Friday, February 21 12:00 NOON- 11:00 PM Executive Council Meeting (lunch and supper served) (Chesapeake Room NB) Saturday, February 22 8:00-9:40 AM Postgraduate Course (Constellation 3:00-5:00 PM Postgraduate Course (Constellation Ballroom A) Ballroom A)

9:40-1 0:00 AM Refreshment Break 6:00-7:00 PM Student Mixer (Maryland Suites-Balti­ 10:00-12:00 NOON Postgraduate Course (Constellation more Room) Ballroom A) 7:00-9:00 PM ASA Welcoming Reception (Atrium 12:00-1 :00 PM Lunch (on your own) Lobby) 7:00-9:00 PM Exhibits Open (Constellation Ball­ I :00-2:40 PM Postgraduate Course (Constellation Ballroom A) rooms E, F) 2:40-3:00 PM Refreshment Break 9:00- 1 I :00 PM Executive Council Meeting (Chesa­ peake Room NB) Sunday, February 23 7:45-8:00 AM Welcome and Opening Remarks 12:00-1 :30 PM Women in Andrology Luncheon (Ches­ (Constellation Ballroom A) apeake Room NB) 8:00-9:00 AM Serono Lecture: "Genetics of Prostate Business Meeting 12:00-12:30 Cancer" Patrick Walsh (Constellation Speaker and Lunch 12:30-1:30 Ballroom A) I :30-3:00 PM Symposium I: "Regulation of Testicu­ 9:00-10:00 AM American Urological Association Lec­ lar Growth and Function" (Constellation ture: "New Medical Treatments of Im­ Ballroom A) potence" Irwin Goldstein (Constella­ Patricia Morris tion Ballroom A) Martin Matzuk 10:00-10:30 AM Refreshment Break/Exhibits 3:00-3:30 PM Refreshment Break/Exhibits (Constellation Ballrooms E, F) (Constellation Ballrooms E, F) 10:30-12:00 NOON Oral Session I: "Genes and Male Repro­ 3:30-4:30 PM Oral Session II: "Calcium Channels duction" (Constellation Ballroom A) and Male Reproduction" (Constellation Ballroom A) 12:00- 1 :30 PM Lune (on your own) � 4:30-6:30 PM Poster Session I (Constellation Ball­ �4·< rooms C, D) \v\wr 7:30-11:00 PM Banquet (National Aquarium) Monday, February 24 7:00-8:00 AM Past Presidents' Breakfast 12:00-1 :30 PM Simultaneous Events: (Pratt/Calvert Rooms) I. Laboratory Science Forum Lunch­ 8:00-9:00 AM Oral Session III: "Sex Accessory Or­ eon: "The Basics of Gamete Cry­ gans" (Constellation Ballroom A) opreservation" (Maryland Suites­

9:00-9:30 AM ASA Lecture: "The Use and Abuse of Columbia Room) Testicular Sperm As Viewed From a 2. Editorial Board Meeting/Luncheon Thirty Year Experience With Epididy- (Executive Boardroom) mal Reconstructive Surgery" I :30-3:00 PM Symposium II: "Estrogen in the Male: Robert Schoysman (Constellation Ball- Hormonal Carcinogenesis, Reproduc­ room A) tive Toxicology, and Role in Normal

9:30-1 0:00 AM Refreshment Break/Exhibits Reproductive Function" (Constellation (Constellation Ballrooms E, F) Ballroom A) E. Mitchell Eddy 0:00-1 :00 AM Pharmacia & Upjohn Lecture: "Sex I I John MacLachlan Chromosome Genes and Spermatogen­ Stephen Safe esis" Colin Bishop (Constellation Ball- room A) 3:00-3:30 PM Refreshment Break/Exhibits (Constellation Ballrooms E, F) 11:00- 12:00 NOON Buckeye State-of-the-Art Lecture: "Re- productive Rescue of Endangered Spe- 3:30-4:30 PM ASA Business Meeting and Awards cies" JoGayle Howard (Constellation Ceremony (Constellation Ballroom A) Ballroom A) 5:00-7:00 PM Poster Session II (Constellation Ball­

12:00-1 :30 PM Lunch (on your own) rooms C, D) 7:00-8:00 PM Student Colloquium "On Human Destiny" Donald Coffey (Maryland Suites-An­ napolis/Baltimore Rooms) Tuesday, February 25 8:00-1 2:00 NOON Andrology Laboratory Workshop (loca- 10:00-12:00 NOON Plenary Session: "Normal and Abnor­ tion to be announced) mal Prostate" 8:00-9:30 AM Oral Session IV: "Andrology and As­ William Nelson sisted Reproductive Techniques" (Con­ Martin Tenniswood stellation Ballroom A) ASA/National Medical Enterprises (NME) State-of-the-Art Lecture: "Gene 9:30-1 0:00 AM Refreshment Break/Exhibits (Constella­ tion Ballrooms E, F) Therapy of Prostate Cancer" Jonathan Simon (Constellation Ballroom A)

12:00-1 :00 PM Lunch (on your own) I :00-5:00 PM Andrology Laboratory Workshop (loca­ tion to be announced) American Society of Andrology Executive Council

President ...... Arnold M. Be Iker, M.D. Secretary ...... Rex A. Hess, Ph.D. Vice President ...... Terry T. Turner. Ph.D. Past President ...... Marie-Claire Orgebin-Crist, Ph.D. Treasurer ...... Terry R. Brown , Ph.D. Business Manager ...... Carol Parlette, CAE

Council Members

- .- Ph.D.; Richard V. Clark. M.D., Ph.D. ; Chri her J. De Jonge, Ph.D., H. C. L.D.; Erma Z. Drobnis, Ph.D. ; :;'::=:-":-::.:Wc;::;;:;:;r ,;, Norman . ec t, .D.; Deborah A. o·Brien. Ph.D.; Jon . Pryor, .D. ; Peter N. Schlegel, M.D.; Steven M. Schrader, Ph.D.; Susan S. Suarez, h.D. ; J. Lis Tenovei:,_ � .D., Ph.D.; Jacquetta . Trasler, M.D., Ph.D.

Committee Chairs

Andrology Laboratories .... Christopher J. De Jonge, Ph.D., H.C.L.D. Long-Range Planning ...... Terry T. Turner. Ph.D. Awards ...... Peter N. Schlegel, M.D. Membership ...... Susan H. Benoff. Ph.D. Constitution and By- Laws ...... Matthew P. Hardy, Ph.D. Nominating ...... Robert E. Chapin, Ph.D. Educational Policy ...... Wayne J.G. Hellstrom. M.D. Postgraduate Course 1997 ...... Robert D. Oates. M.D. Finance ...... Rebecca Z. Sokol, M.D. Postgraduate Course 1998 ...... Stuart S. Howards, Ph.D. Future Meetings ...... Bernard Robaire, Ph.D. Program Committee 1997 ...... Dolores J. Lamb, Ph.D. Industrial Relations ...... Ken Roberts, Ph.D. Program Committee 1998 ...... Barry Hinton. Ph.D. International Liaison ...... Barry Hinton, Ph.D. Publications ...... Lonnie D. Russell, M.S .. Ph.D. Laboratory Science Forum ...... Susan M. Tarchala, B.S. Testis Workshop 1997 ...... Barry R. Zirkin, Ph.D. Liaison ...... Harris M. Nagler, M.D. Student Affairs ...... Grace M. Centola, Ph.D. Local Arrangements 1997 ...... Terry R. Brown, Ph.D. Local Arrangements 1998 ...... Shalender Bhasin, M.D.

Journal of Andrology

Co-Editors-in- Chief ...... Ronald W. Lewis, M.D. Editorial Assistant Denise R. Lecy Donald J. Tindall, Ph.D. International Associate Editors Jorge A. Blaquier, M.D., Ph.D. Associate Editors ...... Glenn R. Cunningham M.D. David M. DeKretser, M.D. David E Katz, Ph.D. Ilpo T. Huhtaniemi, M.D., Ph.D. Gail Prins, Ph.D. Yoshitake Nishimune, M.D., Ph.D.

Editorial Office

Journal of Andro/ogy• Guggenheim 171 I •Mayo Clinic/Foundation• 200 First Street S.W. •Rochester, MN 55905 Office Hours: 8:00 a.m.-5:00 p.m. (Central)•Tel: 507/284-2423 •Fax: 507/284-2384• E- mail: [email protected]

1997 Presidential Message

Welcome to the 22nd annual meeting of the American Society of Andrology! It is with a bittersweet feeling that many members travel to Baltimore for this meeting. We are reminded of the important role played in our society by Tom Chang, who is missed very much. Terry Brown capably assumed Tom's role as local arrangements chairperson despite Terry's duty as our society's treasurer. Terry's ability to spend time working on local arrangements was made easier by the willingness of Gail Prins to continue her duties as treasurer beyond the end of her term. Dorrie Lamb and Bob Oates are to be congratulated for arranging exciting programs for this Annual Meeting and Postgraduate Course, respectively. Our society continues to prosper. Lonnie Russell is in the process of defining the role of the Publications Committee in various endeavors undertaken by our society. Significant improve­ ments in the Joumal of Andrology have been made by Ron Lewis and Don Tindall, and we anticipate further growth of the Joumal under the new editorial direction soon to be provided by David Hamilton and Jon Pryor. Androlog continues to provide a busy forum concerning a variety of reproductive topics, and we are grateful to both Craig Niederberger and Andy Meacham for their continuing efforts with Androlog. Holland-Parlette Associates officially became our society's business office on August I, I 996. The dependable and efficient manner in which Carol Parlette and various members of Holland-Parlette Associates have conducted our business affairs justifies the recom­ mendation of their firm by the Finance Committee, which is chaired by Rebecca Sokol. Each participant in this year's meeting is encouraged to attend the banquet, which will be held in the National Aquarium. The format for the evening will allow ample time both for touring this interesting facility and for interacting with colleagues, while leisurely having supper at various stations along the way. We appreciate the valued support of our organization by the various industrial contributors and exhibitors. I am particularly indebted to the Jewish Hospital HealthCare Services in Louisville, Kentucky both for administrative help with my presidential duties and for generous support of this year's annual meeting. Finally, I want to thank the membership for the opportunity I have had to serve as president of our society. I feel particularly honored to be the first urologist to serve in this capacity. I have enjoyed working with the officers, Council members and committee chairs. Our society is successful because of the unusual dedication of so many members.

Arnold M. Belker President

P- 1 Past Presidents

1975-1977 ...... Emil Steinberger 1986-1987 ...... William D. Odell

1977-1978 ...... Don W. Fawcett 1987-1988 ...... Larry L. Ewing

1978-1979 ...... C. Alvin Paulsen 1988-1989 ...... C. Wayne Bardin

1979-1980 ...... Nancy J. Alexander 1989-1990 ...... Rupert Amann

1980-1981 ...... Philip Treen 1990-1991 ...... Howard Nankin 1981-1982 ...... Richard M. Harrison 1991-1992 ...... David W. Hamilton 1982-1983 ...... Richard J. Sherins 1992-1993 ...... Ronald S. Swerdloff 1983-1984 ...... Andrzej Bartke 1993-1994 ...... Bernard Robaire 1984-1985 ...... Rudi Ansbacher 1994-1995 ...... Glenn R. Cunningham 1985-1986 ...... Anna Steinberger 1995-1996 ...... Marie-Claire Orgebin-Crist

General Information

Headquarters Transportation and Travel Arrangements Hyatt Regency Hotel Baltimore

On the Inner Harbor By Car: From Washington, D.C. & Virginia: Take I-95 300 Light Street North to Baltimore. Exit 53 (395) N Downtown/Inner Baltimore, MD 21202 Harbor. 395 becomes Howard Street, continue 2 blocks Tel: (4 10) 528-1 234 and then turn right on Pratt Street. Follow 4 blocks and

Fax: (410 ) 385-1170 turn right at Light Street. The Hyatt is 1h block on the right. Mail-In Registration American Society of Andrology From Philadelphia/New Jersey/New York: Take 95 74 New Montgomery, Suite 230 South through the Fort McHenry Tunnel and continue on San Francisco, CA 94 105 95 to Exit 53 (395) N Downtown/Inner Harbor. Tel: (4 15) 764-4823 Fax: (415) 764-49 15 From Frederick/West Virginia/Western Maryland: E-mail: 105037.I 1 [email protected] Take 70 East to 695 South to Glen Burnie. Follow 695 to Exit 1 I (95) North to Baltimore. Continue on 95 North On-Site Registration to Exit (53) N Downtown/Inner Harbor. Constellation Foyer Friday, February 21: 3:00 PM-5:00 PM From Pittsburgh, PA: Take Rt. 76 to Rt. 70 East. Follow Saturday, February 22: 7:00 AM-7:00 PM above directions from Frederick. Sunday, February 23: 7:00 AM-5:00 PM Monday, February 24: 7:00 AM-5:00 PM From Annapolis, MD: Take Rt. 50 West to I-97 North Tuesday, February 25: 7:00 AM-12:00 NOON which becomes Rt. 3 North to 695 West. Follow 695 West to 'Exit 11 (85) North to Baltimore. Continue on 95 North Exhibits to Exit 53 (395) N Downtown/Inner Harbor. Constellation Ballrooms E and F. Saturday, February 22: 7:00 PM-9:00 PM From Harrisburg, PA: Take I-83 South to Baltimore. Sunday, February 23: 9:00 AM-5:00 PM Follow 83 South to end and this becomes President St. Monday, February 24: 9:00 AM-5:00 PM Continue 2 blocks and turn right on Lombard St. Follow

7 blocks to Light Street and turn left. The Hyatt is l 1h Slide Preview and Press Room blocks on the right. The Charles Room will be open during the meeting hours (7:30 AM to 6:00 PM) from Saturday, February By Airplane and Car: For those wanting to have per­ 22 to Tuesday, February 25. sonal transportation in the Baltimore Metro area, all major car rental firms are represented at the Baltimore-Wash­ Miscellaneous Meetings ington International Airport. The Calvert or Pratt Rooms can be reserved for com­ For travel from the airport to the hotel, exit the airport mittee meetings or small group meetings (please con­ area via I- l 95W and follow signs to I-95N to Baltimore. tact Sarah Morisseau in the ASA Business Office). From I-95, take exit 53 for I-395N for Downtown/Inner Harbor. I-395 becomes Howard Street at the first traffic Smoking Regulations signal. Continue two blocks and turn right onto Pratt Smoking is not permitted in the meeting rooms or in Street. Follow 4 blocks and turn right onto Light Street. the exhibit and poster areas. The Hyatt is 1/2 block on the right.

P-2 By Taxi or Airport Shuttle Service: Taxi fare from BWI Student Information airport to the meeting hotel is approximately $20. The Air­ port Super Shuttle service is available from the baggage Colloquium Monday, February 24, 1997 from 7:00- claim area. Fares are $JO one-way or $J 5 round-trip. 9:00 PM in the Constellation Ballroom A. A multi-media presentation entitled "On Human Destiny"

Speaker Donald Coffey Travel Arrangements and Hotel Reservations: Arrangements for special airfares can be made by con­ Colloquium California Cryobank, Inc. tacting Ann Adams at Buck Rogers Travel [Tel: (800) Sponsor 580-2472 or Fax: (915) 581-8172]. Mention that you are Mixer Saturday, February 22 from 6:00-7:00 PM with the American Society of Andrology. in the Maryland Suites-Baltimore Room.

Placement Service: A Placement Service for candidates and

employers is available. The Placement Service Board will be Hotel Reservations near the Registration Desk. To register prior to the meeting, contact: Dr. Pasquale Patrizio, Director, Male Infertility Ser­ vice, Division of Human Reproduction, Department of Ob­ stetrics & Gynecology, University of Pennsylvania-School of Hotel reservations should be made using the Hotel Res­ Medicine, 106 Dulles Building, 3400 Spruce Street, Phila­ ervation form in the registration package, or by calling delphia, PA 19 104 (tel: 215-662-2975, fax: 215-349-55 12). the hotel directly at ( 410) 528-1234 or Hyatt Reserva­ tions at (800) 233-1234. To ensure receiving the special Student Awards: A New Investigator Award of $500 and meeting rates, you must identify the group name when five Student Merit Awards of $100 will be presented to stu­ making reservations. dents on the basis of their presentations at the Annual Meet­ Rates and room availability can only be guaranteed ing. The winners will be selected by the Awards Committee. for reservations received by January 20, 1997.

