of leukocytes, excess production in reactive oxygen species (ROS) and consequent onset of oxidative stress (OS). Nevertheless this study agrees with other studies that nicotine exposure may be an additive factor to the impediment of male fertility.
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    A mechanistic study on the mu-opioid receptor in human spermatozoa
    (Stellenbosch : Stellenbosch University, 2021-12) Van Niekerk, Nadia; Du Plessis, Stefan; Marais, Erna; Bongekile, Skosana; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    Background: Infertility remains a complex and often unexplained phenomenon. The effect of opioids on male reproductive parameters is best understood by the suppression of the hypothalamic- pituitary-gonadal (HPG) axis. However, since the discovery of all three opioid receptor types on human spermatozoa, it has been hypothesized that the opioid system plays a regulatory role on sperm functional parameters. Literature regarding the possible mechanisms involved in these regulatory processes remain contradictory and scarce. Therefore, this study aimed to clarify the role of the mu- opioid receptor in human spermatozoa. Methods: 30 semen samples were collected from healthy, human donors of reproductive age. Each sample was divided into 4 equal groups and treated accordingly. The study consisted of a control group, a codeine (opioid agonist) treated group, a naloxone (opioid antagonist) treated group, and a combination (codeine and naloxone) treated group. After a 3 hour incubation period, sperm functional parameters were assessed: Total motility, progressive motility and sperm kinematics were assessed using the Computer-Aided Sperm Analysis (CASA) system. Furthermore, viability, acrosome reaction and DNA fragmentation were investigated. By performing western blot analyses the total expression of A-kinase anchoring protein 3 (AKAP3) and protein kinase C𝛼 (PKC𝛼) were evaluated. Results: No differences in sperm functional parameters were detected. It was observed that codeine influenced the acrosome reaction and DNA fragmentation negatively, and that naloxone seemed to alleviate these effects, but the results were not significant. Interestingly, the study found that naloxone significantly decreased the total protein expression of AKAP3 (1 ± 0.016 vs. 0.766 ± 0.025). Conclusion: In order to clarify the role of opioid receptors on spermatozoa membranes, more research is necessary. The study results suggest that opioids may utilize sperm-specific signalling mechanisms downstream of the opioid receptors located on human spermatozoa. Understanding these mechanisms could prove to be invaluable in enhancing our understanding of infertility.
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    Proteomic analysis of human sperm proteins in relation to sperm motility, morphology and energy metabolism
    (Stellenbosch : University of Stellenbosch, 2010-12) Rapuling, Llewelen; Du Plessis, Stefan; Hattingh, Suzel; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
    ENGLISH ABSTRACT: Male infertility is often associated with impaired sperm motility and morphology (asthenoteratozoospermia) for which there is no specific therapeutic treatment. It has come to light that the modification and expression of human sperm proteins play a crucial role in sperm function. In the present study, we present proteomic data of human spermatozoa in the context of sperm dysfunction. Novel techniques have been used to successfully isolate and identify differences in protein expression on a cellular level associated with asthenoteratozoospermia. In the first part of the study, differences in protein expression within the total sperm proteome were investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=23) and separated into mature and immature sperm populations by 3-layer Percoll gradient centrifugation. Cells were washed and motility and morphology were measured by computer assisted sperm analysis (CASA). For the proteomic investigation cells were lysed and proteins separated by means of two-dimensional gel electrophoresis (2D electrophoresis). PD-Quest was used to identify the differentially expressed proteins. The protein spots of interest were excised and subjected to in-gel digestion. Peptides were separated by High Pressure Liquid Chromatography (HPLC) analysis and amino acid sequences determined by mass spectrophotometry. Proteins were identified by Mascot, using the Swiss Prot database. The results show that the motility (immature; 26.1±1.75% total motile cells vs. mature; 60.93±3.24% total motile cells; p<0.001) and morphology parameters (immature; 64.1±2.75% normal head morphology vs. mature; 87.63±3.24% normal head morphology; p<0.001) of the two populations differed significantly. After 2D electrophoresis, 16 differentially expressed protein spots were identified within the total sperm proteome between the immature and mature sperm populations. 56% of the differentially expressed proteins were more abundant in the immature sperm population compared to the mature sperm population. Functions have been ascribed to these proteins of which only four proteins, namely Tubulin -3C/D chain, Tubulin -2C chain, Outer dense fibre protein 2 and A-Kinase anchoring protein 4 precursor, were directly related to sperm motility and morphology. In the second part of the study the expression of nuclear proteins in human spermatozoa was investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=156) and further separated from the seminal plasma by PureSperm® gradient centrifugation. The immature and mature sperm populations were retrieved and used during further analysis. For the proteomic analysis of nuclear proteins, cells were fractionated into four different subcellular protein fractions, instead of analyzing the whole sperm proteome. The results show that the motility (immature; 32.33±0.51% total motile cells vs. mature; 88.67±0.85% total motile cells; p<0.0001) and morphology parameters (immature; 13.51±0.87% normal head morphology vs. mature; 20.89±1.20% normal head morphology; p<0.0001) of the two populations differ significantly. After 2D electrophoresis, 21 differentially expressed nuclear proteins were identified between the immature and mature sperm populations. 95% of the differentially expressed nuclear proteins were less abundant in the immature population compared to the mature population. Only one nuclear protein namely 78kDa Glucose regulated protein was more abundant in the immature population compared to the mature population. Functions ascribed to these individual proteins were directly related to sperm motility, morphology and energy metabolism. In conclusion,In conclusion, in the current study novel techniques have been employed to investigate protein differences between immature and mature sperm populations. From these results it is evident that protein expression in the total sperm proteome and nuclear protein fraction is significantly different and incomplete in the immature population, compared to mature population. Based on these findings, it is recommended that further studies should be done on human spermatozoa to validate the role of the individual proteins in sperm function. Proteomics is an ideal tool to identify idiopathic causes of male infertility, as it can help to identify novel receptors (and signal transduction pathways) that can be used in the screening of drugs to alleviate sperm dysfunction.