No description available.

No description available.

Normal development and function of the male reproductive tract relies on the crucial balance between androgen and estrogen signalling, furthermore estrogens play an important role in the regulation of spermatogenesis and steroidogenesis. Tamoxifen (TAM) inducible Cre-loxP systems are widely used to study testicular function. TAM is a selective estrogen receptor modulator (SERM), thereby exerting anti- and pro-estrogenic effects. Therefore, it was hypothesised that acute (single dose) TAM administration to the postnatal testis has significant long-term effects on testicular development and adult testis function, questioning its utility in inducible transgenic systems. A suitable Cre line was first validated as a tool to target the postnatal adult Leydig cell (ALC) population. The Nestin-Cre expressing stem Leydig cells (LCs) were demonstrated as a source of a subset of the ALCs. Hence, a TAM inducible Nestin- Cre line was one of the mouse lines employed for further studies. A comprehensive investigation was carried out to assess any short-and long-term testicular phenotype upon administration of high (3mg) and low (1mg, 500ug and 250ug) doses of TAM. These studies were carried out in TAM-inducible Nestin- Cre/ERT2 and PDGFRA-Cre/ERT2 mouse lines as well as C57Bl/6 mice, to ensure that the observations made were independent of transgene effects. High dose TAM treatment resulted in transgene induction, however this also caused short-term spermatogenic arrest, alterations to steroidogenesis and LC number. Spermatogenesis recovers in young adults, but LCs show delayed maturation, suggesting changes in developmental programming of the ALC population. Thus it was concluded that a single dose of TAM in early postnatal life disrupts testicular function in adulthood. Single low doses of TAM did not induce the transgene, but surprisingly also had a long-term impact on ALC development, steroidogenesis and spermatogenesis. Severity of the phenotype worsened with dose concentration, indicating dose dependent impacts of TAM on the testis. Therefore, TAM has adverse impacts on the testis at doses below the threshold of Cre induction. In order to find a substitute for TAM in transgene induction studies, Raloxifene (RAL), another SERM, was hypothesised to induce transgenes with minimal disruption of testicular function. A 3mg dose of RAL did not show the adverse impacts of TAM. However, different dose regimens were assayed to induce the transgene without success, hence ruling out RAL as a substitute for TAM. Given the severity of previously undocumented TAM-induced phenotypes elucidated in these studies, it is evident that the off-target effects of TAM are severely underappreciated and can cause long-term programming effects. These off-target effects are likely to be present in other estrogen responsive tissues. Hence TAM-inducible Cre systems should be used with rigorous controls, to ensure correct conclusions are drawn from results obtained.

No description available.

Abstract not available

The objective of this thesis was to investigate and expand the use of testis tissue xenografting as means of maintaining the developmental potential of donor testis tissue. The objective of the first study was to investigate the effect of donor age on spermatogenesis in canine testis tissue after xenografting into immunodeficient recipient mice. Fragments of testis tissue from 12 dogs of different ages were xenografted under the back skin of mice. Donors were categorized based on testis developmental status at the time of grafting into: less than four months (immature), four to six months (young), and greater than six months of age (adult). The grafts were evaluated at four, six or eight months post-grafting. At four months post-grafting, immature and young groups had higher graft recovery rates (92 ± 5.8 and 88 ± 4.4% versus 69 ± 3.5%; P = 0.001 and P = 0.001), graft weights (34 ± 8.1 and 32 ± 11.0 mg versus 7 ± 2.6 mg; P = 0.001 and P = 0.02), vesicular gland indices (1.1 ± 0.20 and 0.6 ± 0.18% versus 0.1 ± 0.03%; P < 0.0001 and P = 0.02), seminiferous tubule numbers (517 ± 114.8 and 364 ± 161.0 versus 10 ± 5.1; P < 0.0001 and P = 0.03), and larger seminiferous tubular diameters (140 ± 17.8 and 130 ± 3.4 µm versus 55 ± 21.9 µm; P = 0.003 and P = 0.001), compared to adult donor xenografts. Xenografts from immature donors maintained the growth and development for eight months, as exhibited by greater graft weights (17 ± 4.6 mg, P = 0.002), seminiferous tubule numbers (547 ± 210.3, P < 0.01) and tubular diameters (93 ± 15.9 µm, P < 0.0001), and induced greater vesicular gland indices (1.5 ± 0.46%, P = 0.0005), compared to adult donor xenografts. The growth and development of testis tissue xenografts from immature and young donors were not different after eight months (P > 0.05). Young donor xenografts had greater seminiferous tubule number and diameter compared to adult donor xenografts (P = 0.009 and P = 0.004, respectively) at eight months post-grafting. Elongated spermatids were the most advanced germ cell type present at four and eight months post-grafting in the testis grafts of immature and young age groups.<p> The objective of the second study was to evaluate three different strategies to preserve/cryopreserve immature porcine testis tissue. Immature porcine testes were cooled at 4 °C for 24, 48 or 72 hours, and testis tissue fragments were cryopreserved using programmed slow freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol, or vitrified using DMSO or glycerol at 5, 15 or 30 min exposure time. In vitro cell viability was determined by trypan blue exclusion, and in vivo developmental potential was evaluated by xenografting into immunodeficient mice. Compared to fresh tissue, short-term cooling of porcine testis tissue resulted in similar in vitro cell survival rates (93 ± 2.2% for fresh versus 95 ± 0.3, 93 ± 1.7 and 87 ± 4.3% after 24, 48 and 72 hours at 4 °C, respectively; P = 0.74) and in vivo development, with generation of elongated spermatids and sperm after four months of grafting. Cryopreservation of testis tissue with programmed slow freezing using glycerol and vitrification with DMSO (5 min equilibration) or glycerol (5 or 15 min equilibration) did not compromise the developmental competence of xenografts when compared to fresh tissue (control), characterized by the formation of elongated spermatids and sperm.<p> These findings suggest that canine testis tissue from immature donors and cooling of immature porcine testis tissue to refrigerator temperature for up to 72 hours or cryopreservation with slow controlled freezing or vitrification could be suitable methods to restore male fertility following xenografting.