Message from the Local vocative and entertammg multi-media presentation of Donald Coffey entitled "On Human Destiny". Arrangements Committee The Hyatt Regency Hotel is located in the center of downtown Baltimore overlooking the scenic Inner Harbor Welcome to Baltimore's Inner and within walking distance of the National Aquarium, Harbor in February!! Remember, the Maryland Science Center, the Morris Mechanic The­ spring is just around the corner. atre, the Baltimore Arena for indoor sports events, and This, the 22nd Annual Meeting of the shops and restaurants surrounding the harbor area. the ASA, promises to provide a The Meyerhoff Symphony Hall, home to the internation­ stimulating and challenging sci­ ally acclaimed Baltimore Symphony Orchestra, is only a entific program for all attendees. short cab ride away. The University of Maryland Medical Robert Oates and the Postgraduate Center is within several blocks of the hotel and the Johns Course Committee have put to­ Hopkins Medical Institutions can be reached by Metro gether a course on the cutting edge from the downtown Charles Center station. We will have of Technologies and Genetic Advances in Animal and a list of current events, restaurants and directions for trav­ Human Reproduction. Dolores Lamb and the Annual el available at the registration desk. We hope you will Meeting Program Committee have designed an exciting enjoy your visit to Baltimore and please let us know how daily scientific venue comprised of Plenary sessions, oral we can help you during your stay. presentations and posters. Our Corporate Exhibitors will Terry Brown, Ph.D. be displaying and demonstrating the latest technologies Local Arrangements Chairperson and instrumentation in andrology. For our premier social event, we have procured the main pavilion of Baltimore's National Aquarium for a private viewing of the exhibits, Local Arrangements Committee: Partha Banerjee, complete with a fish feeding demonstration and a visit to Angela Brodie, Terry Brown (Chairperson), Adrian the tropical rain forest, which will be combined with Dabs, Brad Lerner, Deborah Ricker, Kevin Whaley, cocktails, hors d'oeuvres, and a sumptuous buffet dinner. and Barry Zirkin. The Student Colloquium will be highlighted by the pro-

P-3 Message from the Program velopment and spermatogenesis, namely the identification of genes on the sex chromosomes. The Buckeye State-of Chairperson the-Art Lecture will feature Dr. JoGayle Howard on the "Reproductive Rescue of Endangered Species". This is a The American Society of Androl­ significant problem facing our world today. ogy consists of a diverse group of A second symposium will focus on a very controversial basic scientists, andrologists, urol­ area in male reproductive medicine, namely the role of ogists, gynecologists, veterinary estrogens in the male. Estrogens have been implicated in scientists and endocrinologists­ the normal function of the testis as described by Dr. E. all with a common interest in Male Mitchell Eddy and in hormonal carcinogenesis as dis­ Reproduction. This year's program cussed by Dr. John McLachlan. Most recently, environ­ committee has attempted to in­ mental estrogens have been implicated in purported de­ clude speakers that will be of gen­ clines of various aspects of human reproductive health. eral interest to both the basic sci­ Dr. Stephen Safe will report on a variety of naturally oc­ entist and the clinician members of our society. curring and synthetic estrogens and anti-estrogens and ad­ The Serano Lecturer is Dr. Patrick Walsh of Johns Hop­ dress the potential of these compounds to act as hormonal kins University, who will discuss " The Genetics of Pros­ disruptors. tate Cancer". Dr. Walsh is a recognized leader in the field The meeting will close with a symposium on the nor­ of both the clinical treatment and the molecular basis of mal and abnormal prostate. The ASNNational Medical prostate cancer. Identification of specific genes involved in Enterprises Lecture will be given by Dr. Jonathan Simon the acquisition or progression of this common urologic tu­ on "Gene Therapy of Prostate Cancer". This area of re­ mor in men will provide important insights into the etiol­ search is particularly timely and addresses a key area of ogy of prostate cancer. In addition, the American Urolog­ research that may provide a potential new therapy for ical Association will sponsor a lecture honoring a promi­ prostate cancer. New markers of prostatic disease can pro­ nent urologist. Dr. Irwin Goldstein, Boston University vide important diagnostic tools to the clinician. Dr. Bill School of Medicine, has been selected to discuss "New Nelson will discuss his most recent studies and provide Medical Treatments of Impotence". Dr. Goldstein is an ex­ attendees with some insight into whether these new mark­ pert in the basic science and clinical treatment of erectile ers help with the diagnosis of human benign prostatic dysfunction. He has also contributed to our understanding hyperplasia and prostate cancer. Dr. Martin Tenniswood of erectile physiology and vasculogenic impotence. will conclude the session with a lecture on androgen reg­ The Sunday symposium is focused upon the regulation ulated prostatic gene expression. This is an area of key of testicular growth and function. Dr. Patricia Morris, interest to many of the andrologists and endocrinologists. from the Population Council of Rockefeller University, will discuss cytokine signal transduction in the testis via Dolores J. Lamb, Ph.D. the JAK-STAT pathway. Testicular cytokines and growth Program Chairperson factors activate tyrosine phosphorylation of STAT pro­ teins, leading to nuclear translocation of these transcrip­ tion factors. Specific receptors thereby induce transcrip­ Continuing Medical Education Credit tional activation of target genes in a cell- and age-specific manner. Targeted deletion of genes and transgenic tech­ The University of Minnesota is accredited by the Ac­ creditation Council for Continuing Medical Education nology has provided important tools for the identification (ACCME) to provide continuing medical education for of key regulatory genes in male reproduction. Dr. Martin physicians. Matzuk, Baylor College of Medicine, has used these tech­ Annual Meeting: niques to evaluate the actions of gonadotropins, inhibins The University designates this continuing education and activins and various other hormones on reproduction. activity as Category I of the Physician's Recognition Dr. Robert Schoysman (Brussels, Belgium) will share Award of the American Medical Association. One credit hour may be claimed for each hour of partici­ his thoughts on the use of testicular sperm in assisted pation up to a maximum of 17.0 hours. reproductive techniques. This talk will be based on his perspective of having performed epididymal reconstruc­ Post Graduate Course: The University of Minnesota designates this continu­ tive surgery for over thirty years. ing medical education activity for 7.0 hours in Cate­ The Pharmacia & Upjohn Lecture, by Dr. Colin Bishop gory 1 of the Physician's Recognition Award of the (Baylor College of Medicine), will focus on an area of American Medical Association. key importance in our understanding of male sexual de-

P-4 Educational Objectives for CME Credit

ASA Annual Meeting By attending the 1997 Annual Meeting, the participants will be able to:

* Discuss the genetics of prostate cancer;

* Describe new medical treatments for impotence;

* Define new advances in the regulation of testicular growth and function;

* Explain sex chromosome genes and spermatogenesis;

* Describe techniques used for reproductive rescue of endangered species;

* Summarize the role of estrogen in normal male reproductive function, hormonal carcinogenesis, and reproductive toxicology;

* List the latest experiments on normal and abnormal prostate.

ASA Postgraduate Course Following this course entitled, "Technological and Genetic Advances in Animal Repro­ duction," the participants will be able to:

* Describe the need for and techniques involved in animal contraception;

* Describe why certain species are experiencing a decline in their reproductive capability;

* Understand the advanced reproductive techniques that are used in veterinary medicine;

* Describe how gene transfer in animals works;

* Describe the genetic mechanisms involved in testicular differentiation and spermatogenesis;

* Understand the molecular and genetic aspects of fertilization and early embryo development, i.e., the role of sperm;

* Gain an appreciation for the concerns regarding the use of testicular spermatozoa and spermatids.

I J:I0-J J:30 AM Questions and Answers 1997 American Society of Andrology All Morning Speakers Postgraduate Course J 1:30-12:30 PM Lunch (on your own) February 22, 1997, Hyatt Regency Baltimore, Maryland J 2:30-12:35 PM Introduction Robert Oates

"Technological and Genetic Advances in 12:35-1:15 PM "Testicular Differentiation From Animal Reproduction'' the Primitive Gonad: Genetic Con­ trol and Morphological Aspects" Course Director: Robert Oates Patricia Donahoe Questions 8:00-8:IO AM Welcome and Introduction Robert Oates I: 15-l :35 PM "Genetic Mechanisms of Azoo­ spermia" 8:I 0-8:50 AM "Contraceptive Techniques in En­ Renee Reijo dangered Animal Species-Why Questions do We Need Them?" Linda Munson I:40-2:00 PM "Advantages of the Human Model in Reproductive Genetic Studies" 8:50-9:30 AM "Reproductive Decline in Animals: Renee Reijo Genetic Aspects" Questions Steven O'Brien 2:05-2:20 PM Refreshment Break 9:30-9:50 AM Refreshment Break 2:20-2:40 PM "The Inheritence of the Sperm 9:50-I0:30 AM "Advanced Reproductive Tech­ Centrosome, the Cell's Microtubule niques in Domestic Animals" Organizing Center, During Human Rupert Amann Fertilization'' Cal Simerly I0:30-l I: IO AM "Gene Transfer in Animals" Questions Robert Wall

P-5 2:45-3:05 "Molecular Reconstitution of the Medicine, Baltimore, MD, "Gene Therapy of Prostate Human Sperm Centrosome: Impli­ Cancer" cations for Diagnosing New Forms of Male Infertility" Symposium I- Cal Simerly Questions " Regulation of Testicular Growth and Function'' 3: 10-3 :30 PM "Paternal Contribution to Early Em­ bryo Development and Growth-Im­ Sunday, February 23, 1 :30 PM portance of Genomic Imprinting" Benjamin Tycko Patricia Morris, The Population Council, Rockefeller Uni­ Questions versity, New York City, NY, "Testicular JAK-STAT Sig­

3:35-3:55 PM "Genomic Imprinting: When it nalling Pathways: Cytokines, Growth Factors, and Sper­ Occurs and Clinical Syndromes matogenesis" Resulting from Imprinting Errors" Martin Matzuk, Baylor College of Medicine, Houston, Benjamin Tycko Texas, "Transgenic Models to Study Male Reproductive Questions Function"

4:00-4:25 PM "Clinical Experience and Ethical Considerations of the Use of Sper­ Symposium matids for IVF/ICSI" 11- Robert Schoysman "Estrogen in the Male: Hormonal Questions Carcinogenesis, Reproductive

4:30-5:00 PM Questions for all speakers Toxicology, and Role in Normal Reproductive Function'' Serono Lecture

Monday, February 24, 1:30 PM Sunday, February 23, 8:00 AM

Patrick Walsh, Johns Hopkins University School of Med­ Edward (Mitch) Eddy, NIEHS/NIH, Research Triangle icine, Baltimore, MD, "Genetics of Prostate Cancer" Park, North Carolina, "Targeted Deletion of the Estrogen Receptor: Effects on the Male Reproductive System"

American Urological Association John McLachlan, Tulane School of Medicine, New Or­ (AUA) Lecture leans, LA, "Hormonal Carcinogenesis and the Male"

Sunday, February 23, 9:00 AM Stephen Safe, Texas A&M, Brenham, TX, "Environmen­ tal Estrogens and the Male" Irwin Goldstein, Boston University School of Medicine, Boston, MA, "New Medical Treatments of Impotence" Pharmacia & Upjohn Lecture ASA Lecture Monday, February 24, 10:00 AM Monday, February 24, 9:00 AM Colin Bishop, Baylor College of Medicine, Houston, Tex­ Robert Schoysman, Brussels, Belgium, "The Use and as, "Sex Chromosome Genes and Spermatogenesis" Abuse of Testicular Sperm As Viewed From a Thirty Year Experience With Epididymal Recontructive Surgery"

Buckeye State-of-the-Art Lecture Program Committee

Monday, February 24, 11:00 AM D. Lamb (Chair), M. Anderson, W. Hellstrom, JoGayle Howard, National Zoological Park, Washington, B. Hinton, R. Oates, K. Roberts, L. Rodriguez-Ri­ D.C., "Reproductive Rescue of Endangered Species" gau, D. Tindall, A. Belker

ASA/National Medical Enterprises Abstract Review Committee (NME) State-of-the-Art Lecture D. Lamb (Chair), S. Benoff, G. Centola, W. Hells­ Tuesday, February 25, 10:00 AM trom, B. Hinton, R. Oates, K. Roberts, L. Rodri­ guez-Rigau, D. Tindall, A. Belker Jonathan Simon, Johns Hopkins University School of

P-6 Serono Award Lectureship Serono Lectureship Recipients

Patrick C. Walsh, M.D. is the Da­ 1980 C. Alvin Paulsen 1988 Roger Guillemin I 98 I Pierre Soupart I 989 Frank S. French vid Hall McConnell Professor and 1982 Kevin J. Catt 1990 David C. Page Director of the Department of Maria L. Dufau 1991 Tony M. Plant Urology at the Johns Hopkins 1983 J. Michael Bedford 1992 Yves Clermont 1984 C. Wayne Bardin 1993 Leroy Hood University School of Medicine 1985 David M. De Kretser 1994 Michael D. Griswold and the Urologist-in-Chief of the 1986 Ronald S. Swerdloff 1995 Marie-Claire Orgebin-Crist 1987 Roger V. Short 1996 Norman B. Hecht James Buchanan Brady Urological Institute of the Johns Hopkins I iy Hospital, Baltimore, Maryland. Distinguished Andrologist Dr. Walsh received his M.D. in 1964 from Case Western Reserve University. From 1964 Award through I 967 he was a resident in surgery and pediatric Brian P. Setchell is the recipient of surgery at the Peter Bent Brigham Hospital and Boston the 1997 Distinguished Androlo­ Children's Hospital. From 1967 through 1971 he was a gist A ward. Dr. Setchell received urology resident at UCLA. In I 974 Dr. Walsh became the his B. V.Sc. with Honors from Syd­ Director and Chairman of Urology at Johns Hopkins ney University and his Ph.D. from where he has worked over the past 22 years perfecting the University of Cambridge in the surgical management of prostate cancer. He is the Ed­ 1957. Dr. Setchell was Veterinary itor-in-Chief of Campbell's Textbook of Urology pub­ Research Officer, Special Veteri­ lished by W.B. Saunders and is a member of the Institute nary Research Officer and subse­ quently Officer-in-Charge of the of Medicine of the National Academy of Sciences. In Nutritional Research Laboratory, Department of Agriculture, June, 1996 Dr. Walsh received the Charles F. Kettering Veterinary Research Station, Glenfield, New South Wales Medal from the General Motors Cancer Research Foun- from 1952-62. He was named Senior Research Scientist in dation for "The Most Outstanding Recent Contributions 1962 and then Principal Research Scientist of the Division to the Diagnosis or Treatment of Cancer". This award of Animal Physiology in Prospect, New South Wales from was shared with Dr. Malcolm A. Bagshaw, Professor 1962-69. He was awarded the title of Senior Principal Sci­ Emeritus of Radiation Oncology, Stanford University. Dr. entific Officer and then Senior Principal Scientific Officer, Walsh has received numerous other awards and citations. Department of Biochemistry, Agricultural Research Coun­ He has served on the Editorial Boards of journals such cil, Institute of Animal Physiology in Babraham, Cambridge from 1969-78. He moved to the Department of Physiology as the Journal of Urology, Fertility and Sterility, the Jour­ and maintained the title of Senior Principal Scientific Officer nal of Andrology and has edited many important Urology in the Agricultural Institute of Animal Physiology at Babra­ textbooks. Among his major original scientific contribu­ ham from I 978-82 where he also served as College Lec­ tions, he was the first to describe the 5a-reductase enzyme turer in Physiology. Since J 982, he has been Professor of deficiency. This work established the embryologic and Animal Sciences, Waite Agricultural Research Institute, physiologic consequences of dihydrotestosterone defi­ University of Adelaide, Glen Osmond, Australia, and Head ciency in man. He established the rat as a model to study of the Department of Animal Sciences from 1982-91. He penile erection. He showed experimentally that cavernous has received numerous awards and honors, including the nerve grafts could be used to restore erectile function. Regnier de Graaf Prize from The Netherlands, was named Goding Lecturer by the Australian Society for Reproductive One study focused upon the identification of an autosomal Biology, Monesi Lecturer at the Fourth Workshop and Ta­ dominant inheritance of Peyronie's disease. He is well pani Vanha-Perttula Memorial Lecturer at the Sixth Euro­ known for his studies on the prostate. Dr. Walsh dem­ pean Workshop on Molecular and Cellular Endocrinology onstrated that benign prostatic hyperplasia could be in­ of the Testis. Dr. Setchell was awarded Doctorate of Sci­ duced in the dog by hormonal manipulation and provided ences (Sc.D.) by Cambridge University in 1977, an unusual the first scientific evidence that estrogen may synergize honor. Dr. Setchell has made several of the contributions with androgen in the induction of BPH, as well as the that we consider the very basis of our understanding of male reversible influence of androgen deprivation on BPH. No­ reproductive physiology. He is credited with discovering the tably, he has developed a nerve-sparing method of radical blood-testis barrier and was the first to elucidate the physi­ ological concept of such a barrier between the testis and prostatectomy that preserves sexual function. Most re­ blood. He was one of the first scientists to investigate and cently, Dr. Walsh was the first to recognize and charac­ attempt isolation of inhibin-when many did not believe terize hereditary prostate cancer. This may lead to insight that inhibin existed. Of note, Dr. Setchell wrote the first into the genetic mechanisms responsible for prostate can­ article to appear in the Joumal of Andrology, Volume 1, cer. page 1. The publisher of Male Reproduction, Volume 17 of

