Theses - Faculty of Medicine
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Item In vitro differentiation of human male germ cells from spermatogonial stem cells derived from umbilical cord blood(University of Kelaniya, 2017) Dissanayake, D.M.A.B.Rationale: Spermatogenesis is a unique, complex and orchestrated process that requires a period of amplification of spermatogonial stem cells (SSCs proliferation), the reduction, division (meiosis), and the morphological transformation of round spermatids into spermatozoa (spermiogenesis). Objective: Our objective was to recapitulate this process in vitro using umbilical cord blood derived mesenchymal stem cells (UCB-MSCs). Methodology: Blood was collected from the veins of umbilical cords and, mono nuclear cells (MNCs) were separated using the density gradient centrifugation over Ficoll-Hypaque. The separated cells were propagated in monolayer culture system. Cells were characterized using MSCs specific markers, and differentiation potential in to adipogenic and osteogenic lineages. At the second passage cells were induced with 10 flM all trans retinoic acid (ATRA) for two weeks. Stage specific genes expressed or suppressed at pre-meiotic, meiotic and post-meiotic stages were detected using RT-PCR technique. Possibility of arising primitive stages of SSCs were determined by alkaline phosphatase activity (ALP) and GPR ] 25 gene expression. Putative germ cells were cultured with rat Sertoli cells for further four weeks. Chromosome spreading was used to observe meiotic reduction. Development of advanced stages of germ cells were detected using periodic acid Schiff stain and aniline blue stain. Results: The number offibroblastoid colony forming units (CFU-f) was very low in UCB samRles. However, the rate of proliferation was high, and reached 70% confluence within six weeks. Cells were positive for MSCs and pluripotent makers; CD44, CD 73, CD 90, CD 105 and OCT4. OCT4 a stem cell marker, and PLZF an early SSCs marker, were suppressed during the induction period. Expression of other germ cell markers; pre-meiotic (STRAS), meiotic (SCP3) and post-meiotic (ACR, PRMl) were increased. Mild ALP activity and lack ofGPR ]25 expression confirmed the absence of PGCs and early stages of SSCs. Around ] 5% transformed in to haploid cells after induction. Different stages of acrosome formation and replacement of histone from protamine were observed in post meiotic cells. Conclusion: Human cord blood derived MSCs can be differentiated in to male germ cells up to round spermatids stage without genetic manipulations. Further studies are needed to improve the culture techniques to transform spermatids into mature and functional sperm.Item Effects of zinc on human semen quality and sexual behaviour of male rats(University of Kelaniya, 2008) Dissanayake, D.M.A.B.The main aim of this study was to evaluate the effects of serum and seminal plasma zinc levels on semen quality of a subfertile male population. At the same time, effects of zinc on different aspects of male reproduction were studied. The study was carried out as a prospective hospital and laboratory based study. Semen samples from 152 males were analyzed. Seminal plasma and serum levels of zinc, serum hormone levels, seminal plasma fructose and neutral a- glucosidase levels of same males were also measured. Relationship between seminal plasma zinc and semen quality was observed using two markers; zinc concentration and total zinc per ejaculate (Total zinc). Effects of zinc on various functions of spermatozoa were studied in-vitro and, the effects on sexual competence of males were observed using a rat model. Of the 152 semen samples 55 (36 %) were normozoospermic and 97 (64 %) were pathozoospermic. The mean (SD) serum and seminal plasma zinc concentrations of the population were 0.94 (ig/ml (0.36) and 121.87 (ig/ml (69.13) respectively. Seminal plasma total zinc was significantly low in samples with low volume and hyperviscosity compared to samples with normal volume and viscosity; 139.72 ]ig (73.72) vs. 377.40 (ig (231.06), p < 0.01 for volume and 220.06 jag (144.09) vs. 336.34 |Ag (236.33), p < 0.05 for viscosity. Conversely significantly high amount of total zinc was found in low viability group compared to normal; 437.67 ug (283.88) vs. 305.15 (ig (221.19), p < 0.05. Percentage of pathozoospermics and volume abnormalities were significantly higher in abnormal total seminal zinc group compared to normal (pathozoospermics, 27 % vs. 7.3 % and volume abnormalities, 55.3 % vs. 8.8 %, p < 0.05). Mean zinc concentratiorfwas significantly high in Asthenozoospermics compared to normal motile group; 138.11 u.g/ml (83.92) vs. 110.69 11 fig/ml (54.59), p < 0.05. Significantly positive correlations were found between total seminal plasma zinc and volume (r = 0.53, p < 0.01) as well as total sperm count (r = 0.21, p < 0.05). whereas correlation between seminal zinc and pH was inverse (r = -0,193, p < 0.05 for zinc concentration and r = -0.280, p < 0.01 for total zinc). In contrast serum zinc levels correlated positively with seminal plasma pH (r = 0.167, p < 0.05). Gonadotropin levels were significantly high in azoospermics compared to normozoospennics; LH - 12.82 mlU/ml (11.82) vs. 5.90 mlU/ml (2.78), FSH - 19.69 mlU/ml (9.93) vs. 4.18 mlU/ml (2.78), p < 0.05). FSH level was inversely correlated with sperm concentration (r = -0.203, p < 0.05) and total sperm count (r = -0.206, p < 0.05). There was an inverse correlation between seminal plasma zinc concentration and serum PRL levels (r = -0.198, p < 0.05). Serum zinc concentration showed a negative correlation with serum T levels (r = -0.207, p < 0.05). Both fructose concentrations and total fructose were significantly low in abnormal volume group compared to normal; 15.30 u.mol/ml (1.52) vs. 44.27 umol/ml (2.44) for fructose concentration and 19.06 umol (2.39) vs. 160.63 umol (16.0) for total fructose, p < 0,0001. Mean fructose concentration was significantly high in oligozoospermic group compared to normal; 45.33 umol/ml (5.02) vs. 35.07 umol/ml (2.39). p < 0.05. Total neutral a -glucosidase activity was significantly low in low volume group compared to normal; 55.37 mU (8.79) vs. 140.93 mU (15.36). p < 0.0001. Seminal plasma total zinc positively correlated with total fructose (r = 0.378, p < 0.001), and NAG (r = 0.247. p < 0.001). In-vitro incorporation of zinc. > 5.0 umol/ml into the processed sperm samples and, > 10 umol/zinc into unprocessed sperm samples, caused an impairment of the progressive motility of sperms. Incorporation of 1.2 umol/ml of zinc into sperm culture medium exerts a significantly beneficial effect on sperm recovery rate. The mean post wash sperm concentration showed an increase in the 1.2 umol/ml of zinc added group compared to the zinc devoid sample; 21.87 million/ml (6.61) vs. 18.34 million/ml (9.73), p < 0.05. The percentage of hyperactwated sperm also increased in 1.2 umol/ml zinc added group compared to zinc devoid group; 46.70 % (3.80) vs. 38.83 % (3.56), p < 0.05. In behavioural studies, supplementation of zinc (5 mg/day) for two weeks led to a prolonged ejaculatory latency; 711.6 Sec. (85.47) vs. 489.50 Sec. (67.66), p < 0.05 and an increase in penile thrusting compared to controls; 52.80 Sec (11.28) vs. 26.50 Sec (6.17), p< 0.05. Similarly the PRL and T levels were significantly increased after the treatment period compared to hormone levels before the treatment; PRL - 7.22 ng/dl (3.68) vs. 2.90 ng/dl (0.34) and T - 8.21 ng/ml (6.09; vs. 2.39 ng/ml (1.79), p < 0.05. In conclusion, this study revealed that zinc is beneficial in male reproduction in different aspects.