Comparative Survival of Human Spermatozoa in Media Supplemented With Polyvinyl Alcohol or Human Serum Albumin

Objectives: Zinc ions play an important role in testis development and spermatogenesis. Little is known about the expression of function of transporters involved in testicular zinc homeostasis. In the present work the expression of the zinc transporter ZnT-1 was characterized in mouse testicular slices and compared with level of chelatable zinc of methalthionins. Materials and Methods: Immunoblot analysis of total testis homogenate employed an antibody against the C-terminal domain of ZnT-1. Relative levels of ZnT-1 were quantified from the ECL stained imunoblots using NIH Image. Immunostaining of murine testis slices with the ZnT-1 antibody was visualized with Cy2 conjugated goat-anti rabbit IgG in the fluorescent microscope. Staining of chelatable zinc was performed using Tim’s stain. Results: Using an antibody against the C-terminal domain of ZnT-1 stained a single 60 kDa band, corresponding to the expected molecular weight of ZnT-1. Immunostaining of murine testis slices with the ZnT-1 antibody demonstrated an intense expression of ZnT-1 in Leydig cells. Along the seminiferous tubules, the expression of ZnT-1 was high in spermatogonia and luminal spermatozoa but much lower in spermatids and Sertoli cells. In contrast to the expression pattern of ZnT-1, levels of methalothionins and chelateable zinc were high in spermatids and low in luminal spermatozoa and spermatogonia. Conclusions: This work represents the first immunohistochemical characterization of a zinc transporter in the testis. Furthermore, the inverse relationship between the expression of ZnT-1 and the levels of chelatable zinc and methalothionins suggest that ZnT-1 expression correlates with decreasing Zni in distinct cell types in the seminiferous tubules and suggests that it plays a role during spermatogenesis and spermiogenesis. P-479 Are Some Males in the General Population at a Higher Risk for Y-Chromosome Deletions Leading to Male Infertility? L. QuintanaMurci1, C. Krausz1, M. Jobling2, K. McElreavy1. 1Immunoge´ne´tique Humaine, Institut Pasteur, Paris, 2University of Leicester, UK. Objectives: The human Y-chromosome is susceptible to the loss of genetic material leading to microdeletion formation. Microdeletions have been used to define three non overlapping regions (AZFa, AZFb and AZFc) that are recurrently deleted in infertile males. However, little is known about the mechanisms underlying deletion formation, or if some Y chromosome are susceptible to deletions. Design: Y-chromosome haplotypes were defined in more than 50 males of north-western European origin who carried Y-microdeletions. The defined haplotypes were compared to a control group of the same ethnic and geographic origin in the framework of the European Biodiversity Project. Materials and Methods: All patients were tested for 10 biallelic markers (SRY-1532, SRY-2627, SRY-8229, M9, 92R7, YAP, LLY22g, Tat, sY81, DYS257) and the distribution of the defined haplogroups was compared to a control population. Results: No evidence of a difference in Y-haplotype distribution was observed between the microdeleted and control population. Conclusion: The absence of correlation between Y-chromosome backgrounds and deletion formation may reflect a genuine lack of correlation or it may be due to the resolution of the markers used to define the haplotypes. The study is being extended to include the analysis of the Y-specific MSY1 minisatellite. The analysis of this informative marker could determine if some Y-chromosome structures are either more susceptible to, or protective against deletion formation. P-480 Comparative Survival of Human Spermatozoa in Media Supplemented With Polyvinyl Alcohol or Human Serum Albumin. M. H. Javed, M. Geisman, M. A. Shaikh, C. Ruberto, A. P. Del Valle. ReproMed Ltd/AVR Andrology Inc., Toronto, Canada. Objectives: Human Serum Albumin (HSA) is a vital component of media used for human sperm cryopreservation. Risks associated with HSA are composition variability among different lots, availability problems, adverse reactions and disease transmission. An experiment was designed to replace HSA with polyvinyl alcohol (PVA) and to compare 2 commercially available media. The objective of this study was to compare human sperm viability and kinematics in Tyrode’s media supplemented with either PVA or HSA and modified human tubal fluid 1 HSA (mHTF).

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Design: Human sperm survival was compared in 3 groups: (1) fresh semen, (2) Standard cryopreserved semen and (3) Pre-washed cryopreserved semen. The media were supplemented either with PVA or HSA. Untreated specimens served as a control. Materials and Methods: For each of the 3 groups semen from healthy men (N521) was aliquoted into 4 treatments: (a) untreated semen (b) 1:1 dilution with Tyrode’s solution containing 1 mg/ml HSA, (c) 1:1 dilution with Tyrode’s solution 1 1 mg/ml PVA and (d) 24- and 48-h by Hamilton Thorne Computer Assisted Sperm Analysis System. Motility, straight-line velocity (VSL), curvilinear velocity (VCL), linearity (LIN), average path velocity (VAP), beat cross frequency (BCF) and amplitude of lateral head (ALH) were recorded. Results: Motility (Mean 6 SD) after 5-h in treatments a, b, c and d was 40 6 12, 35 6 14, 40 6 13 and 36 6 13 in Group 1; 12 6 7, 10 6 8, 10 6 9 and 10 6 7 in Group 2 and 8 6 5, 8 6 3, 8 6 5 and 7 6 4 % in Group 3, respectively. The differences were non-significant (P,0.05). At 24 and 48-h best motility was observed in treatment d of Group 1. Mean motility at 24-h was 4, 5, 3 and 9 and at 48-h 1, 0, 0, and 3% for treatments a, b, c and d, respectively. Decline in motility over 5-h period was faster in untreated specimens for all 3 groups. Among groups decline in motility was faster in Group 2 in all treatments. Motility decline among treatments b, c and d was similar (P,0.01). Kinematics after 5-hr in various treatments were similar. VSL after 5-h in Group 1 for treatments a, b, c, and d, was 30.8 6 7.1, 34.5 6 9.8, 36.3 6 9.9 and 34.8 6 9.5; in Group 2, 25.7 6 2.4, 26.4 6 5.9, 26.4 6 4.1, and 25.1 6 3.9 and in Group 3, 21.5 6 2.9, 29.3 6 1.7, 27.8 6 7.5, and 22.8 6 2.5, respectively. VAP after 5-h in Group 1 for treatments a, b, c, and d, was 40.3 6 6.0, 43.0 6 8.1, 45.3 6 9.1 and 43.7 6 10.0; in Group 2, 31.6 6 3.0, 34.4 6 5.4, 33.8 6 3.4, and 32.1 6 3.5 and in Group 3, 27.1 6 2.8, 36.9 6 7, 34.3 6 7.9, and 29.6 6 2.9, respectively. Conclusion: (1) Comparable survival and kinematics observed in media supplemented with PVA indicated that it was non-toxic to human sperm and could be a suitable replacement for HSA. (2) Sperm motility declined faster in untreated specimens. (3) At 24 and 48-h best motility was observed in mHTF. Further experiments are being conducted to investigate suitability of PVA supplementation in sperm cryopreservation media.

REPRODUCTIVE ENDOCRINOLOGY Wednesday, October 25, 2000 P-481 Bidirectional Effect of Transforming Growth Factor-b1 (TGF-b1) and Stimulatory Effect of Platelet-Derived Growth Factor (PDGF) on Mitogenesis of Human Myometrial and Leiomyoma Cells. I. Sozen, E. Kovanci, A. Arici. Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT. Objectives: TGF-b1 is the prototype of a bidirectional regulator of cell growth that can either inhibit or stimulate the proliferation of smooth muscle cells. These effects of TGF-b1 are dependent on the type and differentiation state of the target cell, the concentration of TGF-b1, and the presence of other growth factors. At least a part of TGF-b1-mediated stimulation of growth is associated with increased production of PDGF, which is another potent mitogen for smooth muscle cells. We have previously shown the elevated expression of TGF-b1 in the leiomyomatous myometrium and the increased active to latent TGF-b1 ratio under the influence of ovarian hormones. In the present study, we investigated the mitogenic effects of TGF-b1, anti-TGF-b antibody, and PDGF on myometrial and leiomyoma smooth muscle cells. Design: We conducted in vitro experiments in which the cell proliferation of cultured human myometrial and leiomyoma cells treated with TGF-b1, anti-TGF-b antibody or PDGF was assessed through [3H] thymidine incorporation method. Materials and Methods: After myometrial and leiomyoma cells in culture were incubated with TGF-b1 (0.01–1 ng/mL), anti-TGF-b antibody (0.01– 10 ng/mL) or PDGF (10 ng/mL) in serum- and phenol red-free medium for 24 –96 h, [3H] thymidine (1 mci/well) was added. 4 –24 h thereafter, cells were harvested using automated cell harvester and radioactivity was quantified by liquid scintillation spectrophotometry. Results: TGF-b1, at lower concentrations, induced an increase (2–3 fold; p,0.05) in thymidine incorporation, which disappeared at higher concentrations, compared to control. This increase was more prominent and

Vol. 74, No. 3, Suppl. 1, September 2000