P-7 the Benchmark Papers in Human Physiology Series, de­ subsequently Research Assistant Professor at Vanderbilt scribed him as follows, "A true Renaissance man, Dr. Setch­ in the Department of Cell Biology from 199 1-94. Dr. ell is fluent in several languages, played bassoon and con­ Cornwall travelled to Lubbock, Texas in 1994, where she trabassoon for many years in the Cambridge Philharmonic currently serves in an appointment as Assistant Professor Society Orchestra and is a student of both general and med­ in the Department of Cell Biology and Biochemistry at ical history-in short, an ideal individual to guide us the Texas Tech University Health Sciences Center. Dr. through the significant history of male reproductive biolo­ gy." Dr. Setchell has translated the entire works of Regnier Cornwall has worked extensively in the area of sperm de Graaf on male and female reproduction (published by maturation and studied the acquisition of sperm motility the Journal of Reproduction & Fertility as Supplement 17). that occurs in the epididymis. Her pre-doctoral work at He has also translated the original paper by Sertoli, describ­ The Johns Hopkins University was performed under the ing the somatic supporting cells of the seminiferous tubules. direction of Dr. Thomas S.K. Chang and Dr. Larry Ewing. Dr. Setchell has served on, or currently serves as, a member She continues to investigate epididymal biology, using of the Council of Management for Biology of Reproduction, molecular techniques that have allowed the isolation of a Council of Management for the Journal of Reproduction novel and reproductive-specific gene, CRES (cystatin-re­ and Fertility, the Editorial Boards of Molecular & Cellular lated epididymal-spermatogenic). Her work has involved Endocrinology, Reproduction, Fertility and Development, and Reproduction, Nutrition and Development. Dr. Setchell not only isolating this gene but characterizing its expres­ is recognized for having and trying out new ideas that may sion pattern. The highly transient nature of protein ex­ not have been accepted science at the time of their inves­ pression in the male reproductive tract suggests that tigation. He is credited for having imaginative insight into CRES may play a specialized role in epididymal and tes­ male reproductive physiology and has applied careful sci­ ticular function. Ongoing NIH support allows her to fur­ ence to further our understanding of testicular function. He ther evaluate the role of this important, highly conserved has supervised at least twenty Ph.D. candidates for Cam­ gene, CRES, in the process of sperm development and bridge and Adelaide Universities as well as the National maturation. Dr. Cornwall has received numerous student Council for Academic Awards of the United Kingdom. For research awards from the American Society of Androlo­ his unique and original contributions to andrology that are considered the basis for our understanding of testicular gy, and she has been an invited presenter at ASA Post­ physiology, his active enthusiasm for research and teaching, graduate Courses, the Gordon Research Conference, and as well as his support of young scientists entering the study recognized by an invitation to write a review on epidid­ of andrology, the Society is delighted to award Dr. Brian P. ymal-specific gene expression in the Journal of Androl­ Setchell with the 1997 Distinguished Andrologist Award. ogy. She serves as a member of the Editorial Board of the Journal of Andrology, member of the Student Affairs Committee, Program Committee and Long-Range Plan­ Distinguished Andrologists ning Committees for the American Society of Andrology. 1976 Roy 0. Greep 1986 Alfred D. Jost Dr. Cornwall serves as an excellent role model for young M. C. Chang 1987 Emil Steinberger scientists, especially women in andrology. Her profes­ 1977 Roberto E. Mancini 1988 Yves W. Clermont 1978 Robert J. Hotchkiss 1989 C. Alvin Paulsen sional, yet warm interactions with colleagues and students 1979 Thaddeus Mann 1990 Marie-Claire Orgebin-Crist are remarked upon universally by any scientist who has 1980 John MacLeod 1991 Philip Troen 1981 Alexander Albert 1992 C. Wayne Bardin had the privilege to work with her. Her scientific presen­ 1982 Eugenia Rosemberg 1993 Anna Steinberger tations are always clear and precise. For her research, her 1983 Kristen B. D. Eik-Nes 1994 Richard J. Sherins 1984 Mortimer B. Lipsett 1995 Rupert P. Amann character, and continued contributions to andrology, the 1985 Robert H. Foote 1996 1. Michael Bedford American Society of Andrology is delighted to award Dr. Gail A. Cornwall with the 1997 Young Andrologist Award.

Young Andrologist A ward Young Andrologists Gail A. Cornwall is the recipient 1990 Luis Rodriguez-Rigau of the 1997 Young Andrologist 1982 L.J.D. Zaneveld 1983 William B. Neaves 1991 Patricia M. Saling Award. Dr. Cornwall received her 1984 Lonnie D. Russell 1992 Gary R. Klinefelter B.S. and M.S. from Brigham 1985 Bruce D. Schanbacher 1993 Robert Chapin 1986 Stephen J. Winters 1994 Wayne J.G. Hellstrom Young University and her Ph.D. in 1987 Ilpo T. Huhtaniemi 1995 Christopher 1. De Jonge Reproductive Biology from The 1988 Larry Johnson 1996 Paul S. Cooke Johns Hopkins University School 1989 Barry T. Hinton

of Hygiene and Public Health in Sponsored by the Texas Institute for Reproductive Medicine 1988. She was an NIH NRSA fel- and Endocrinology, P.A. low at Vanderbilt University and

P-8 mittee for the Center for Population Research, as a mem­ Distinguished Service A ward ber of the Population Research Committee for NICHD, as a member of the Reproductive Biology Study Section, Marie-Claire Orgebin-Crist is the as a Key Consultant to the NICHD Five Year Research recipient of the 1997 Distin­ Plan in 1980, as well as a member of the Five Year Re­ guished Service Award. Dr. Or­ search Plan for the Reproductive Sciences Branch of the gebin-Crist received her License Center for Population Research, NICHD, in 1996. Dr. Or­ of Natural Sciences and License of gebin-Crist has also served the World Health Organiza­ Biology from the University of tion, as a member of the task force on methods for the Paris in 1957 and her Doctorate of Regulation of Male Fertility (1972-75) and ad hoc mem­ Sciences from the University of ber of the Review Committee on Resources for Research Lyon, in France, in 1961. After ( J 985-90). She has served or continues to serve as a l ' post-doctoral work in the Depart­ member of the Editorial Boards of Andrologia, Biology ment of Biochemistry, Faculty of Medicine in Paris as of Reproduction, Journal of Andrology, and International well as the National Center for Scientific Research in Par­ Journal ofAndrology, as well as Associate Editor for Ga­ is, she served as a Research Associate at The Population mete Research, Molecular Reproduction & Development Council in New York in 1962-63 and went to Vanderbilt and Revue Maghrebine d'Endocrinolgie-Diabete et Re­ University, Nashville, Tennessee in 1963. Dr. Orgebin­ production. One of her greatest contributions to androl­ Crist has remained at Vanderbilt since that time, starting ogy has been in the training and support of numerous as a Research Associate in 1963, was promoted to Re­ Postdoctoral Fellows, Visiting Scientists, and Ph.D. stu­ search Instructor in 964, to the level of Assistant Pro­ I dents. Many of her former students have gone on to be fessor in 1966, and to Associate Professor in 1970. Dr. active contributors to the American Society of Andrology Orgebin-Crist was awarded full Professorship and named and leaders in the field of andrology. Not only as she as occupant of the Lucius E. Burch Chair of Reproductive made extensive contributions to andrology service, train­ Biology and Director of the Division as well as Center ing and research, but Dr. Orgebin-Crist has also fostered for Reproductive Biology Research in 1973. She was also new scientists by making them feel welcome into the fam­ named as a Professor in the Department of Cell Biology ily of andrologists. For her active service, enthusiastic in 1975. Dr. Orgebin-Crist has had a distinguished re­ support and extensive involvement in andrology, the So­ search career, with invaluable contributions to our under­ ciety is delighted to award Dr. Marie-Claire Orgebin-Crist standing of the function and relevance of the epididymis. with the Distinguished Service Award. She received the Distinguished Andrologist Award from the American Society of Andrology in 1990, and served as the second editor of the Journal of Andrology. She was Distinguished Service A ward the Serono Lecturer in 1995 and is currently the past Pres­ ident of the Society, having served as President from Recipients 1995-96 and Vice President from 1994-95. Dr. Orgebin­ 1994 C. Alvin Paulsen 1996 Philip Troen Crist was recipient of the Presidential Award of ASA in 1995 Andrzej Bartke 1982, made an honorary member of the Canadian Fertility & Andrology Society in 1985, and received the Distin­ guished Scientist A ward from the American Society for Reproductive Medicine (formerly known as the American New Investigator Award Fertility Society) in J 996. She has served as chair of the ASA Nominations Committee (1981 -83), Program Com­ Recipient will be announced at the Awards Ceremony on mittee (1977-78), and International Liaison Committee Monday, February 24, 1997 at 3:30 PM. ( 1993-95), as well as the Executive Council from 1978- 81. In addition, Dr. Orgebin-Crist has served on Publi­ cations (1975-76), Finance (1980-81), Membership New Investigator A ward Recipients

(1978-79), Nominations (1975-76) and Program 1983 Thomas T. Tarter 1990 Donna 0. Bunch Committees ( 1980-81) for the ASA. Dr. Orgebin-Crist 1984 Peter S. Albertson 1991 Robert Viger has also been very active in the International Society of Randall S. Zane 1992 John Kirby 1986 Mark A. Hadley 1993 Michael A. Palladino Andrology, currently serving on both the Executive 1987 Peter Grosser 1994 Linda R. Johnson Council (since 1989) and Program Organizing Commit­ 1988 Stuart E. Ravnik 1995 Mehdi A. Akhondi tees (since J 993). Her involvement with the Society for 1989 Tracy L. Rankin 1996 Wei Gu Daniel B. Rudolph the Study of Reproduction has also been extensive. She has played an important role for the National Institutes of Sponsored by the West Michigan Reproductive Institute, P.C. Health, serving as member of the Contract Review Com-

P-9 The American Society of Andrology wishes to Supporters of the 1997 Annual thank the following organizations for their generous support: Meeting & Postgraduate Course

Gold Club Abbott Laboratories Alza Pharmaceuticals A minimum $10,000 contribution to an ASA American Urological Association Endowment Fund California Cryobank, Inc. Genetics & IVF Institute Buckeye Urology and Andrology, Inc. Jewish Hospital HealthCare Services, Louisville, Kentucky Silver Club Merck & Co., Inc. Pfizer U.S. Pharmaceuticals Group minimum contribution to an A $5,000 ASA Anna Steinberger, Ph.D. Endowment Fund

National Medical Enterprises We st Michigan Reproductive Institute, P.C.

Sustaining Sponsor

A minimum $500 contribution to ASA fo r five or more years

Hamilton Thome Research Pharmacia & Upjohn Serano Laboratories, Inc. Texas Institute for Reproductive Medicine and Endocrinology, P.A.

Future Meetings l\1arch 28-31, 1998 23rd Annual Meeting of the American Society of Andrology and Postgraduate Course Hyatt Regency Long Beach, Long Beach, California, USA Contact: ASA Executive Offices 74 New Montgomery, Suite 230, San Francisco, California 94105 phone (4 15) 764-4823; fax (415) 764-49 15; e-mail [email protected].

April 9-12 or April 16-19, 1999 24th Annual Meeting of the American Society of Andrology and Postgraduate Course Chicago, Illinois, USA Contact: ASA Executive Offices 74 New Montgomery, Suite 230, San Francisco, California 94 105 phone (415) 764-4823; fax (415) 764-49 15; e-mail [email protected].

P- 10 Women in Andrology Luncheon

"Successful Mentoring"

Chairs: Donna Vo gel, Ph.D. and Dolores Lamb, Ph.D.

Speaker: Anna Steinberger, Ph.D.

Date: Sunday, February 23, 1997

Time: 12:00-12:30 PM (Business Meeting) 12:30-1 :30 PM (Speaker and Lunch)

Place: Chesapeake Room AIB

(Registration fe e: $22 (includes lunch)-see Registration Form)

Laboratory Science Forum

"The Basics of Gamete Cryopreservation"

Chair: Susan Tarchala, B.S., T.S.

Speaker: Grace Centola, Ph.D. University of Rochester

Date: Monday, February 24, 1997

Time: 12:00-1:30 PM

Place: Maryland Suites-Columbia Room

(Lunch available for $20-See Registration Form)

Participants will learn about donor selection and screening, cryopreservation, client depositor versus donor and receive an update on oocyte and embryo cryopreservation and donation.

Student Colloquium (Sponsored by: California Cryobank, Inc., Los Angeles, CA)

"On Human Destiny"

Chair: Grace Centola, Ph.D.

Speaker: Donald Coffey, Ph.D. Johns Hopkins School of Medicine

Date: Monday, February 24, 1997

Time: 7:00-8:30 PM

Place: Maryland Suites-Annapolis/Baltimore Rooms

P- 11 Andrology Laboratory Workshop

(Hosted by: 1997 Annual Andrology Laboratories Committee (ALC) in Conjunction with the American Society of Andrology)

"Moving Beyond Boundaries: Clinical Andrology in the 21st Century"

Director: Christopher De Jonge, Ph.D., H.C.L.D.

Chairs: Christopher Barratt, Ph.D., Sheffield, UK Pasquale Patrizio, M.D., Philadelphia, PA

Topics: "Introduction to Molecular Biology: The Language & Methods" "Recombinant Protein Te chnology in the Andrology Lab" "Molecular Biology of Signal Transduction Mechanisms in Sperm-Zona Binding" "Clinical Application of Molecular Biology: Cystic Fibrosis Mutations, Y-Chromosome Deletions and the Future" "DNA Breakage: Why is it Important for the Andrologist?"

Date: Tuesday, February 25, 1997

Time: 8:00 AM-5 :00 PM

Place: Location to be announced

Course Description Molecular genetic techniques are greatly advancing our understanding of many events taking place during the reproductive process. However, for many, the language and the methodologies of these techniques remain obscure. This course is specifically designed to provide the key background information to facilitate the understanding, the current use and the potential applications of molecular biology techniques in the field of andrology. The course is structured in a morning session where the participants will be introduced to the basic language and to the methodologies currently in use (i.e., PCR for DNA and RNA cloning, recombinant DNA, testis libraries, in situ hybridization, etc.). The afternoon session will cover the clinical application of these techniques. Finally, to enable attendees to verify their understanding of the topics covered, the participants will be organized in small groups and assigned each with a specific genetic problem to be solved with the help of the faculty.

Course Objectives After attending this course, the participant will be able to: l. Understand the language and the methodologies used in molecular biology; 2. Describe the application, power and short falls of each technique; 3. Interpret current literature in genetic andrology; 4. Identify new areas of research in both basic science and clinical andrology.

The course is specifically designed for andrologists, whether clinicians or laboratory directors, biologists, technicians, researchers and students.

Registration fe e is $125 (ASA Student Member) or $150 (Student Nonmember) and $175 (ASA Member) or $200 (Nonmember), with a late fee of $25 after January 20, 1997.

P- 12 SOCIAL/MEETING EVENTS AT A GLANCE

Friday, February 21, 1997 ------

12:00 NOON-1 1:00 PM Executive Council Meeting (Chesapeake Room A/B)

Saturday, February 22, 1997 ------

8:00 AM-5 :00 PM Postgraduate Course (Constellation Ballroom A)

6:00 PM-7:00 PM Student Mixer (Maryland Suites-Baltimore Room)

7:00 PM-9:00 PM We lcoming Reception (Atrium Lobby) Exhibits Open (Constellation Ballrooms E, F)

9:00 PM-1 1:00 PM Executive Council Meeting (Chesapeake Room A/B)

Sunday, February 23, 1997 ------

7:45 AM-8:00 AM President's Welcome and Opening Remarks from Program and Local Arrangements Chairpersons (Constellation Ballroom A)

8:00 AM-4:30 PM ASA Meeting (Constellation Ballroom A)

8:00-9:00 AM Serono Lecture: "Genetics of Prostate Cancer" (Constellation Ballroom A)

9:00 AM-5 :00 PM Exhibits Open (Constellation Ballrooms E, F)

9:00-10:00 AM American Urological Association Lecture: "New Medical Treatments of Impotence" (Constellation Ballroom A)

10:00-10:30 AM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

10:30-12:00 NOON Oral Session I: "Genes and Male Reproduction" (Constellation Ballroom A)

12:00 NOON-1 :30 PM Lunch (on your own)

12:00 NOON-1:30 PM Wo men in Andrology Luncheon (Chesapeake Room A/B) Business Meeting 12:00 NOON-12:30 PM Speaker and Lunch 12:30-1 :30 PM

1 :30-3:00 PM Symposium I: "Regulation of Testicular Growth and Function" (Constellation Ballroom A)

3:00-3 :30 PM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

3:30-4:30 PM Oral Session II: "Calcium Channels and Male Reproduction" (Constellation Ballroom A)

4:30 PM-6:30 PM Poster Session I (Constellation Ballrooms C, D)

7:30 PM-11:00 PM Evening Social and Buffet Dinner (National Aquarium)

P- 13 l\'londay, February 24, 1997 ------

7:00 AM-8:00 AM Past-Presidents' Breakfast (Pratt/Calvert Rooms)

8:00 AM-4:30 PM ASA Meeting (Constellation Ballroom A)

8:00-9:00 AM Oral Session III: "Sex Accessory Organs" (Constellation Ballroom A)

9:00 AM-5 :00 PM Exhibits Open (Constellation Ballrooms E, F)

9:00-9:30 AM ASA Lecture: "The Use and Abuse of Te sticular Sperm As Viewed From A Thirty Year Experience With Epididymal Reconstructive Surgery" (Constellation Ballroom A)

9:30-10:00 AM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

10:00-1 1:00 AM Pharmacia & Upjohn Lecture: "Sex Chromosome Genes and Spermatogenesis" (Constellation Ballroom A)

11:00-1 2:00 NOON Buckeye State-of-the-Art Lecture: "Reproductive Rescue of Endangered Species" (Constellation Ballroom A)

12:00-1 :30 PM Lunch (on your own)

12:00 NOON-1:30 PM Simultaneous Events:

Editorial Board Meeting/Luncheon (Executive Boardroom) Laboratory Science Forum Luncheon (Maryland Suites-Columbia Room)

1:30-3:00 PM Symposium II: "Estrogen in the Male: Hormonal Carcinogenesis, Toxicology, and Role in Normal Reproductive Function" (Constellation Ballroom A)

3:00-3:30 PM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

* 3:30 PM-4:30 PM ASA Business Meeting/Awards Ceremony (Constellation Ballroom A)

4:30 PM-5:00 PM Exhibitors Prize Drawing (Constellation Ballrooms E, F)

5:00 PM-7:00 PM Poster Session II (Constellation Ballrooms C, D)

7:00 PM-8 :00 PM Student Colloquium "On Human Destiny" (Maryland Suites-Annapolis/ Baltimore Rooms)

Tuesday, February 25, 1997 ------

8:00-12:00 NOON Andrology Laboratory Workshop (location to be announced)

8:00-9:30 AM Oral Session IV: "Andrology and Assisted Reproductive Techniques" (Constellation Ballroom A)

9:30-10:00 AM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

10:00-12:00 NOON Plenary Session: "Normal and Abnormal Prostate" ASA/National Medical Enterprises State-of-the-Art Lecture: "Gene Therapy of Prostate Cancer" (Constellation Ballroom A)

12:00-1:00 PM Lunch (on your own)

1:00-5 :00 PM Andrology Laboratory Workshop (location to be announced)

P-14 22nd Annual Meeting

Sunday, February 23

7:45-8:00 AM Welcome and Opening Remarks (Constellation Ballroom A) Arnold Belker, President Te rry Brown, Local Arrangements Chairperson

8:00-9:00 AM Serono Lecture (Constellation Ballroom A) Patrick Wa lsh "Genetics of Prostate Cancer" Chairperson: Dolores Lamb

9:00-I 0:00 AM AUA Lecture (Constellation Ballroom A) Irwin Goldstein "New Medical Treatments of Impotence" Chairperson: Wayne Hellstrom

10:00-10:30 AM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

10:30-12:00 NOON Oral Session I Genes & Male Reproduction (Constellation Ballroom A) Chairpersons: Shalender Bhasin, Craig Niederberger

10:30 AM 1 cDNA cloning, chromosomal mapping, and tissue-distribution of the human axonemal dynein light chain gene: A candidate gene for the immotile cilia syndrome I K. Kastury, W.E. Taylor, S. Arver, C.E. Fisher, M. Gutierrez, S. Bhasin- 10:45 AM 2 Y-Deletions in men with severe oligospermia I E.J. Meuleman, J.A. Kremer, J.H. Tuerlings 11:00 AM 3 Hst-7: A male sterility mutation perturbing sperm motility, ftagellar assembly, and mi­ tochondrial sheath differentiation I S.H. Pilder, P. Olds-Clarke, J.M. Orth 11: 15 AM 4 Dysplasia of the fibrous sheath and dynein deficiency, a cytoskeletal abnormality of hu­ man spermatazoa I H.E. Chemes 11:30 AM 5 Interaction of sterility factors in the t haplotype prevents sperm penetration of the zona­ free egg IP.Olds-Cla rke, L. H. Johnson, S.H. Pilder 11:45 AM 6 Cloning and characterization of the human SCPl gene encoding a major component of meiotic synaptonemal complexes IN. Kondah, Y. Nishina, M. Koga, K. Uchida, H. Tanaka, M. Kitamura, K. Matsumiya, Y. Nishimune, A. Okuyama.

12:00-1:30 PM Lunch (on your own) 12:00-1:30 PM Women in Andrology Luncheon (Chesapeake Room NB) Anna Steinberger "Successful Mentoring" Chairpersons : Donna Vogel, Dolores Lamb ' Business Meeting 12:00-12:30 \u'V tf)j\ Speaker and Lunch 12:30-1 :30

1:30-3:00 PM Symposium I: "Regulation of Testicular Growth and Function" (Ballroom A) Chairpersons: Ken Roberts, Michael Griswold Patricia Morris "Testicular JAK-STAT Signalling Pathways: Cytokines, Growth Factors, and Spermatogenesis" Martin Matzuk "Transgenic Models to Study Male Reproductive Function"

3:00-3:30 PM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

3:30-4:30 PM Oral Session II Calcium Channels and Male Reproduction (Ballroom A) Chairpersons: Susan Benoff, Joel Marmar

3:30 PM 7 Investigation into the mechanism underlying infertility associated with calcium (Ca1+) channel blockers I A. Jacob, LR. Hurley, A. Hershlag, S. Benoff 3:45 PM 8 Isolation and characterization of the rat testes-specific voltage dependent calcium (Ca1+) channel (VDCC) I L.O. Goodwin, N.B. Leeds, S. Benoff 4:00 PM 9 A challenge to the concept that the use of calcium channel blockers cause reversible male infertility I D. Katsoff, J.H. Check, D.J. Check 4:15 PM 10 Identification of fu nctional progesterone receptors on the surface of human spermatozoa I G. Forti, M. Maggi, E. Baldi, M. Luconi, L. Bonaccorsi

P-15 4:30-6:30 PM POSTER SESSION I-Includes Student Award Candidates (Constellation Ballrooms C, D)

Sperm

� Impact of abnormal semen parameters on the genetic status of human spermatozoa I L. T. ({_/Colomb ero, J. Hariprashad, F. Moy, Z. Rosenwaks, G.D. Palermo 12 Signal transduction by novel sperm chemoreceptors as a mechanism for mammalian sperm-egg chemotaxis I L.D. Walensky, M. Ruat, R. Bakin, G.V. Ronnett, S.H. Snyder 13 Calcium flux through sperm inositol 1,4,5-trisphosphate receptors is implicated in trig­ gering the mammalian acrosome reaction I L.D. Walensky, S.H. Snyder 14 Effect of sodium nitroprusside on sperm motility: A dose-response curve I F.G. Martins, H.M. Nagler, S. Goldberg, N. Virji 15 Effect of tumor necrosis factor (TNF-a) and interferon (IFN-j') on human sperm motility, viability, and motion parameters I H.C. Champion, L.S. Estrada, R. Wang, A. Baratta, M. Rajaskaran, S.C. Sikka, W.J.G. Hellstrom 16 Antioxidant capacity of seminal plasma compared to synthetic and biological free radical scavengers I J.S. Armstrong, M. Rajaskaran, A. Baratta, W.J.G. Hellstrom, S.C. Sikka 17 Do peptides stimulate sperm fu nction? I R.Wang, C.T. Da Ros, M. Barcelos, H.Champion, S.C. Sikka, W.J.G. Hellstrom 18 The relationship between serum testosterone and nutritional status in patients with non­ small cell lung cancer I E.J. Maher, S. O'Reilly, D. Hoover, D.S. Ettinger, J. Whalen, A.S. Dobs 19 Transesterification of human sperm phospholipids with exogenous fatty acid coenzyme A thioesters I J.G. Alvarez, S. Balgobin 20 A role for SGP-1 in binding of sperm to eggs I R.H. Hammerstedt, P. Cramer, G.F. Barbato, R.P. Amann

Epididymis

® Epididymal protein pattern in infertile ornidazole treated rats I A. Wagenfeld, C.H. Yeung, T. G. Cooper 22 Immunoexpression of FAS (CD95) ligand (FasL) in the rat testis and epididymis I F.C. Griffin, D.F. Cameron 23 Oxidative stress differentially regulates expression of gamma-glutamyl transpeptidase mRNA in the initial segment of the rat epididymis I C.M. Markey, D.B. Rudolph, J.C. Labus, B.T. Hinton 24 Identification and expression of the ETS transcription factor polyomavirus enhancer ac­ tivator 3 (PEA3) in rat epididymis, and its regulation by testicular factors I Z.J. Lan, M.A. Palladino, D.B. Rudolph, J.C. Labus, B.T. Hinton 25 The status of cauda epididymal sperm DNA following partial sympathetic denervation I f:':\ M. Schwartz, D.D. Ricker � Epithelial cell pathology of the epididymis in adult and elderly men I J. Regadera, F. Martinez-Garcia, P. Cobo, J. Palacios, M. Nistal

- Environment and Reproduction

27 Urinary creatine as a biomarker for lead induced testicular damage I S.R. Skaggs, W.J. Moorman, D.D. Sharpnack, S.M. Schrader 28 The use of silastic condoms in an exposure assessment occupational field study I S.M. Schrader, T. Arbuckle, L. Turner, L. Ritter 29 Sertoli cells in culture and mRNA differential display provide a sensitive early warning assay system to detect xenobiotics IV. Syed, W. Gu, N.B. Hecht A morphological study of the epididymis and testis of rats UChA and UChB submitted to experimental chronic alcoholism I E. Bustos-Obregon, F.E. Martinez, M. Martinez, V.H.A. Cagnon, P.J. Garcia, C.R. Padovani, H.R. Contreras 31 No decline in Sertoli cell number or function in men over the last 16 years I L. Johnson, J.J. Barnard 32 No decline in spermatogenic potential in a group of North American men over the last 16 years IL. Johnson, J.J. Barnard, H. Meguerditchian 33 Decline in Leydig cell volume in a group of North American men over the last 16 years I L. Johnson, S.W. Brown, J.J. Barnard

P- 16 Impotence

34 Proadrenomedullin N-terminal 20 peptide (PAMP), a novel product of the adrenomedul­ lin gene, induces penile erection in the cat I H.C. Champion, R. Wa ng, W. E. Fitzgerald, W. A. Murphy, D.H. Coy, P.J. Kadowitz, W.J .G. Hellstrom 35 Age-related alterations in plasma nitrite and nitrate levels in impotent men I M. Rajase­ karan, W.J. Hellstrom, S.C. Sikka 36 Penile erection induced by transurethral administration of novel nitric oxide donors I W.J .G. Hellstrom, R. Wang, H.C. Champion, S.C. Sikka, L.K. Keeffer, P.Doherty 37 cx1-Blockers enhance penile erection effect of PGE1 in cats I R. Wang, H. Champion, S.C. Sikka, P. Doherty, W.J.G. Hellstrom 38 ATP'YS, a potent purinergic agonist, induces penile erection in the cat I H.C. Champion, R. Wang, D.G. Lambert, P.J. Kadowitz, W.J .G. Hellstrom 39 Galanin induces penile erection in the cat I H.C. Champion, R. Wang, W. A. Murphy, D.H. Coy, P.J. Kadowitz, W. J.G. Hellstrom 40 Androgen regulation of rat penile erection I C.M. Reilly, V. S. Stopper, T. M. Mills

Prostate

41 Assessment of the male accessory glands of infertile men with kallikrein hK2 in seminal plasma I R.R. Tremblay, E. Coulombe, G. Frenette, J.Y. Dube 42 The effects of spinal cord injury on TRPM and androgen receptor mRNA in the prostate of the rat I H.F.S. Huang, M-T Li, T. A. Linsenmeyer, J.E. Ottenweller, L.M. Pogach, R.J. Irwin 43 Isolation and purification of basal and secretory epithelial cells from the ratventral pros­ tate IN. Ravindranath, M. Dym Intraprostatic injection of zinc salt as a treatment for benign prostatic hyperplasia I M. ® Wang, J. Cao, M.S. Fahim 45 Osteopenia in men with prostate cancer treated with androgen deprivation I D.A. Cook, M. Eisenberger, C.R. Schneyer, D. Hoover, A.S. Dobs 46 The regulation of prolactin and EGF receptor mRNAs in human prostate cancer cell line (LNCaP) by LHRH I J-Y, Liang, J.N. Rao, J.F. Wilber, P. Feng 47 Androgen ablation induced cell kinetic changes in a rat prostate tumor I M.L. Meistrich, R.A. White, C. Sikes, D.L. Joon, C.S. Wu, M. Hasegawa, N.H.A. Terry, G.K. Zagars, A. Pollack 48 The cynomolgus monkey prostate under physiological and hypogonadal conditions: an ultrasonographic study I A. Kamischke, H.M. Behre, G.F. We inbauer, E. Nieschlag

Testis

49 Developmental shift of inhibin or inhibin related proteins producing site in fe tal through infantile rat testes I J. Noguchi, H. Hikono, S. Sato, K. Kikuchi, A. Shimada, H. Kaneko, Y. Hasegawa, G. Watanabe, K. Tay a 50 Local testicular control mechanism enabled the total number of Sertoli cells found in an intact rat to be exceeded I L. Johnson, K.L. Kunde, G.E. Keillor, A.J. Kaschmitter 51 Effects of exercise on testosterone and nitric oxide production in the rat testis I V.J.

Meskaitis, F.S. Harman, J.S. Volek, B.C. Nindl, W.J. Kraemer, D. Weinstock, D.R. Deaver 52 Paternal age affects progeny outcome in the Brown Norway Rat IV. Serre, B. Robaire 53 Stage-specific loss of germ cells in the rat after a single exposure to heat occurs by apop­ tosis I Y. H. Lue, A.P.S. Hikim, P. Im, K.S. Tiang, T. Bui, A. Leung, R. Swerdloff, C. Wang 54 Biochemical characterization of Sertoli cell (SC) membrane proteins that are likely to be involved in specialized junctional complex (JC) formation ID Mruk, W.M. Lee, C.Y. Cheng 55 TPX-1 gene is specifically transcribed by normal human germ cells during the late stage of spermatogenesis IN. Tokuda, S. Matsuzaki, K. Nomoto, J. Kumazawa 56 Sertoli cell cytoskeletal and related proteins, and cx2-macroglobulin, are not involved in inhibited spermiation (l.S.) I R.N. Wine, R.E. Chapin 57 5-Azacytidine treatment initiated during meiotic development of male germ cells results in abnormal embryo development I T.E. Doerksen, J.M. Trasler 58 Expression of cyclin H and cyclin-dependent kinase 7 in the mouse testis I J.T. McGaughy, S.E. Ravnik 59 Effect of thyroid hormone on testicular development and mRNA levels of thyroid hor­ mone receptors in the testes of immature and adult rats I J.N. Rao, J-Y. Liang, J.F. Wilber, P. Feng

P- 17 60 Identification of two kinetically distinct activities of 11�-hydroxysteroid dehydrogenase in rat Leydig cells I R.S. Ge, H.B. Gao, V. Lakshmi, G.L. Gunsalus, M.P. Hardy

7:30- 11:00 PM Banquet (National Aquarium)

Monday, February 24

7:00-8:00 AM Past President's Breakfast (Pratt/Calvert Rooms)

Oral Session III Sex Accessory Organs (Constellation Ballroom A) Chairpersons: Barry Hinton, Donald Tindall

8:00 AM 61 Nitric oxide as a factor in growth of the canine prostate I J.K. Crone, S.L. Chamness, T. S.K. Chang, J.D. Strandberg, A.L. Burnett 8: 15 AM 62 Synergism of estradiol and testosterone on brown Norway rat prostate growth I P. P. Ba­ nerjee, S. Banerjee, B.R. Zirkin, T. R. Brown 8:30 AM 63 Loss of testicular factors causes changes in the stability and transcription rate of gamma­ glutamyl transpeptidase mRNA-IV in the initial segment of the rat epididymis I D.B. Rudolph, B.T. Hinton 8:45 AM 64 Ductuli efferentes in estrogen receptor knock-out mice do not reabsorb luminal fluid I R.A. Hess, D. Bunick, J. Bahr, D.B. Lubahn

9:00-9:30 AM ASA Lecture (Constellation Ballroom A) Robert Schoysman "The Use and Abuse of Testicular Sperm As Viewed From A Thirty Year Experience With Epididymal Reconstructive Surgery" Chairperson: Arnold Belker

9:30-10:00 AM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

10:00-1 1:00 AM Pharmacia & Upjohn Lecture (Constellation Ballroom A) Colin Bishop "Sex Chromosome Genes and Spermatogenesis" Chairperson: Peter Schlegel

11:00-12:00 NOON Buckeye State-of-the-Art Lecture (Constellation Ballroom A) JoGayle Howard "Reproductive Rescue of Endangered Species" Chairperson: Terry Brown

12: 0-1:30 PM Lunch (on your own) 2:00-1:30 PM Simultaneous Events 1. Laboratory Science Forum (Maryland Suites-Baltimore Room) Grace Centola "The Basics of Gamete Cryopreservation" Chairperson: Grace Centola - 2. Editorial Board Meeting/Luncheon (Executive Boardroom)

Symposium II (Constellation Ballroom A) "Estrogen in the Male: Hormonal Carcinogenesis, Toxicology, and Role in Normal Re­ productive Function" Chairperson: Rebecca Sokol t f

3:00-3:30 PM Refreshment Break/Exhibits (Constellation Ballrooms E, F) E0 ASA Business Meeting and A wards Ceremony (Constellation Ballroom A)

P- 18 5:00-7:00 PM POSTER SESSION II (Constellation Ballrooms C, D)

Animal Science

65 Effect of testicular biopsy and triethylenemelamine on testis function and semen quality in the dog I R.H. Foote 66 Sensitivity of domestic cat sperm to cold-induced acrosomal damage I B.S. Pukazhenthi, K.M. Pell, D.E. Wildt, J.G. Howard 67 Seasonal comparison of semen parameters in captive cheetahs I B.S. Durrant, J.K. Yamada, S.E. Millard, C.D. Burch, D.M. Zimmerman 68 Ej aculate characteristics of the black-handed spider, southern black howler and diana monkey I J.A. Long, N. Lamberski, A.H. Shoemaker 69 Repeated follicular stimulation in the endangered lion-tailed macaque (Macaca silenus) with recombinant human gonadotrophins I M. Cranfield, N. Berger, J. Liu, J. Smart, D. Summers 70 The use of intracytoplasmic sperm injection (ICSI) to enhance the conservation of the endangered lion-tailed macaque (LTM) (Macaca silenus) IM. Cranfield, J. Liu, D. Summers, J. Smart, N. Berger 71 Increased fe rtility in turkeys resulting from semen donor selection I A.M. Donoghue 72 Characterization of human and turkey spermiophages and identification of turkey sper­ miophage precursor cells using a monoclonal antibody I F.C. Derrick Jr., B.P. Pooser, T.R. Scott, R.J. Thurston, N. Korn 73 Sperm-binding assay to detect males likely to have relatively low fe rtility I R.H. Ham­ merstedt, S.P. Gill, R.P. Amann, P.C. Cramer, G.F. Barbato 74 Testicular morphology and gonadotropin receptors reflect age-related alterations in func­ tion I M.A. Ottinger, N. Thompson, M.R. Bakst, K. Kubakawa, K. Kubakawa, M. Kikuchi, S. Ishii 75 Variability and power calculations of viability estimates in dutch belted rabbit sperm I M.J. Breitenstein, S.M. Schrader, S.D. Simon 76 Role of inhibin in the regulation of FSH secretion in immature bulls I H. Kaneko, J. Noguchi, K. Kikuchi, A. Shimada, G. Watanabe, K. Taya, Y. Hasegawa, K. Okuda

Sperm Processing

77 Comparison of monopercoll sperm separation with two-layer percoll method I K. Seifarth, D. Garlak, L. Cordek, C. Fitzhugh, A.J. Thomas, Jr., A. Agarwal 78 Microscopic sperm preparation might be a novel approach for intracytoplasmic sperm injection (ICSI) I A. Hossain, S. Barik, B. Rizk, S. Lynn Jr., I. Thorneycroft 79 Isolate® as a new method for sperm separation: A preliminary study I S.M. Tarchala, R.G. Rawlins 80 Changes in sperm morphology after preparation for in vitro fe rtilization (IVF) I D.S. Karabinus, C. Racowsky, L.A. Rudzitis, T.J. Gelety 81 Processing method of frozen donor sperm correlates with final sperm parameters and intrauterine insemination pregnancy outcome I S.K. Jindal, K.J. Lipetz 82 Frozen human sperm maintains motility following processing and refreezing I P.J. Jones, L. Thanh, J. Schulman, E. Fugger 83 Post-thaw plasma membrane integrity (PMI) of mouse sperm cryopreserved in media containing polyols and/or trehalose or raffinose I K.A. Thompson, E.E. Noiles, B.T. Storey 84 Treatment of human or bull sperm with a synthetic peptide increases binding to egg­ membrane substrate I R.P. Amann, R.H. Hammerstedt, R.B. Shabanowitz 85 Gender preselection in mammals: An update on flow cytometric X/Y sperm sorting I L.A. Johnson, G.R. We lch, W. Rens

Clinical Andrology

86 Vasectomy, vasovasostomy and MESA I ICSI: Is it the fu ture triad of vasectomised man who regrets vasectomy? M.T.W.T. Lock, H. Roshani 87 Evaluation of men with varicoceles for IVF, ICSI or other options ID. Glazier, J.L. Marmar, M. Gibbs, S.L. Corson 88 Micronuclei and other nuclear abnormalities in buccal mucosa in people exposed to tes­ tosterone or gonadotrophines IM. Diaz, 0. Torres-Bugarin, E. Vargas, M.P. Ramierz-Munoz, J. Sanchez-Corona, G. Zuniga 89 Rheumatologic disease in male hypogonadism I. Relationship with sexual hormones I R. Tap ia-Serrano, F.J. Balderas, M.E. Fonseca, C. Lara, A. Beltran, A. Fraga

P- 19 90 Testicular microlithiasis (TML) in men with azoospermia and severe oligospermia I I. H. Hirsch, R.l. Feld, L.G. Gomella, P. T. McCue 91 Are sperm motion parameters influenced by varicocele ligation? I l.H. Hirsch, M.T. Ismail, J. Sedor 92 Effects of Mullerian Inhibiting Substance on human sperm survival I Y. Siow, M.E. Fallat, F. A. Amin, S.C. Yoffe, A. M. Belker 93 Intrauterine insemination for cervical or male factor problems in women >40 have very poor viable pregnancy rates I J.H. Check, D. Lurie, M. Peymer, D. Katsoff, R. Long 94 Absence of glucose decreases human fe rtilization and sperm movement characteristics in vitro I M.M. Mahadevan, M.M. Miller, D.M. Moutos 95 Improvement in semen quality and frequency of spontaneous acrosome reaction after pentoxifylline addition in normozoospermic men I S.C. Esteves, R.K. Sharma, A.J. Thomas, A. Agarwal 96 Effect of peritoneal fluid on sperm motion characteristics and acrosome reaction in wom­ en with endometriosis I Y. Wang, R.K. Sharma, T. Falcone, A. Agarwal 97 Toxicity of lubricants on sperm I K.O. Pomeroy, W. Savage, M. Zimmer, D.V. Moffitt, J.H. Mattox 98 Vasectomy-related infertility: A common and expensive medical problem I A.M. Jequier 99 Progressive motility in computer-assisted sperm analysis (CASA) I V.L. Slott, S.D. Perreault 100 Conditioned media from cumulus cell cultures stimulate in vitro acrosome reaction I G.M. Centola, S.P. Weisensel, V. L. Lewis 101 Mitochondrial DNA, semen quality and clinical assessment I A.M. Jequier, R. Martin, D. Mehmet, J. Goldblatt, J.M. Cummins 102 CASA characterization of hyperactivated motility in hamster sperm I S.D. Perreault, S.C. Jeffay, D.F. Katz 103 A new category of infertility due to sperm-specific abnormality of CD46 IM. Kitamura, K. Matsumiya, M. Yamanaka, A. Okuyama 104 Creatine kinase (CK)-M isoform in human sperm : isolation of CK-M and development of a CK-M specific antibody I G. Huszar, L. Vigue

Contraception

105 Vaginal delivery of new formulations of nonoxynol-9 co-precipitated with polyvinylpyr­ rolidone: asessment of two formulation-delivery systems on the onset of pregnancy in rabbits I P.M. Zavos, J.R. Correa 106 Assessment of spermicidal efficacy of new formulations of nonoxynol-9 coprecipitated with polyvinylpyrrolidone in rabbits: comparison between two formulation-delivery systems I P.M. Zavos, J .R. Correa 107 Effects of an inhibitor of adenylate cyclase (AC) on sperm proacrosin activation IT. Toda, Y. Yamamota, I. Miyagawa, P.M. Zavos, N. Sofikitis 108 Cyproterone acetate (CPA) plus testosterone enanthate (TE) 100 mg/week induces a dose­ dependent suppression of spermatogenesis in normal men I M.C. Meriggiola, C. Flamigni, W.J. Bremner 109 Triptolide: A post-testicular antifertility agent I Y. H. Lue, A.PS. Hikim, A. Leung, S. Bar­ avarian, C.Wang, R.S. Swerdloff

Male Infertility Diagnosis

110 Test kit for the determination of sperm viability I J.G. Alvarez, S. Balgobin, R.D. Powers, G. Davis 111 Test kit forthe determination of leukocyte count in semen I 1.G. Alvarez, S. Balgobin, R.D. Powers, G. Davis 112 The sperm penetration assay: an important tool in the age of ICSI I J.D. Wininger 113 The fe rtility score test kit® detects high sperm density but is not as sensitive for low density I E.V. Younglai, G. Tuke, J.A. Collins, A. Vawda 114 Correlation between mannose ligand receptor expression, acrosome reaction, Kruger's morphology and sperm motility in normozoospermic men I R.K. Sharma, A.J. Thomas Jr., A. Agarwal 115 Role of electron microscopy in the evaluation of male infertility during the era of ICSI I D. Carbone, J. McMahon, H. Levin, A.J. Thomas Jr., A. Agarwal 116 Sperm fu nction tests in the assessment of semen quality I R.S. Sidhu, R.K. Sharma, Y. Wang, A.J. Thomas Jr., A. Agarwal

P-20 117 Differences in the manual and CASA results in semen specimens before and after freezing I R.S. Sidhu, R.K. Sharma, A.J. Thomas, A. Agarwal 118 Lipid peroxidation in cryopreserved semen from cancer patients I Y. Wang, R.K. Sharma, A.J. Thomas, A. Agarwal 119 Assessment of the relationship of sperm morphology with seminal and other clinical con­ ditions of the patients I A. Hossain, B. Rizk, R. Selukar, D. Bhaumik, C. Huff, J. Dyer­ William, R. Ye oman, I. Thorneycroft 120 Correlation of hypoosmotic swelling (HOS) test with vitality staining, motility and sper­ matic morphology I A. Arredondo-Pineda, C. Bravo-Gatica, P Troncoso-Torrez, R. Ta pia­ Serrano

7:00-8:00 PM Student Colloquium (Maryland Suites-Annapolis/Baltimore Rooms) "On Human Destiny" Donald Coffey Chairperson: Grace Centola

Tuesday, February 25

8:00-9:30 AM Simultaneous Event Oral Session IV (Ballroom A) Andrology and Assisted Reproduction Techniques Chairpersons: Luis-Rodriguez-Rigau, Juan Alvarez

8:00 AM 121 Different techniques to identify and label human round spermatids I L.T. Colombero, PN. Schlegel, M. Feliciano, M.L. Papale, M. Goldstein, Z. Rosenwaks, G.D. Palermo 8:15 AM 122 Cryopreservation of sperm collected by TESE provides adequate post-thaw viability and successful IVF-ICSI pregnancies I R. Dolgina, G. Wo lf, P Studney, B. Kaplan, C. Nieder­ berger, L. Ross, G.S. Prins

8:30 AM 123 Successful intercontinental genome resource banking and artificial insemination with cry­ opreserved sperm in cheetahs. J.G. Howard, T.L. Roth, W. F. Swanson, J.L. Buff, M. Bush, J. Grisham, L. Marker-Kraus, D. Kraus, D.E. Wildt

8:45 AM 124 Interpretation of CASA data using distribution based and multivariate statistical methods I D. Higdon, M. Lavine, J. Liu, S. Perreault, V. Slott, D. Katz 9:00 AM 125 Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine sperm I D.L. Garner, C.A. Thomas, H.W. Jorg, J.M. DeJarnette, C.E. Marshall

9: 15 AM 126 Lower concentration of sperm used for oocyte insemination for in vitro fe rtilization (IVF) may result in higher pregnancy rates: A preliminary study I J.H. Check, C. Hourani, K. Benfer, A. Baker

9:30-10:00 AM Refreshment Break/Exhibits (Constellation Ballrooms E, F)

10:00-12:00 NOON Plenary Session "Normal and Abnormal Prostate" (Ballroom A) Chairpersons: Gail Prins, Glenn Cunningham

ASA/National Medical Enterprises (NME) State-of-the-Art Lecture "Gene Therapy of Prostate Cancer" Jonathan Simon

"Novel Markers of Benign and Malignant Prostatic Disease-Do They Help in Diagno­ sis?" Wi lliam Nelson

"Androgen Regulated Prostatic Gene Expression" Martin Te nniswood

8:00-12:00 NOON Simultaneous Event Andrology Laboratory Workshop (location to be announced) "Moving Beyond Boundaries: Clinical Andrology in the 21st Century" Chairperson: Christopher De Jonge Program Directors: Christopher Barratt, Pasquale Patrizio

8:00-9: 15 AM "Introduction to Molecular Biology I: The Language" Ines Moretti-Roj as

9: 15-10: JO AM "Introduction to Molecular Biology II: The Methods" Ines Moretti-Rojas

JO: J0-10:30 AM Break

P-2 1 10:30-1 1: 15 AM "Recombinant Protein Technology in the Andrology Lab" Christopher Barratt

11:15-12:00 AM "The Use of Molecular Biology to Define Molecules Involved in Gamete Recognition and Signal Transduction During Fertilization" Greg Kopf

12:00 NOON Lunch (on your own)

1 :30-2:30 PM "Clinical Application of Molecular Biology: Cystic Fibrosis Mutations, Y-Chromosome Deletions and the Future" Mary Jo Kent-First, Pasquale Patrizio

2:30-3: 15 PM "DNA Breakage: Why is it Important for the Andrologists?" Donald Evenson

3: 15-3:30 PM Break

3:30-4:00 PM Break Out Session-Group Problem Solving 1. How to set up a service screening for Y-chromosome deletions for men attending ART clinics. 2. How to set up a testis library and what is the most effective way to screen the library 3. A purified protein of interest is supplied to your lab. How do you go about cloning the gene.