The objective of this thesis was to investigate and expand the use of testis tissue xenografting as means of maintaining the developmental potential of donor testis tissue. The objective of the first study was to investigate the effect of donor age on spermatogenesis in canine testis tissue after xenografting into immunodeficient recipient mice. Fragments of testis tissue from 12 dogs of different ages were xenografted under the back skin of mice. Donors were categorized based on testis developmental status at the time of grafting into: less than four months (immature), four to six months (young), and greater than six months of age (adult). The grafts were evaluated at four, six or eight months post-grafting. At four months post-grafting, immature and young groups had higher graft recovery rates (92 ± 5.8 and 88 ± 4.4% versus 69 ± 3.5%; P = 0.001 and P = 0.001), graft weights (34 ± 8.1 and 32 ± 11.0 mg versus 7 ± 2.6 mg; P = 0.001 and P = 0.02), vesicular gland indices (1.1 ± 0.20 and 0.6 ± 0.18% versus 0.1 ± 0.03%; P < 0.0001 and P = 0.02), seminiferous tubule numbers (517 ± 114.8 and 364 ± 161.0 versus 10 ± 5.1; P < 0.0001 and P = 0.03), and larger seminiferous tubular diameters (140 ± 17.8 and 130 ± 3.4 µm versus 55 ± 21.9 µm; P = 0.003 and P = 0.001), compared to adult donor xenografts. Xenografts from immature donors maintained the growth and development for eight months, as exhibited by greater graft weights (17 ± 4.6 mg, P = 0.002), seminiferous tubule numbers (547 ± 210.3, P < 0.01) and tubular diameters (93 ± 15.9 µm, P < 0.0001), and induced greater vesicular gland indices (1.5 ± 0.46%, P = 0.0005), compared to adult donor xenografts. The growth and development of testis tissue xenografts from immature and young donors were not different after eight months (P > 0.05). Young donor xenografts had greater seminiferous tubule number and diameter compared to adult donor xenografts (P = 0.009 and P = 0.004, respectively) at eight months post-grafting. Elongated spermatids were the most advanced germ cell type present at four and eight months post-grafting in the testis grafts of immature and young age groups.<p> The objective of the second study was to evaluate three different strategies to preserve/cryopreserve immature porcine testis tissue. Immature porcine testes were cooled at 4 °C for 24, 48 or 72 hours, and testis tissue fragments were cryopreserved using programmed slow freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol, or vitrified using DMSO or glycerol at 5, 15 or 30 min exposure time. In vitro cell viability was determined by trypan blue exclusion, and in vivo developmental potential was evaluated by xenografting into immunodeficient mice. Compared to fresh tissue, short-term cooling of porcine testis tissue resulted in similar in vitro cell survival rates (93 ± 2.2% for fresh versus 95 ± 0.3, 93 ± 1.7 and 87 ± 4.3% after 24, 48 and 72 hours at 4 °C, respectively; P = 0.74) and in vivo development, with generation of elongated spermatids and sperm after four months of grafting. Cryopreservation of testis tissue with programmed slow freezing using glycerol and vitrification with DMSO (5 min equilibration) or glycerol (5 or 15 min equilibration) did not compromise the developmental competence of xenografts when compared to fresh tissue (control), characterized by the formation of elongated spermatids and sperm.<p> These findings suggest that canine testis tissue from immature donors and cooling of immature porcine testis tissue to refrigerator temperature for up to 72 hours or cryopreservation with slow controlled freezing or vitrification could be suitable methods to restore male fertility following xenografting.

No description available.

Delicate epigenetic modifications are essential for production of spermatids during spermatogenesis. DNA methyltransferase 3 (DNMT3), the enzymes involved in adding a methyl group to unmodified DNA, contains three members: DNMT3A, DNMT3B and DNMT3L. The latter lacks methyltransferase activity, but was closely associated with spermatogenesis in many reports. According to the presentation of mature spermatids in testis, azoospermia could be separated into obstructive and non-obstructive categories. Non-obstructive azoospermia is spermatogenesis defective, germ cells absent in seminiferous tube is the most serious type. Therefore, we would like to find out if there are differential expression of DNMT3 family transcripts in testes of azoospermia patients from infertility clinic. Using RT-PCR and qPCR, we found only 5 (29.4%) expressed DNMT3L in 17 non-obstructive patients, whereas all 20 obstructive patients expressed. Both groups were similar in expression levels of DNMT3A and DNMT3B. Nuclei of spermatogonia and spermatocyte were the main immunohisto-chemical localization of DNMT3L protein. Lost of germ cells should be the cause of undetectable DNMT3L expression in azoospermia patients. By this founding, it could serve as an indicator for ability of male germ cell culture in further applications of assisted reproduction.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy