published_or_final_version / Biological Sciences / Master / Master of Philosophy

abstract / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

No description available.

Protein phosphorylation is a central regulatory mechanism in countless cellular processes. Deletion of the PP1 serine/threonine phosphatase gene Ppp1cc in mice results in male infertility due to a severe impairment in spermatogenesis. This disruption in spermatogenesis is hypothesized to arise due to a deficiency of the testis specific Ppp1cc isoform PPP1CC2. To learn more about the function of PPP1CC2 in spermatogenesis, we have employed several proteomic approaches aimed at identifying both regulatory proteins and substrates that interact with PPP1CC2 in the testis. First, we created transgenic mouse embryonic stem cell lines expressing a tandem affinity tagged version of PPP1CC2. Tandem affinity purification using these cell lines identified a number of known PP1 interacting proteins, and one novel interactor DDOST (dolichyl-di-phosphooligosaccharide-protein glycotransferase) which we hypothesize to have a role in spermatogenesis. In a second approach, we conducted GST pull down assays from mouse testis lysates to identify PPP1CC2 interacting proteins. TSSK1 (testis-specific serine kinase 1) was identified as a novel PPP1CC2 interacting protein. We then demonstrated that TSSK1 interacts with PPP1CC2 in an indirect manner via a common interacting protein TSKS (testis-specific serine kinase substrate). Binding of TSKS to PPP1CC2 is regulated via phosphorylation of a PP1 docking motif on the TSKS surface, and localization of TSSK1 and TSKS in the testis is disrupted in Ppp1cc mutants. Finally, to identify candidate substrates of PPP1CC2 in the testis, we conducted a comparative phosphoproteomic analysis and identified 33 different peptides that were hyperphosphorylated in the testis of 3 week old Ppp1cc knockout mice. Amongst these candidate substrates are several proteins essential for mouse spermatogenesis—HMGA1 (high mobility group AT-hook 1), HSPA4 (heat shock protein 4), YBX2 (Y box protein 2) and SYCP2 (synaptonemal complex protein 2). Taken together, our results suggest that PPP1CC2 interacts with a number of different proteins in the testis, and is likely to play a role at several different stages of spermatogenesis, in both meiotic and post-meiotic spermatogenic cells.

Protein phosphorylation is a central regulatory mechanism in countless cellular processes. Deletion of the PP1 serine/threonine phosphatase gene Ppp1cc in mice results in male infertility due to a severe impairment in spermatogenesis. This disruption in spermatogenesis is hypothesized to arise due to a deficiency of the testis specific Ppp1cc isoform PPP1CC2. To learn more about the function of PPP1CC2 in spermatogenesis, we have employed several proteomic approaches aimed at identifying both regulatory proteins and substrates that interact with PPP1CC2 in the testis. First, we created transgenic mouse embryonic stem cell lines expressing a tandem affinity tagged version of PPP1CC2. Tandem affinity purification using these cell lines identified a number of known PP1 interacting proteins, and one novel interactor DDOST (dolichyl-di-phosphooligosaccharide-protein glycotransferase) which we hypothesize to have a role in spermatogenesis. In a second approach, we conducted GST pull down assays from mouse testis lysates to identify PPP1CC2 interacting proteins. TSSK1 (testis-specific serine kinase 1) was identified as a novel PPP1CC2 interacting protein. We then demonstrated that TSSK1 interacts with PPP1CC2 in an indirect manner via a common interacting protein TSKS (testis-specific serine kinase substrate). Binding of TSKS to PPP1CC2 is regulated via phosphorylation of a PP1 docking motif on the TSKS surface, and localization of TSSK1 and TSKS in the testis is disrupted in Ppp1cc mutants. Finally, to identify candidate substrates of PPP1CC2 in the testis, we conducted a comparative phosphoproteomic analysis and identified 33 different peptides that were hyperphosphorylated in the testis of 3 week old Ppp1cc knockout mice. Amongst these candidate substrates are several proteins essential for mouse spermatogenesis—HMGA1 (high mobility group AT-hook 1), HSPA4 (heat shock protein 4), YBX2 (Y box protein 2) and SYCP2 (synaptonemal complex protein 2). Taken together, our results suggest that PPP1CC2 interacts with a number of different proteins in the testis, and is likely to play a role at several different stages of spermatogenesis, in both meiotic and post-meiotic spermatogenic cells.

Fertility in human males is negatively correlated with obesity and diabetes mellitus (DM). Whether CLA influences obesity and DM associated infertility has not been studied. Seven-week-old male obese and DM-2 mice (db/db, n=40) were randomized to either an 8.5% (w/w) fat diet of a CLA isomer (0.4%, w/w) or a control diet for 6 weeks. Lean (n=10) mice were fed a control diet. The db/db mice fed a control diet displayed increased abnormal morphology, decreased sperm concentration. They also had decreased cst and cgt gene expression, despite increased seminolipid concentration, and decreased expression of genes involved in spermatogenesis compared to their lean counterparts. CLA isomers increased sperm number and normal sperm morphology, influenced seminolipid concentration, seminolipid enzyme gene expression and significantly increased Ccna1 gene expression. Seminolipid and genes of spermatogenesis appear to factor into DM and obesity induced reproductive dysfunction. Dietary CLA isomers appear to increase functionally viable sperm and thereby improve fertility.

No description available.

Fertility in human males is negatively correlated with obesity and diabetes mellitus (DM). Whether CLA influences obesity and DM associated infertility has not been studied. Seven-week-old male obese and DM-2 mice (db/db, n=40) were randomized to either an 8.5% (w/w) fat diet of a CLA isomer (0.4%, w/w) or a control diet for 6 weeks. Lean (n=10) mice were fed a control diet. The db/db mice fed a control diet displayed increased abnormal morphology, decreased sperm concentration. They also had decreased cst and cgt gene expression, despite increased seminolipid concentration, and decreased expression of genes involved in spermatogenesis compared to their lean counterparts. CLA isomers increased sperm number and normal sperm morphology, influenced seminolipid concentration, seminolipid enzyme gene expression and significantly increased Ccna1 gene expression. Seminolipid and genes of spermatogenesis appear to factor into DM and obesity induced reproductive dysfunction. Dietary CLA isomers appear to increase functionally viable sperm and thereby improve fertility.

Some stallions are expected to perform athletically and breed contemporarily. Athletic activity has the potential, especially during the summer months, to induce thermal stress to the testes, resulting in reduced reproductive capability due to decreased sperm quality and libido. There is concern in the horse industry about what level of exercise, if any, affects the reproductive capability of a stallion. Thermal stress associated with training and exercise may impact sperm quality and the future reproductive capability of the stallion. The goal of this study was to determine the effect of strenuous exercise on stallion sperm quality. The objectives were to measure changes in body and scrotal temperatures following strenuous exercise and sperm quality following strenuous exercise. Miniature Horse stallions (n = 7), implanted with subdermal thermosensory devices in the subcutaneous neck and scrotal tissue, were assigned to treatment group based on age and semen quality. Exercising stallions (EX; n = 3) were exercised 4 d/wk for 90 min for 12 wk, while non-exercising stallions (CN) were tied in the shade. Semen was collected from stallions for 5 consecutive days every 4 wk to evaluate semen quality (raw, 24 h and 48 h cooled). Subcutaneous scrotal (SQST), rectal (RT) and neck (NT) temperatures were recorded along with heart rate. Spermatozoa data were normally distributed; therefore, they were subjected to parametric analysis by repeated measures (wk) using the PROC MIXED procedure (SAS v 9.1; SAS Inst. Inc., Cary, NC). Model included treatment (CN or EX), time (wk 0, 4, 8, or 12), and stallion as the subject of the repeated measures. Compared to the CN group, EX stallions had elevated temperatures (avg RT 39.27 vs 37.07 degrees, NT 39.77 vs 37.44 degrees C, and SQST 34.90 vs 33.40 degrees C; P < 0.0001). There was no difference in sperm quality between treatment groups (P > 0.05). In this study, strenuous exercise in Miniature Horse stallions, did not affect sperm quality. This suggests that anecdotal reports of reduced sperm quality in stallions in training may have other causes other than elevated scrotal and body temperature. While previous studies have illustrated that prolonged insulation of the testes reduces semen quality, strenuously exercising stallions for up to 90 min under hot and humid ambient conditions may not be harmful to spermatogenesis.