4:00-4:45 PM Group Discussion of Problems

4:45-5:00 PM Concluding Remarks

P-22 Index to Authors of Abstracts

A Cheng, C. Y., 54 G Johnson, L.H., 5 Cobo, P., 26 Jones, P.J., 82 Agarwal, A., 77, 95, 96, 114, Collins, J .A., I 13 Gao, H.-B., 60 H.irg, H. W., 125 115, 116, 117, 118 Colombero, LT., 11, 121 Garcia, P.J., 30 Alvarez, J.G., 19, I IO, 111 Contreras, H.R., 30 Garlak, D., 77 Amann, R.P., 20, 73, 84 Cook, D.A., 45 Gamer, D.L., 125 Amin, F. A., Ge, R.-S., 92 Cooper, T. G., 21 60 K Arbuckle, T. , 28 Cordek, L., 77 Gelety, T. J., 80 Armstrong, J.S., 16 Correa, J.R., 105, 106 Gibbs, M., 87 Kadowitz, P.J., 34, 38, 39 Arredondo-Pineda, A., 120 Corson, S.L., 87 Gill, S.P., 73 Kamischke, A., 48 Arver, S., I Coulombe, E., 41 Glazier, D., 87 Kaneko, H., 49, 76 Coy, D.H., 34, 39 Goldberg, S., 14 Kaplan, B., 122 Cramer, P., 20 Goldblatt, J., IOI Karabinus, D.S., 80 Cramer, P.C., 73 Goldstein, M., 121 Kaschmitter, A.J., 50 Cranfield, M., 69, 70 Gomella, LG., 90 Kastrop, P., 86 B Kastury, K., Crone, J.K., 6 I Goodwin, LO., 8 I Katsoff, D., Bahr, J., Cummins, J.M., IOI Griffin, F.C., 22 9, 93 64 Katz, D., Baker, A., Grisham, J., 123 124 126 Katz, D.F., Bakin, R., Gu, W., 29 102 12 Keeffer, L.K., Bakst, M.R., Gunsalus, G.L., 60 36 74 Keillor, G.E., Baldi, E., Gutierrez, M., I 50 10 Kikuchi, K., Balgobin, S., D 49, 76 19, I 10, 111 Kikuchi, M., Banerjee, P.P., 74 62 Da Ros, C.T., Kitamura, M., Banerjee, S., 17 6, 103 62 Davis, G., Koga, M., Baratta, A., 110, 111 6 15, 16 Deaver, D.R., H Kondoh, N., Baravarian, S., 51 6 109 DeJamette, J.M., Korn, N., Barbato, G.F., 125 Hammerstedt, R.H., 20, 73, 84 72 20, 73 Derrick, F.C., Jr., Kraemer, W.J., Barcelos, M., 72 Hardy, M.P., 60 5 I 17 Dfaz, M., Kraus, D., Barik, S., 88 Hariprashad, J., I I 123 78 Dobs, A.S., Kremer, J.A., Barnard, J.J., 18, 45 Harman, F.S., 5 I 2 3 I, 32, 33 Doerksen, T. E., Kubakawa, K., Behre, H.M., 57 Hasegawa, M., 47 74 48 Doherty, P., Kumazawa, J., Belker, A.M., 36, 37 Hasegawa, Y. , 49, 76 55 92 Dolgina, R., Kunde, K.L., Beltran, A., 122 Hecht, N.B., 29 50 89 Donoghue, A.M., Benfer, K., 71 Hellstrom, W.J.G., 15, 16, 17, I 26 Dube, J.Y., Benoff, S., 41 34, 35, 36, 37, 38, 39 7, 8 Durrant, B.S., Berger, N., 67 Hershlag, A., 7 69, 70 Dyer-William, J., Bhasin, S., 119 Hess, R.A., 64 L I Dym, M., Bhaumik, D., 43 Higdon, D., I 19 124 Labus, J.C., Bonaccorsi, L., Hikono, H., 23, 24 I 0 49 Lakshmi, V., Bravo-Gatica, C., Hinton, B.T., 60 I 20 23, 24, 63 Lamberski, N., Breitenstein, M.J., Hirsch, l.H., 68 75 90, 91 Lambert, D.G., Bremner, W.J., Hoover, D., 38 108 18, 45 Lan, Z.J., Brown, S.W., E Hossain, A., 24 33 78, 119 Lara, C., Brown, T.R., Hourani, C., 89 62 Eisenberger, M., 126 Lavine, M., Buff, J.L., 45 Howard, J.G., 124 123 Esteves, S.C., 66, 123 Lee, W.M., Bui, T. , 95 Huang, H.F.S., 54 53 Estrada, LS., 42 Leeds, N.B., Bunick, D., 15 Huff, C., 8 64 Ettinger, D.S., 119 Leung, A., Burch, C.D., 18 Hurley, I.R., 53, 109 67 7 Levin, H., Burnett, A.L., Huszar, G., 115 6 I 104 Lewis, V.L., Bush, M., I 00 123 Li, M.-T. , Bustos-Obregon, E., 42 30 Liang, J .-Y. , 46, 59 F Lim Joon, D., 47 I Linsenmeyer, T.A., 42 Fahim, M.S., 44 Li petz, K.J ., Im, P., 8 I Falcone, T. , 96 53 Liu, J., c Irwin, R.J., 69, 70, 124 Fallat, M.E., 92 42 Lock, M.T.W.T., Ishii, S., 86 Cagnon, V. H.A., 30 Feld, R.I., 90 74 Long, J.A., Ismail, M.T. , 68 Cameron, D.F., 22 Feliciano, M., 121 91 Long, R., 93 Cao, J., 44 Feng, P.,46, 59 Lubahn, D.B., 64 Carbone, D., I 15 Fisher, C.E., I Luconi, M., 10 Centola, G.M., 100 Fitzgerald, W.E., 34 Lue, Y. H., 53, !09 Chamness, S.L., 61 Fitzhugh, C., 77 J Lurie, D., 93 Champion, H., 17, 37 Flamigni, C., I 08 Lynn, S., Jr., 78 Champion, H.C., 15, 34, 36, Fonseca, M.E., 89 Jacob, A., 7 38, 39 Foote, R.H., 65 Jeffay, S.C., I 02 Chang, T. S.K., 61 Forti, G., 10 Jequier, A.M., 98, 101 Chapin, R.E., 56 Fraga, A., 89 Jimenez-Balderas, F.J., 89 M Check, D.J., 9 Frenette, G., 41 Jindal, S.K., 81 Check, J.H., 9, 93, 126 Froman, D.P., 7 I Johnson, L., 3 I, 32, 33, 50 Maggi, M., 10 Chemes, H.E., 4 Fugger, E., 82 Johnson, L.A., 85 Mahadevan, M.M., 94

P-23 Maher, E.J., Selukar, R., 18 p 119 u Marker-Kraus, L., 123 Serre, V. , 52 Markey, C.M., 23 Padovani, C.R., 30 Seya, T. , 103 Uchida, K., 6 Marmar, J.L., 87 Palacios, J., 26 Shabanowitz, R.B., 84 Marshall, C.E., 125 Palermo, G.D., 11, 121 Sharma, R.K., 95, 96, 114, Martinez, EE., 30 Palladino, M.A., 24 116, 117, 118 Martinez, M., 30 Papale, M.L., 121 Sharpnack, D.D., 27 v Martinez-Garcia, E, 26 Pell, K.M., 66 Shimada, A., 49, 76 Perreault, S.D., Martin, R., I 0 I 99, 102, 124 Shoemaker, A.H., 68 Vargas, E., 88 Peymer, M., Martinis, EG., 14 93 Sidhu, R.S., I 16, I 17 Vawda, A., 113 Pilder, S.H., Matsumiya, K., 6, 103 3, 5 Sikes, C., 47 Vigue, L., I 04 Pogach, L.M., Matsuzaki, S., 55 42 Sikka, S.C., 15, 16, 17, 35, 36, Virji, N., 14 Pollack, A., Mattox, J.H., 97 47 37 Volek, J.S., 51 McCue, P.T., 90 Pomeroy, K.O., 97 Simon, S.D., 75 Pooser, B.P., McGaughy, J.T., 58 72 Sinha Hikim, A.P., 53, 109 McMahon, J., Powers, R.D., 115 110, 111 Siow, Y., 92 Prins, G.S., Meguerditchian, H., 32 122 Skaggs, S.R., 27 Pukazhenthi, B.S., Mehmet, D., IOI 66 Slott, V., 124 w Meistrich, M.L., 47 Slott, V.L., 99 Wagenfeld, A., Meriggiola, M.C., 21 I 08 Smart, J., 69, 70 Wa lensky, L.D., Meskaitis, VJ ., 12, 13 51 Snyder, S.H., 12, 13 Wang, C., Meuleman, E.J., 53, 109 2 R Sofikitis, N., 107 Wang, M., Millard, S.E., 44 67 Stopper, V. S., Wang, R., Racowsky, C., 40 15, 17, 34, 36, 37, Miller, M.M., 94 80 Storey, B.T. , Mills, T. M., Rajasekaran, M., 83 38, 39 40 35 Strandberg, J.D., Wang, Miyagawa, I., Rajaskaran, M., 61 Y., 96, 116, 118 107 15, 16 Studney, P., Watanabe, G., Moffitt, D.V., Ramirez-Munoz, M.P., 122 49, 76 97 88 Summers, D., Weinbauer, G.E, Moorman, W.J., Rao, J.N., 69, 70 48 27 46, 59 Swanson, W.E, We instock, D., Moutos, D.M., Ravindranath, N., 123 51 94 43 Swerdloff, R.S., We isensel, S.P., Moy, E, Ravnik, S.E., 53, I 09 100 II 58 Syed, V. , We lch, G.R., Mruk, D., Rawlins, R.G., 29 85 54 79 Whalen, J., Murphy, W.A., Regadera, J., 18 34, 39 26 White, R.A., Reilly, C.M., 47 40 Wilber, J.E. Rens, W., 46, 59 85 Wildt, D.E., Ricker, D.D., 66, 123 25 Wine, R.N., Ritter, L., 56 28 T Wininger, J.D., Rizk, B., I 12 78, 119 Wo lf, G., N Robaire, B., 122 52 Tanaka, H., Wu, C.S., Ronnett, G.V., 6 47 Nagler, H.M., 12 Tapia-Serrano, R., 14 Rosenwaks, Z., 89, 120 Niederberger, C., 11, 121 Tarchala, S.M., 122 Roshani, H., 79 Nieschlag, E., 86 Taya, K., 48 Ross, L., 49, 76 Nindl, B.C., 122 Taylor, W.E., 51 Roth, T. L., I Nishimune, Y. , 123 y 6 Ruat, M., Terry, N.H.A., 47 Nishina, Y. , 12 6 Rudolph, D.B., Thanh, L., 82 Yamada, J.K., 67 Nistal, M., 23, 24, 63 26 Rudzitis, L.A., Thomas, A.J., 77, 95, 114, Yamamoto, Y. , I 07 Noguchi, J., 80 49, 76 115, 116, 117, 118 Yamanaka, M., 103 Noiles, E.E., 83 Thomas, C.A., 125 Ye oman, R., 119 Nomoto, K., 55 Thompson, K.A., 83 Yeung, C.H., 21 Thompson, N., 74 Yaffe, S.C., 92 s Thorneycroft, I., 78, 119 Younglai, E.V., 113 Sanchez-Corona, J., 88 Thurston, R.J., 72 Sato, S., 49 Tiang, K.S., 53 0 Savage, W. , 97 Toda, T. , 107 Schlegel, P.N., To kuda, N., 55 121 z Okuda, K., 76 Schneyer, C.R., 45 Torres-Bugarin, 0., 88 Okuyama, A., 6, I 03 Schrader, S.M., 27, 28, 75 Trasler, J.M., 57 Zagars, G.K., 47 Olds-Clarke, P., 3, 5 Schulman, J., 82 Tremblay, R.R., 41 Zavos, P.M., 105, 106, 107 O'Reilly, S., 18 Schwartz, M., 25 Troncoso-Torrez, P., 120 Zimmer, M., 97 Orth, J.M., 3 Scott, T. R., 72 Tuerlings, J.H., 2 Zimmerman, D.M., 67 Ottenweller, J.E., 42 Sedor, J., 91 Tuke, G., 113 Zirkin, B.R., 62 Ottinger, M.A., 74 Seifarth, K., 77 Turner, L., 28 Zuniga, G., 88

P-24 Journal of Andrology •January/February 1997

l � cDNA CLON!NG. CHROMOSOMAL Mi\PPfNG. AND TISSUE J,... 3 Hsr-7: A MALE STERILITY MUTATION PERTURBING SPERM DISTRIBUTION AXONEMAL T Of' THE HUMAN DYNEIN LIGH "1'- MOTILITY, FLAGELLAR ASSEMBLY, AND MITOCHONDRIAL SHEATII GENE: A CANDIDATE THE IMMOTILE DIFFERENTIATION CHAIN GENE FOR A CE foher, S.H. Pilder, P. Olds-Clarke, and J.M. Orth, Depanment of Anatomy CILIA SYNDROME. �. WE Taylor, S rver, M Gutierrez. Cell Biology, Temple University School of Medicine, Philadelphia, PA S B as n. Endocrinology, Departm�nt of & h i Division of 19140. . l Medic ine ChJrlcs R. Drew Uniwrsity, Los Ange es, CA lmmotilc Syndr m is chJracterized recurrent smus Cilia o <: !ICS) b) Hs r-7 is a novel mouse mutation, mapping to the I haplotype region of u N and l ng infections, bronch1c�tJsis, and sperm immotility. asal .:ilia chromosome 17, a large portion of the chromosome known to include several cause and sperm taih in patients \\ith!CS exh ibit a variety of ulrrasm1ctural genetic factors which defects in sperm function and/or differentiation. Male laboracory mice hecerozygous for the wildtype (+) allele an allele defects, but or shortening of d n n is and derived absence the inner y d arms che from another mouse species, M. sp retus (s), are sterile as the result of poor commonest. hnmotile mutant strains of Chlamydoml'nJs. a sperm motilicy. Slighrl� but significantly less s/+sperm are motile relative to to thos<� biflagcllated algae, ha,·e ulcrastructura l defects similar seer. in +I+ concrol sperm, and the mutant sperm exhibit a net velocity of about half that and t !CS; further, splice-site mutations in the inner dynein arm gene (p2SJ of +I+ sperm. Male mice heterozygous for the s allele a haplotype allele have only abouc half the number of motile sperm as the +I+ control animals. are associated i p i ed Tl agcllar moti l i . We hypothe�iz�d that (r) with m a r ty and these motile sperm exhibit so little forward movement that it is below the the che human homolog of Clamydomonas dyncin p28 gene would b� decectable limit of the sperm motion analysis system. d h ICS. an attractive can idate for patients wit Accordingly, we c l,m eJ Lighc microscope observations of sl+ cauda epididymal sperm reveal thac most appear normal or near normal. However, similar observations of the lengch cDNA and ge omi.: clone by screening of sit fu ll n appropriate sperm reveaj a number of visible morphological defects in nearly half the sperm libraries and databases, che DNA th"' using c sequence of including UJidpiece bubbles, iil)o nen.ed tails, nqnna!. bfads anoched � Chlamydomonas p:?8 The e c des bp J/ g"ne. hu"1an cDNA (hp:?S) n o a 92 1 �rentiated "�ge lla". (reminiscent of the previously reported Hsr - ' '1\" trnnscripr with an open reading frame of 25 7 am ino acids. Using homo� et al. 1993, Devel. Biol: 159, 631-642), and many

Y-DELETIONS IN MEN WITH SEVERE OLIGOSPERMIA. 4 DYSPLASIA OF THE FIBROUS SHEATH AND DYNEIN 2 DEFICIENCY, A CYTOSKELETAL ABNORMALITY OF HUMAN E.J. Meuleman, J.A. Kremer·, J.H. Tuerlings", De artments of p SPERMATOZOA . H.E.Chemes, Laboratory of Testicular Urology, Obstetrics Gynaecology and Human Genetics, & Pathology . Buenos Aires Children 's Hospital . Argentina. University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, In 1987 we described the dysp lasia of the fibrous The Netherlands. sheath as a serious defect of human spermatozoa in five sterile patients with severe asthenozoospermia. We have The introduction of ICSI offered a successful treatment option for gathered a large series of 34 patients and defined the clinical and seminal features of this syndrome . The subfertile male with severe oligospermia, although the etiology of patients are young males in their third and forth decades the disorder remains unclear in most cases. Recently, micro· of life , consulting for primary sterility . Seven of them deletions in the AZF region of the Y chromosome have been have chronic sinusitis , bronchitis, and or bronchiectasis detected in men with azoospermia or severe oligospermia. from early childhood. Two of the patients were brothers (not twins ). Seminal features include rigid, short and thick flagella, extreme asthenozoospermia, and various In this study we investigated the prevalence of microdeletions in degrees of necrozoospermia. On ultrastructural the AZFc region of the Y. chromosome in our ICSI population (by examination the conunon characteristic is a serious PCR analysis) and looked for clinical differences between the men distortion of the fibrous sheath (FS) with hypertrophy and without the deletion. Blood was drawn from 154 men, who and hyperp lasia of its constitutive elements, frequent deficiencies of mitochondr ial assembly at the middle were waiting for ICSI treatment: 24 azoospermic men, 98 piece and axonemal anomalies like missing dynein arms and oligospermic and 32 normospermic men. (after previous lack of central doublets (9+0 axonemes ). The patients can fertilization failure). Chromosome analysis showed 4 Klinefelters be divided in two subgroups defined by the prevalence of in the azoospermic group and two Klinefelters in the oligospermic the fibrous sheath anomalies in their spermatozoa. The group. One translocation was observed in the oligospermic group. complete form (n=2 1) shows 0,7±1,4% total motility (TM) , Microdeletions in the AZFc region were present in 7 of the SB with 0% fast forward motility (FFM) . All spermatozoa have severe FS alterations . In the incomplete form (n=l3 ) TM oligospermic men (7%). None of these 7 men had abnormal was 7,9±7 ,3% and FFM 0,6±1,4%. Tail alterations were findings on andrologic history and examination. No microdeletions similar, but there were 20-30% of spermatozoa with normal were found in the azoospermic and normospermic group. fibrous sheaths . Dead spermatozoa ranged between 4 and We conclude that microdeletions in the AZFc region of the Y­ 75%, with ll patients showing values higher than 40% . chromosome are frequently found in men with severe oligospermia There were no spontaneous or IVF pregnancies . ICSI has been recently performed with success in 2 casee. Lack of and with no other causal factors. We recommend DNA screening dynein arms was found in bronchial cilia in the two (and genetic counseling) in this population of subfertile men. patients with respiratory symptoms in which a biopsy was performed. They constitute a variant of the immot ile cilia syndrome . Regardless of various treatments, seminal and clinical characteristics remained stable in all the patients which were followed for different periods of time . The features described configure a henot e that _suggests a genetic origin of the syndrome .

P-25 TWENTY-SECOND ANNUAL MEETING

!NVESTIG-\TION INTO INFERTILITY S INTERACTION OF STERJLITY FACTORS IN THE t HAPLOTYPE 7 1t!E :vlE�HANISM UNDERLYING ASSOCIATED WITH CALC !illvl 1,Ca· CHANNEL BLOCKERS A Jacob•, IR PREVENTS SPERM PENETRATION OF THE ZONA-FREE EGG A "J C Hurley, Henhlag• and S. Benoff D1v1sion of Human Reproduction, North Shore P. Olds- larke, L.H. Johnson and S.H. Pilder, Departmentof Anatomy & Uru\'ers1t) Ho;p11al, Manhasset. NY Cell Biology, Temple University School of Medicine, Phila., PA 19140. Chmcal dosa£" of hpoph1hc ca2+ entry antagonists ("Ca2+ channel blockers" CCBs) such as lhe prototype dili)dropyndille rufed1pme, correlate with infertility m The t haplotype includes several genetic factors which cause defects men wilh normosperrruc semen (Fert1L Sten! 1994. 62.606-6 17) These drug; in spermfu nction in fe rtilization and which are linked together within four suppress !he surface expression of mannose receptors (MR). zona recogmt1on m'OleCu les:- b\ raising lhe free cholesterol content of lhe sperm plasma membrane large inverted regions on mouse c omosom 17. Sperm from mice carrying hr e However, bas

P-26 Journal of Andrology • January/February 1997

9 A CHALLENGE TO THE CONCEPT THAT THE USE OF 11 IMPACT Of ABNORMAL SEMEN PARAMETERS ON THE GENETIC CALCIUM CHANNEL BLOCKERS CA USE REVERSIBLE STATUS Of HU�IAN SPERMATOZOA LT. Cotombero'. J. H:iripr sh F. Moy•. Rosenwaks•. G.D. Palermo. The Center MALE INFERTILITY D. Katsoff, J.H. Check and DJ. Check'; UMDNJ, a ad•. Z. for Reproductive Infertility. The New York Hospital-Cornell M1% normal forms number of below <500,000 blockers. Pregnancies occurred in 5/11 fe male partners. The median and Finally, when the total normal motile spermatozoa fell incidence mean ages were 33 and 33.8.±.·US for those achieving pregnancy vs 35 and (the threshold indicator level for !CS!). the of chromosomal abnormalities 1.5% in (P<0.001). 36.3.±.5.68 for non-conceivers. Oligospermia and/ or asthenospermia was was 2.6% vs. samples above that threshold In subfertile men, (P<0.001), present in 5/6 males not achieving pregnancy. Other contributing factors the most frequent abnormality was diploidy this being most evident (P«l.001). included tubal factor (n = 1), endometriosis (n = 1), advanced maternal age of where morphology ':Vas SI% Infertile higher incidence spenn 2_42 (n=2), and ovulation abnormalities (n =l). These data demonstrate that Conclusions: men have a of chromosomal among even in vivo pregnancies are possible despite the use of calcium channel anomalies, which diploidy appears to be the most common. Therefore, in have severely parameters. blockers by m1le partners. Whether the PR is reduced or not would have to patients undergoing !CS! who compromised semen prenatal diagnosis should be answered by a much larger prospective study. be performed.

IO IDENTIFICATION OF FUNCTIONAL PROGESTERO'E 12 SIG:-.:ALTRA:-.:SDLCTIO\ BY SOVEL SPER\1 CHE\10RECEPTORS AS A RECEPTORS ON THE SURFACE OF HUMAN SPERMATOZO.\. \lECHANlS\l FOR \IA\l\IALIA\ SPER\1-EGG CHE\!OTAXIS. L.D. Walenskv. \1. Ruat•. R. Bakin'. G.V. Ronn t:ni,

P-27 TWENTY-SECOND ANNUAL MEETING

1 3 CALCIL''.l FLUX THROl!GH SPER.\l INOSITOL l.�.5-TR!SPHOSPHATE 15 EFFECT OF TDIOR NECROSIS FACTOR (TNF-a)AND RECEPTORS IS l'.!PL!CATED IN TRIGGERING THE \!AMMAL!AN ACROSOME INTERFERON (IFN -y) ON HUMAN SPERM MOTILITY, VIABILITY, REACTION. L.D. Walensky and S.H. Snyder'. Department of Pharmacology and MOTION PARAMETERS. Molecular Sciences. Johns Hopkins University School oi '.\ledicine. Baltimore. �ID. AND on L. S. R. HWl!er C. Champi • . Estrada•, Wang.,A. Baratta•, M. Rajaskaran., S. SikkA, Calcium tlux. is required for the 3Crosomc= reaction of mammalian s�rm. The C. W. J. G. Hellstrom, Tulane University School of Medicine, New acrosome reJction is an e.'tocytotic event triggered by egg binding. which results in J Orleans LA local. drammic rise in sperm intracellular calcium. CJ!cium·dependent membrane fusion results in the release of i:nzymc:s th:u facilitate spenn penetr:nion through the Male genital tract infections and non-specific inflammatory cotxlitions may be zona pellucida during fertilization. We in1,·estigated whether mammalian sperm associated with WJexplain«J infertility_ Previous studies have shown the presence possess inositol 1 A,5-trisphosphate recept0rs OP3Rsl and upstream componenis of of cytokines such as tumor necrosis fa ctor -a (TNF-a) AND INTERFERON -y the phosphoinositide signalling system that may participate in calcium dynamics (IFN-y) in thesemen of infe rtile men. However, the mechanism of effect of these Peptide antibodies colocalized Gaq/11 and the Bl isoiorm of phospholipase C !PLCJ cytokines on human spennfunction is not known. The present study was to the anterior acrosomal region of rodent sperm. In siru hybridization using type­ Wldertaken toinvestigate lhein-vitro effects of TNF-a and INF-y on human sperm specific 1P3R riboprobes demonstrated a distinct distnbullon of 1P3R ISOforms in the motion parameters and viability. spermatogenic cells of rat testis. Scatchard analysis of 3H-!P3 binding to rat sperm Washed spenn from normal volunteers (n=9) were incubated in the membranes revealed a curvilinear plot w1th high aifinity !K =26 nM. B .=30 d ma presence/absence of -tt (lµg/ml) plus IFN-y (0.1 µg/ml). The sperm pmoUmg) and low affinity \K =l.6 µM. B .=550 pmol/mg) sites. Western TNF d ma motility, viability and video sequences were recortled at different time intervals 0, blotting with a polyclonal antibody directed against purified brain !P3R identtfied J 30, 60, and 180 minutes. The sperm motion parameters were analyzed using 260 kD band in 1% Triton X-100 extracts of mature rat. hamster. mouse and do• sperm. In each species. 1P3R immunostaining localized to the acrosomal cap computer assisted semen analysis. � IFN lmmunoelectron microscopy revealed that 1P3Rs specifically decorate the outer Our results showed a time-

14 EFFECTOF SODIUM NITROPRUSSIDE ON SPERM M011U1Y: A DOSE­ 16 ANTIOXIDANT CAPACITY OF SEMINAL PLASMA COMPARED RESPONSE CURVE. FG Martinis". HM Nagler. S Ooldberg", N V"uj i. TO SYNTHETIC AND �!OLOGICAL FREE RADICAL SCAVENGERS. S. Departmeac of Urology, Beth Isruel Medical Center, New York,NY. J. Annstrong•, M. Rajaskanui, A. Baratta•, W. 1- O. Hellstrom and S.C. Sikka. Tulane Andrology Section, Tulane University Medical Center, New Nitric oxide (NO) ha.s been sbawn IO improve the maintenancespena of motility Orleans, LA. in ctyopreserved human sperm aswell asincrease the yield of motilo spermusing swim-up methods. However, an ition o! motility by NO ac higher inhib sperm Reactive oxygen species (ROS) are known to iolullit sperm motility aiid coaceatratioas has beea reported.This suggesu thacNO may exerc its effect also cause damage to the sperm membrane. Antioxidants in semen such as oa motility ia a dose superoxidedismulase (SOD), catalaseand glutathione protect the sperm cells depeadeac manner. To investigate this hypothesis, ,,.. generated doso-respomo from ROS damage. this srudy we compared the ROS scavenging efficacy evaluate tho effects of NO oa motility over wido range of Ia cum:s IO sperm a of plasma anlioxidants towards an vitro superoxide anion coaceacratioas usingsodium aitropnmide as tho NO doaor. seminal withother in generation system. Superoxide anion was generated using a xanthine oxidase/ Motilo obtained from (our fertilo donors e ) and sperm knowa (two samples ach hypoxanthine system. Measurement was made using a luminol induced iafertilo patientswero separated oa a Percoll padieat. Sodium aitroprussido five chemiluminescence assay in a lurninometer for 20 minutes. SOD, catalase , was added in decreasing coaceacratioas IO tho washed semen aliquots IOachieve ascotbic acid (AA) and pentoxyfylline were compared to a 1 :SO dilution of fiaalcoaceauatioas intho tubooC lltla'IO � M.S perm coaceaaatioa in acho tubowas 20 mill/mL A coauoltubo was maiacained withouc sodiumaitropnmide. seminal plasma in phosphate buffer (pH 8. 7) for their abilities to scavenge Motility was evaluated aft.er 2 hou� incubation at rt' C. superoxide anion. Seminal plasmaquenched the chemiluminescence response Spmu motility ia thocoauol ranged from 71'684'6. IO High coaceacratioos of (peak relative light units obtained within 3 to 4 minutes of initiation of sodium aitropiussido (lltla' and 1.XlO" M) wero iahill iiory IO sperm motility reaction) in a dose dependent manner ranging from 24 % to 15 % of the peak. resulting ia liaal motility of 1'6 IO39'6. Al the coaceauation o! l.xlO"M. sperm SOD similarly quenched the peak chemiluminescense in a dose-

P-28 Journal of Andrology •January/February 1997

TRANSESTERIFICATION OF HUMAN SPERM 1 7 DO PEPTIDES STil\'lULATE SPERM FUNCTION? 19 PHOSPHOLIPIDS WITH EXOGENOUS FATTY ACID COENZYME THIOESTERS. J.G. Alrnrez and S. Balgobin*. Dept. of Qb, Gyn, Beth Run Wang, Carlos T. Da Ros*, Maryano Barcelos, • Hunter Champion,* A Israel IVF, Han·ard Suresh C. Sikka, Wayne J. G. Hellstrom, Tulane CJ.iversity School of Hospital.Boston Medical School, Boston, MA Medicine, New Orleans, La. Human spermatozoa undergo a process of lipid remodeling dunng llllgrauon from the capuJ lo the auia epididymis. This lipid remodeling invoh·es a Piruitary adenylate cyclase-activating polypeptide (PACAP) binding sites decrease m palmitic (16.0) and oleic (18: I) acids and a concomitant increase in aracludoruc (20:4) and docosahe:.aenoic acids (22:6). We recelltly reported (�Joi have been found on spermatozoa tails. Since P ACAP in..•eases intracellular Reprod Dev, 42:334-46, that human sperm can incorporate picomolar cyclic adenosine monophosphate (cAMP) which mediates sperm motion, this I 995) concentrations of ewgenous fatty acids and that this incorporation is mediated b) srudy investigated the effects of PACAP27, PACAP38. and VIP on sperm the phospholipid remodeling pathway. The objective of this srudy was lQ investigate motility and other motion parameters using th.e CASA system. the ab11i1y of hwnan sperm lo transeslerify micromolar concentrations of fatty acids r Cryopreserved human sperm samples (n = 15) were thawed and th en into the phospholipid pool f om e:.ogenous fatty acid CoA thioesters. A total of 12 semen samples were obtained from 12 healthy sperm donors. All samples were incubated with 140 nMOL/ml of PACAP27, PACAP38, and VIP, normal by WHO criteria. The seminal plasma was removed by centrifugation al respectively. Video sequences were recorded at 0, 30, 120, and 180 min 60, 600g for 8 min and the resulting pellet resuspended in BWW mediwn containing foranalysis of spermmotion parameters using the Cell Tnck Sperm Analysis 5mg ml of BSA. The sperm suspensions were then incubated at 37°C in the System. Sperm viability was analyzed by 0.5 % eosin at the same time. stilll presence of 0.2-5 µM oleo) I CoA for up lo 4 h. Aliquots of the sperm suspensions Our results showed that PACAP27 and VIP had no significant stimulatory wereremo• ·cd al I h intervals, centrifugedal 800g for 8 min and the resulting pellet effect on sperm motility, viability and on motion pal"...:neters (straight-line extracted with 20 vol of chloroform-methanol I: I. The phospholipid fraction was velocity,curvilin ear velocity, lateral head displacement !lid mean linearity). then isolated by aminopropyl column chromatography, the fatty acids methylated using BF3-methanol , and the resulting fatty acid methyl esters analyzed by gas Surprisingly, PACAP 38 caused immediate agglutinationand death of sperm chromatography. Incorporation of oleic acid into the phospholipid pool fol lowed as demonstrated by sperm motility and viability. first order kinetics at oleo) I CoA concentrations between 0.2 and 2 µM and zero This srudy shows that PACAP27 and VIP have no nmulatory effect on order kinencs at concentrations > 2 µM. The mean concentration of phospholipid post-thaw sperm function at the given doses. The d::trimental effect of 18: J, 20:4 and 22:6 in control samples was 15 ± 2, 9 ± 2 and 52 ± 7 µg/ 108 cells, PACAP38 on sperm needs further investigation. respecti,.cly, and that obtained after 4 h of incubation in the presence of > 2 p�! oleo) I CoA was 35 ± 4, 5 ::: 0.8 and 28 ± 3 µg/ 108 cells, respectively. These differences were statistically significant (P < 0.001). These results indicate that human sperm have (i) the ability to transesterify micromolar concentrations of fany acids into the phospholipid pool frome:.ogenous fatty acid CoA thioesters, and that (ii)transestcrificatioii of fatty acids from e:.ogenous fatty acid CoA thioeslers results in a decrease in endogenous phospholipid 20:4 and 22:6 suggesting that this incorporation is mediated by the phospholipid remodeling pathway.

20 18 THE RELATIONSHIP BETWEEN SERUM TESTOSJERONE A ROLE FOR SGP-1 fN BfNDfNG OF SPERM TO EGGS AND NUTRITIONAL STATUS IN PATIENTS WITH NON-SMALL CELL R.H. Hammerstedt, P Cramer*, G.F Barbato* and R. P. Amann, LUNG CANCER Biochemistry Program, The Pennsylvania State University, E.J. Maher•, S. O'Reilly•, D. Hoover•, D.S. Ettinger•, J. Whalen•, University Park PA and BioPore Inc, State College PA A.S. Dobs, The Johns Hopkins University, Baltimore, MD. In the lung cancer population, malnutrition and weight loss are SGP- 1 is a highly conserved molecule secreted by Senoli cells and poor prognostic factors with limited successful therapeutic epithelial cells of the excurrent ducts in rats, bound to sperm, but of interventions available. In addition, hypogonadism, manifest by unknown function; the protein sequence is identical to prosaposin. reduced testosterone levels, has been reported in 43-66% of cancer Compared to fresh sperm, frozen-thawed rooster sperm are oflow patients. Testosterone (Tl has anabolic effects on the body and is fe nility and a low percentage bind in vitro to an egg membrane important in maintaining weight and muscle mass. We hypothesize substrate (assay modified from BOR 50 Suppl I: 128. 1994 ) semi­ . A that in patients with unresectable non-small-cell lung cancer (NSCLC) p.urified extract (UPSEBP = native bioactive molecule) prepared from the degree of hypogonadism correlates with nutritional status and supernatant of thawed rooster sperm restored binding. Intuitive quality of life, including sexual functioning. This cross-sectional study deductions led to probing Western blots with polyclonal antibody of men and women with unresectable NSCLC assessed serum against rat SGP-1; a - I OkD band stained. Pre-treating semi-purified gonadal hormones, nutritional status, and quality of life. Patients were UPSEBP with antibody before use to treat sperm eliminated divided into two groups based on nutritional status as assessed by a enhanced binding. Studies with saposins A-D, antibodies against combination of six nutritional factors: albumin, % usual body weight, saposins, and synthetic sequences for ponions of rat SGP- 1 led to % ideal body weight, body mass index, upper arm muscle area, and conclusions that UPSEBP was not a known saposin, but was a % lean body mass. Patients' gonadal state was assessed by blood fragment of SGP- 1. A synthetic 60-AA sequence with high analysis of free and total T, FSH, LH, prolactin, and estradiol. Quality bioactivity (FertPlusTM peptide) was identified, based on enhanced in of life measures included the Functional Assessment of Cancer vitro binding of rooster to the egg-membrane substrate. When sperm Therapy Scale and Deragotis Interview for Sexual Function. from individual roosterswere treated with either UPSEBP or Preliminary data from the initial 20 patients (1 6 male, 4 female) FertPlusTM peptide and used for Al, both maximum fe rtility and indicate that malnutrition and hypogonadism are prevalent. The mean duration of fe rtile life span increased. We speculate that UPSEBP is a decline in male patients' usual body weight was 7% (range -25 to proteolytic cleavage product of SGP- 1, formation of which is + 10%). The mean total T for male patients was 336ng/dl ( +/-204). facilitated by an alternative post-translational foldingand proteolysis Five of the 1 5 (33 % ) male patients had total testosterone levels of SGP- 1 relative those of prosaposin. We concluded that UPSEBP is below the anticipated values for patients of their age and gender. highly conserved across species, ubiquitous in semen fromcommon Accrual is ongoing to evaluate the hypothesis that hypogonadism is animals, can be eluted from sperm by freeze-thawing, normally associated with weight loss and quality of life, and ultimately to becomes bound to the sperm plasma membrane over the head, evaluate the potential role of testosterone or other anabolic therapy facilitates initial sperm-egg binding, and may be in suboptimal in this population. amounts on sperm from some males. Deficiencies can be restored by in vitro treatment of sperm with FertPlusTM peptide prior to Al. MD G is d JS O e provided reagents; USDA92-92202-073 r wol & ' Bri n

