P-132 Varicocele ⴛ heat stress: Expression of the heat shock protein 70 –2 (HSP70 –2) in the sperm cells. S. Lima, A. Cedenho Sr., P. Hassun Sr., R. Bertolla Sr., S. Oehninger Sr., M. Srougi Sr. Federal University of Sao Paulo, Sao Paulo, Brazil; Jones Institute for Reproductive Medicine, Norfolk, VA. OBJECTIVE: The importance of testicular thermoregulation is evidenced by the fact that even slight temperature elevations, such as those caused by varicocele, may inhibit spermatogenesis. Heat stress suppresses sperm cell maturation, thus decreasing sperm output and overall semen quality. Recent studies have demonstrated that the heat shock proteins (HSPs), particularly HSP70 –2, significantly influence the acquisition of thermal tolerance in cells. The purpose of this study is to evaluate mRNA expression of the HSP70 –2 gene in ejaculated spermatozoa of thirty adolescents with varicocele grades II and III (experimental group), and 15 adolescents without varicocele (control group). DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Semen was obtained by masturbation following 2– 4 days of sexual abstinence. Seminal analysis was performed according to the World Health Organization criteria and morphology was evaluated by strict criteria. Total RNA was extracted from sperm ejaculate samples using TRIzol reagent (Gibco-BRL威). First-strand cDNA synthesis from RNA was catalyzed by Superscript II RT (Gibco-BRL威). Semiquantitative RT-PCR analysis was performed using relative expression level of the HSP70 –2 gene according to the expression level of the housekeeping ␤ -actin gene. Statistical analysis of the data were performed using Student’s t-test (p⬍0.05). RESULTS: According to these data, the HSP70 –2 gene showed a higher expression level in adolescents with varicocele and normozoospermia, when compared to adolescents with varicocele and oligozoospermia and to the control group (p⬍0.05). CONCLUSION: This study demonstrates that HSP70 –2 gene expression is up-regulated in sperm from normozoospermic patients with varicocele, which in turn suggests that it could be a candidate as a molecular marker for the acquisition of thermal tolerance in spermatozoa. Supported by: CNPq
P-133 Male infertility and heat shock protein 70 –2 (HSP70 –2) expression. S. Lima, A. Cedenho Sr., M. Cenedeze, R. Bertolla Sr., S. Oehninger Sr., M. Srougi Sr. Federal University of Sao Paulo, Sao Paulo, Brazil; Jones Institute for Reproductive Medicine, Norfolk, VA. OBJECTIVE: Oligozoospermic men generally possess a higher rate of sperm with abnormal morphology or low progressive motility, which leads to lower fertility. These oligozoospermic men are candidates for intracytoplasmic sperm injection (ICSI). The presence of HSP70 –2 is correlated, in spermatogenesis, with maturity, function, and fertility. Dysfunctional expression of regulated genes may result in abnormal spermatogenesis. The purpose of this study is to evaluate mRNA expression of the HSP70 –2 gene in ejaculated spermatozoa of ten oligozoospermic men (study group) and 10 normozoospermic men (control group). DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Semen was obtained by masturbation following 2– 4 days of sexual abstinence. Seminal analysis was performed according to the World Health Organization criteria and morphology was evaluated by strict criteria. Total RNA was extracted from ejaculated sperm samples using TRIzol reagent (Gibco-BRL威). First-strand cDNA synthesis from RNA was catalyzed by Superscript II RT (Gibco-BRL威). Semiquantitative RT-PCR analysis was performed using relative expression level of the HSP70 –2 gene according to the expression level of the housekeeping ␤ -actin gene. Blood samples were obtained by venipuncture for karyotype tests and hormonal analysis (FSH, LH and testosterone). Statistical analysis of the data was performed using Student’s t-test (p⬍0.05). RESULTS: According to these data, all patients possessed a normal karyotype (46XY), no significant hormonal differences were found between the two groups, but the semi-quantitative RT-PCR analysis of HSP70 –2 gene showed a lower expression level in infertile men, when compared to the control group (p⬍0.05). CONCLUSION: This study demonstrates that HSP70 –2 gene expression
FERTILITY & STERILITY威
is down-regulated in sperm from oligozoospermic patients, which in turn suggests that it could be associated with the pathogenesis of male infertility. Supported by: None.
P-134 Over-expression of IL-10 in testicular tissues of immature as compared to adult mice; induction by lipopolysaccharide. M. M. Huleihel, C. Akfeld, M. Abu Elhija, K. Bahat, E. Lunenfeld. Ben-Gurion University of the Negev, Beer-Sheva, Israel; Ben-Gurion University of the Negev and Sorok University Medical Center, Beer-Sheva, Israel. OBJECTIVE: To evaluate the expression levels and the cellular origin of IL-10 in testicular tissue of sexually immature and adult mice, under physiological and pathological conditions. DESIGN: In vivo and in vitro study MATERIALS AND METHODS: Eight weeks or two-weeks old Balb/c mice were intraperitonealy (i.p) injected with saline (control) or LPS (2, 20, 100 mg/100 ml/mouse). Three hours or 16 hours after the injection, testes were collected, tunica albugina was removed, and testes were formalin-fixed and paraffin-embedded for immunohistochemical staining (IHC) or homogenized for homogenates preparing or for RNA extraction. Leydig cells (LC) were separated from testes of 8 week old mice by Percoll gradient. LC cultures (2.5 ⫻ 104 cells/0.5ml/well) were incubated in the absence or presence of LPS (0.1, 1 or 5 mg/ml) or LH (0.1, 1 or 5 IU/ml) for 3 and 16 hours. The levels of IL-10 were examined in testicular homogenates and Leydig cell supernatants by specific ELISA. RESULTS: The levels of IL-10 were significantly higher in testicular tissue of sexually immature than mature mice (as examined by ELISA and RNA expression). Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that the main producing cells are interstitial cells (Leydig cells and macrophages) and germ cells. Interstitial cells express higher levels of IL-10 than germ cells. Peritubular cells of sexually immature and adult mice did not express IL-10. Intraperitoneal injection of LPS (2,20 or 100 mg/ml/mouse) for 3 hours or 24 hours were significantly (p⬍ 0.05) increased the levels of IL-10 in the homogenates, IHC and RNA expression of both adult and sexually immature mice. This increase was dose- and time-course dependent manner. Leydig cell cultures constitutively produced IL-10, however stimulation with LH or LPS did not affect this capacity. CONCLUSION: Our results demonstrate for the first time the expression of IL-10 in Leydig and germ cells of testicular tissues from adult and sexually immature mice. The over-expression of IL-10 in immature mice as compared to adults may indicate the involvement of IL-10 in spermatogenesis and the possibility of its regulation by gonadotropins and testosterone. In addition, up-regulation of IL-10 levels following systemic infection/ inflammation (LPS) may suggest its involvement in the regulation of immune response in the testis (regulation Fas/Fas ligand system and/or cytokine expression). Thus, testicular IL-10 should be considered in male fertility. Supported by: Israel Ministry of Health, Jerusalem, Israel.
P-135 Capillary electrophoresis of seminal plasma links cell-free DNA in semen to sperm quality. P. J. Chan, J. S. Chou, J. D. Jacobson, W. C. Patton, J. U. Corselli, A. King. Loma Linda University, Loma Linda, CA. OBJECTIVE: (i) To determine the presence of low and high molecular weight cell-free DNA in seminal plasma and: (ii) To correlate the amounts of cell-free DNA with sperm parameters. DESIGN: The capillary electrophoresis (CE) method was modified to provide a rapid assay for cell-free DNA in semen obtained from 25 male patients attending the fertility clinic. The results were correlated to important sperm function parameters. MATERIALS AND METHODS: Advanced semen analyses were performed on 25 specimens. Fresh and frozen-thawed seminal plasma were each separately pipetted inside glass micro- hematocrit capillary tubes (internal diameter 1.2 mm, wall 0.2mm) half-filled with a 2.4 % agarose gel and sealed with Liquasonic conducting gel. Control capillary tubes with either buffer, female sera or HaeIII-phiX DNA markers were also included. Horizontal electrophoresis (100 volts, 24 mins, TAE buffer)was carried out
and the agarose column from each capillary tube was stained in Sybr-Gold (5 mins), rinsed, and analyzed under UV-epifluorescence. Digital images from gel sections corresponding to high (12 Kb) and low (1 Kb) molecular weight DNA fragments were analyzed. Results were expressed as a ratio of the DNA intensity to the control and significance tested using linear regression and Student’s t-test. RESULTS: The amount of low molecular weight cell-free DNA was directly correlated to rapid progression, curvilinear velocity (⬎ 40 /sec), normal strict morphology and the sperm penetration assay capacitation index. In contrast, cell-free DNA quantity was negatively correlated to post-wash hyperactivation. Sperm concentration was not related to cell-free DNA quantity. Freezing semen did not increase cell-free DNA but decreased the low molecular weight cell-free DNA. CONCLUSION: Cell-free DNA present in semen was related to important sperm parameters linked to normal sperm function. The data suggested the possible use of low molecular weight cell-free DNA as a marker of semen quality. This study reports on the novel finding of cell-free DNA released along with sperm during each ejaculation. Supported by: None
were performed by fusing ACT or its major identified variant with the GAL-4 activation domain and CREM with the GAL-4 binding domain. Plasmids containing one of the ACT fusions, CREM, and a Lac-Z reporter were transfected into the ABY37 yeast strain and tested for interaction. Values were recorded in Miller units. Four independently derived wild type and variant cell lines were tested. RESULTS: A total of eight single nucleotide polymorphisms (SNPs) were identified in the ACT gene. Six of the polymorphisms fell within the exon regions and four resulted in amino acid changes. Five of the polymorphisms segregate into three distinct, conserved patterns termed haplotypes 1,2, and 3. Yeast two-hybrid assays designed to test the interaction between haplotypes 1 (wild type) or 3 with CREM showed a 45% reduction in the interaction between haplotype 3 and CREM compared with haplotype 1. CONCLUSION: All identified polymorphisms that resulted in an amino acid change were in conserved regions of the gene. Three of the amino acid changes fell in the putative protein binding regions, which may explain the reduced interaction of haplotype 3 with CREM. Since all three haplotypes were present in both the fertile and infertile study populations, it is not expected that the reduced interactions of haplotype 3 with CREM is directly responsible for the infertility of the men homozygous for that allele. In combination with other factors it is possible that it could contribute to a reduction of spermatogenesis. A drop off in linkage disequilibrium on either side of this region suggests that natural selection may be maintaining the different haplotypes for a purpose. Supported by: None P-137 Adult human testicular tissue with impaired spermatogenesis survives as ectopic xenograft. P. Patrizio, A. Honaramooz, S. Schlatt, I. Dobrinski. Yale University Fertility Center, New Haven, CT; Center Animal Transgenesis and Germ Cell Research, University of Pennsylvania, PA; University Pittsburgh School Medicine, Cell Biology, Pittsburgh, PA.
P-136 Identification of three haplotypes within the Activator of CREM in the Testis (ACT) gene and their implications for fertility. G. L. Christensen, I. P. Ivanov, J. F. Atkins, D. T. Carrell. University of Utah, School of Medicine, Salt Lake City, UT. OBJECTIVE: A large number of genes have now been identified with a specific role in spermatogenesis. One of these, the four-and-a-half LIM domain ACT, is a phosphorylation-independent coactivator of CREM. The purpose of this study was to screen the ACT gene in a group of men with severe male factor infertility for alterations and test the identified alterations with a yeast two-hybrid assay to determine if they introduced a functional change. DESIGN: A prospective study in which the DNA from consenting, severely oligozoospermic (less than 5 M/ml) or azoospermic patients and fertile controls was evaluated by direct sequencing for mutations in the ACT gene. Individuals who had been diagnosed with any condition or treatment connected with infertility (e.g., cystic fibrosis, varicocele, chemotherapy, etc.) were not included. MATERIALS AND METHODS: A total of 96 azoospermic or severely oligozoospermic males and 69 men of known paternity were screened for alterations in the ACT gene. Three sets of PCR primers were developed to amplify the 5 exons of ACT and its promoter region. All reactions were optimized to give clean, ample quantities of DNA. Additional internal primers were designed to allow sequencing in both the forward and reverse directions. Sequencing was conducted on an ABI 3700 capillary sequencer. Sequence trace files generated by the ABI 3700 were subsequently assembled using Phrap program software and evaluated for alterations using both the Phred and Consed sequence analysis programs. Yeast two-hybrid assays
OBJECTIVE: To provide a laboratory model to evaluate survival and maintenance of adult human spermatogenesis from men with a variety of pathological conditions. DESIGN: Research study (IRB-approved) of transplantation by xenografting of human testicular biopsies in immunodeficient mice. MATERIALS AND METHODS: A total of 8 testicular biopsies, obtained from four men diagnosed with obstructive azoospermia and one man with maturation arrest, and multiple testicular samples from two testes (orchiectomy specimen) of a patient undergoing a sex reversal operation after prolonged hormonal suppression, were used for the experiments. The specimens were grafted into castrated, six-week old male, immunodeficient ICR SCID mice. At the time of grafting a piece of testicular tissue was also fixed for histological exam and to serve as a reference for graft development. The remaining fragments of testis tissue were inserted subcutaneously into the dorsal skin and analyzed at different time intervals (between 1 and 19 weeks). At the time of analysis, the following were documented: a) histology; b) measurement of the diameter of the seminiferous tubules; c) maintenance or development of spermatogenesis. RESULTS: The xenografts from each donor survived and all established blood supply; however, grafts from donors with complete spermatogenesis at the time of grafting showed consistently massive degenerative changes of the germinal epithelium and progressive decline and disappearance of spermatogenic activity. On the other hand, from 2 and up to 19 weeks, all the testis grafts of patients exhibiting absent spermatogenesis maintained their tubular morphology and showed almost no degenerative changes. CONCLUSION: These pioneering observations suggest that: a) adult human testis grafts from patients with complete spermatogenesis undergo progressive degenerative changes in xenografts; b) testis grafts of patients with less advanced spermatogenesis (histologically comparable to prepubertal boys) are more likely to survive. These findings provide strong support to the hypothesis that, as already demonstrated in domestic animal species, xenografting could be used to initiate and maintain spermatogenesis in grafts obtained from prepubertal boys prior to gonadotoxic treatments. Supported by: None P-138 The effect of an environmental toxin, perfluorooctanoic acid, on cryopreserved-thawed, human sperm. S. K. Dahl, J. C. Robins, R. S. Soman,
Vol. 82, Suppl. 2, September 2004