P-29 TWENTY-SECOND ANNUAL MEETING

230XIDATIVE STRESS DIFFERENTIALLY REGULATES EXPRESSION OF 21 EPIDIDYMAL PROTEIN PATTER.i \l IN INFERTILE ORNIDAZOLE TREATED RATS GAMMA-GLUTAMYL TRA:\SPEPTIDASE mRNA IN THE INITIAL A. Wagenfeld0, C. H. Yeung", T.G Cooper, Institute for Reproductive Medicine of SEGMENT OF THE RAT EPIDIDYMIS. C. M. Markey*, D. B. Rudolph. J. C. the University, Miinster, Germany Labus* & B. T. Hinton. Department of Cell Biology, University of Virginia Health Sciences Center, Charlottes' 1lle, VA 22908. To investigate proteins involved in the fertilization process male rats were treated for I 0 days with the antifertility agent ornidazole at a dosage of 400mgfkg/d. This Oxygen radicals have a powerful .:ytotoxic effect on spermatozoa and have been short time to infertility suggests a direct action on the epididymis. CASA analysis implicated in sperm dysfunction and male infertility. Recent evidence suggests that showed reduced velocity parameters of spermatozoa from the distal regions of the the epididymis utilizes the antioxidant glutathione to protect maturing sperm against epididymis and two sperm enzymes of the glycolytic pathway - triose phosphate oxidative stress. One enzyme essential to the metabolism of glutathione is isomerase and glyceraldehyde 3-phosphate dchydrogenase • had markedly reduced gamma·glutamyl transpeptidase (GGT). We have previously shown that multiple activities. We have studied the effect of ornidazole treatment of rats on the protein forms of GGTmRNA (II-IV) are di ffe rentially expressed in the epididymis and that pattern in epididymal fluid and on sperm. Epididymal fluid was flushed out with they are regulated by androgens and/or testicular factor(s). The aims of this study PBS from the cauda and corpus regions of the epididymis by tubule cannulation and were (I) to establish conditions in which oxygen radicals are generated, and perfusion. Fluid and sperm were separated by centrifugation through 5% Ficoll and i11 •·itro (2) to determine if oxidative stress regulates the expression of GGTmRNAs II-IV in sperm proteins extracted with -!Om.\! N-octylglucopyranoside. They were analysed the initial segment of the epididymis. Initial segments were collected from adult by 2D gel electrophoresis and silver-staining. Both corpus and cauda fluid protein male rats and incubated for l .5h. 3.5h and 6.5h in culture media to which oxygen patterns from infertile animals revealed the existence of a new protein that was not radical-generating compounds, h) poxanthine and xanthine oxidase, were added in found in control animals. Its molecular weight was -25kDa with a pl of - 5.8. varying concentrations. By 6.5h. incubation of tissue in high oxidizing conditions From the intensity of staining the protein was present greater amounts in the in caused a -50% decrease in ATP concentration, and a concomitant -60% decrease in cauda than corpus fluid. Incubation of tissue from the e ididymal caput. corpus and � reduced glutathione levels and -200% increase in oxidized glutathione levels cauda regions from ornidazole fed infertile rats with 5S-mcthionine followed by compared to controls. These data demonstrate a biological response to superoxide fluorography of the radioactive gels gave no hint that this extra protein was a secretory product of the epididymal epithelium. This was supported by the absence anion and hydrogen peroxide confirming the generation of oxygen radicals within the of the protein in corpus fluid of sperm-free epididymidcs of efferent duct ligated, experimental tissue. RNase pro1ection assays using [321P labeled probes of the ornidazole treated rats. Analysis of proteins from cauda sperm revealed the 5'UTR of GGTmRNA 11,IIl,IV demonstrated a -70'1c increase in the expression of existence of the new protein in fertile rats and less of it associated with sperm from GGTmRNA II yet only a -20% increase in the expression ofGGTmRNA III and IV infertile rats. There seems to be an influence of ornidazole in the corpus and cauda compared to controls under these same conditions. These results support the epididymidis on the nature of sperm proteins during their transit through the hypothesis that expression of GGTmRNA II, in particular, is regulated by the epididymal tract. In fluid from the rete testis there was no extra protein and no oxidative status of the initial segment in the epididymis. This study contributes to difference between infertile and fertile rats. Blotting of the protein and MALDI­ the growing body of evidence that the epididymis plays a critical role in protection analysis revealed a MW of 20,420 = 120 Da. Digestion of the protein and partial of sperm from oxygen radicals and other xenobiotics. Further studies are underway sequence analysis of 17 amino acids demonstrated no homology to any sequenced to examine the mechanisms by which oxidative stress upregulates GGTmRNA II. protein. This protein may be critical in the fertility of male rats. Supported by NIH grant HD32979 (BTH), NIH P30-HD28934, and training grant NIHT32-DK07642 (DBR).

12 IMMUNOEXPRESSION OF FAS (CD95) LIGAND 2� IDENTIFICATION AND EXPRESSION OF THE £TS TRANSCRIPTION (FasL) IN THE RAT TESTIS AND EPIDIDYMIS F.C. FACTOR POLYOMAVIRUS ENHANCER ACTIV . \TOR 3 (PEA3) 1:-1 RAT Griffin and D.F. Cameron , University of South EPIDIDYMIS, AND ITS REGULATION BY TESTICLl.AR FACTORS. Z.J. Florida College of Medicine , Tampa, FL 33612 Lan*, M.A. Palladino, D.B. Rudolph, J.C. Labus* & BT. Hinton. Dept of Coll Biology, University of Virginia Health Sci. Center. Charlottesville, VA 22908. FasL is a type II transmembrane protein of the tumor necrosis factor (TNF) family that has The promoter region of rat gamma glutamyl transpep1idase (GGn mRNA-IV been extensively studied in lymphoid tissue . contains at least five exact matched PEA3 motifs (5'-AGGAAG-3'). Our previous It is associated with down-regulation of the studies have shown that multiple forms of GGT mR.'>As (ll·IV) are expressed 10 immune response by inducing apoptosis of the epididymis and one form. mR.i\IA IV, is highly expressed in the initial segment activated lymphocytes . Although the RNA and is regulated by testicular factors. We hypothesize that PEA3 fu nctions to message for FasL has been identified in the regulate rat GGT mR.i\IA-IV expression and that its expression is also regulated by rat testis , its protein expression and testicular factors. The goals of this study were (I) to ex:imine PEA3 protein level cellular distribution has not yet been and mRNA expression along the epididymal duct, (2) to determine DNA binding reported . Normal and is chemic testes and activity of PEA3 to promoter IV of the rat GGT gene. and (3) to determine if norma l epididymides were fixed in 10% NBF and expression of PEA3 mRNA is regulated by testicular factors. Western blot analysis paraffin imbedded . Sections (7µ.) were showed that a 62 kDa protein was detected in the nuclear extracts from the initial immunostained with the ABC (Vector Labs) segment in greater levels compared to the distal epididymal regions. Electrophoretic immunoperoxidase bridge utilizing rabbit mobility shift assay (EMSA) showed that nuclear extractS isolated from the initial polyclonal antibody for FasL (Santa Cruz segment specifically bound to the native PEA3 motif derived from the promoter Biotechno logies) and DAB and TB as chromogenic region of GGT mRNA IV.This DNA protein complex contained the 62 kDa PEA3 substrates . Controls included first antibody protein as demonstrated by EMSA and Southwestern analysis using PEA3 mAbs deletion , antibody absorption and splenic and a catenated PEA3 motif probe. Northern blot analysis and RNase protection tissue . Immunostaining was apparent on basal assays showed that a 2.4 kb PEA3 mRNA was highly expressed in the initial compartment germ cells , Sertoli cells and segment and its expression was regulated by testicular factors. PEA3 mRNA levels epididymal epithelium . Generation of reaction were reduced to 68% and 85% of sham-operated control levels after 12 h and 24 h product appeared greater in the ischemic efferent duct ligation respectively. Our findings support the hypothesis that PEA3 testis when compared to the normal testis . is involved in the regulation of rat GGT mRNA-IV in the initial segment, and that Results suggest that cell death in the its expression is regulated by testicular factors. Further work is underway to testis , induced by ischemia, may be associated identify the testicular factors and the mechanisms by which the expression of with the FasL apoptotic pathway . Additionally, PEA3 and GGT genes are regulated in the rat epididymis. the expression of FasL in the testis and the ep ididymis suggests a mechanism of immune Supported by N.l.H. grants HDI8257 & HD32979 (BTH), P30-HD2893-I, and regulation in these two organs . traininggrant T32-DK07642 (DBR).

P-30 Journal of Andrology •January/February 1997

URL"IARY CREATINE AS A �::> THE STATUS OF CAUDA EPIDIDYMAL SPERM DNA FOLLOWING 2 7 BIOMARKERFOR PARTIAL SYMPATHETIC DENERVATION. M. Schwanz• and D.D. Ricker, LEAD INDUCEDTESTICULAR DAMAGE. Dcparunent of Biological Science, York College of Pennsylrnma.York, PA. S.R. Skaggs•, W.J. Moorman•,D.D. Sharpnack•, and S.M Schrader NIOSH, 4676 Columbia Parkway, Cincinnati, OH 45226 Previous studies in the rat have demonstrated that the loss of caud.Jepididymal sympatheuc innervation via the Inferior Mesenteric Ganglion (l'.-.lGJad\·erscly Inrecent years there bas beena growing concern about emironmental and industrial affects sperm transport, maturation and storage. Moreover, our lab has shO\m exposure to a wide variety of chemicals and/or compounds which affect the male that \\rule cauda epidid}mal sperm from IMG-denervated rats can fe rtilize reproductive system. Semen studies have been the most common means for OOC}tes,the resultingembryos fail to develop successfully. The obJecti\·e of this determining a male's ability to produceviable sperm capable of fertilizing an ovum. normallye1. aluate spermparameters (count, motility, morphometry, studywas toexamine whether this impaired embryodevelopment "as related to These studies and morphology), andbiochemical assays. Timbrell ct al. (fox. & Ecotox. News, 1(1), 4- changes in cauda sperm DNA following the loss of epidid)mal �mpathetic 14, 1994) states thatthe biomarkm for toxic effects on the male reproductive system innervation. For this investigation, we utilized adult male Sprague-Dawley rats arc mostly invasive or the samples arcnot easy tocollect and suggcsu that a urinary (n=l6 total) \\hich had undergone either a sham (control) procedur� or surgical biOIIlall=for testicular damage would be extremely valuable. Whileurinary crcatine !MG removal 1 week previously. Spermatozoa were collected from excised isnot trnditionallymeasured as a marker for testiculardamage, an increase in urinary creatine reported to coincide with testicular damage induced by cadmium cauda epididymal tubules and were subjected to three distinct experiments in bas been order to detect potential DNA breaks: Acridine Orange fluores�nt labeling, (Nicholsonct al. MolPbarmacol, 36, 398-404, 1989) and2-m ethoxyethanol (fimbrell ct al. Int. Symp. Health Glycol Ethers, Nancy, France, 1994). However, no Apotag immunostaining, and a DNA fragmentation assay. The results of each Haz. reports of anincrease in urinary crcatinc levels due to lead induced testicular damage procedure consistently revealed that lMG-denervation produced no detectable have beenreported. of Aspart a larger proj ect to study the rcproducti\'e effecu of lead DNA strand breaks in cauda epididymal spermatozoa. a separate set of In in in the rabbit urinary analysis of cceatine, crcatinine, their ratio, and histological vitro e:-q1eriments, the rate of nuclear decondensation was measured using cauda evaluation of the testis was conducted to determine ifcceatine might serve as a useful epididymal spermatozoa from sham operated and IMG-denervatedrats. Cauda biomarker of lead induced testicular damage. Rabbits were given subcutaneous injections oflcad acetate in 5% dextrose to achieve dose levels ofO.O ..1g/dl, 20 µgldl, epidid::vmal spermatozoa were treatedwith 10 mM DTT and 0.5° • SOS for 30 minutes and samples were taken every 5 minutes. DNA was then stained with 40 µgldl, and 80 µgldl. Histologic evaluation of the testis showed altered Hoechst 33258 and nuclear area was measuredunder fluorescence microscopy. spcnniogenesis characterized by a dose response increase in defective elongate spennatids. However, a decrease intesticular weight was not found. Assays were The results showed that, under theseconditions, cauda epididymal ;perm nuclei perfonnedto dctcnnincthere if was a dose response increase in crcatine and crcatinine fromll'v1G-

IN AN EXPOSt"R.E -· EPITHELIAL CELL PATHOLOGY OF THE EPIDIDY\llS 28 THE USE OF SILASTIC CONDOMS I' .\Dl'LT A:-iD ELDERLY "1 EN ASSESSJ\epididymitis. BC are predominant cells. The K expression is variable. 4% were found to ineligible (not planning to spray pesticides under stu &...u ndary 10 obstruction. diverttculi 1s frequently observed. be dy). Ii.- Transi1ional Metaplasta (TM) and Squamous Metaplasia (SM) are fr

P-3 1 TWENTY-SECOND ANNUAL MEETING

19 SERTOLI CELLS IN CULTURE AND mRNA 3 I NO DECLINE IN SERTOLI CELL NUMBER FUNCTIONOR IN MEN OVER THE LAST YEARS. DIFFERENTIAL DISPLAY PROVIDE A SENSITIVE 16 L. Johnson, and J.J. Barnard. EARLY WARNING ASSAY SYSTEM TO DETECT Dept. of Vet. Anatomy & Public Hlth., Texas Veterinary Medical Center, XENOBIOTICS Texas A&M University, College Station, TX. Dept. of Pathology, University Viqar Syed,• Wei Gu* and Norman B. Hecht, Department of of Texas Southwestern Medical Center, Dallas, TX. Biology, Tufts University, Medford, 02155, USA MA Reports of diminished spenn counts and reduced seminal quality in humans We have developed a sensitive early detection method to worldwide have raised concerns for the effect of fe tal exposure to estrogen­ like compounds on malfonnationof the male reproductive tract and reduced investigate the effects oflow doses of potential testicular toxicants Sertoli cell number. Reduced spennatogenic potential in aging humans is long beforedecreases in sperm count would be seen. Sertoli cells related to a decline in Scrtoli cell number. The objective was to detennine if were used as an early warning system to monitor morphological Sertoli cell number and function as measured by the number of pachytene and biochemical changes induced by two different xenobiotics­ primaryspennatocytes or spennatids with spherical nuclei accommodated by cadmium acetate and polychlorinated biphenyls (PCBs). Sertoli a single Sertoli cell have declined in recent years. Testes obtained at autopsy cells begin to round, vacuolize and detach from their substrate inDallas were fixedwith glutaraldehyde, furtherfixed in osmium, embedded within 24 hours of culture in the presence of cadmium at in Epon, sectioned at 0.5 um, stained with toluidine blue, andevaluated by concentrations of0.5 to 1.0 µM. The mixture of PCBs used in the stereo logy for volume density of seminiferous rubules. Numbers of Sertoli present study was shown to have estrogenic activity, which causes cells and genn cells were detennined in homogenates of fixed testicular reproductive disorders including reduction in fertility. Sertoli cells parenchyma using phase-contrast cytometry. For years '80, '86, and '96, cultured for 24 hours in the presence of the PCBs (3 3' 4 4' respectively, several men (13, 8, and 21) of similarage (31 ±2, 31 ±3, and tetrachlorobiphenyl, 2 2' 4 6 6' pentachlorobiphenyl, and 2 2' 3 3' 28 ±Iyrs.) were used. Estimated paired testicular parenchymal weight (46 4 5 5' 6 6' nonachlorobiphenyl) at concentrations of 1.0-2.0 µM ± 3, 43 ± 7, and 42 ± 2 g), volume of seminiferous rubules/man (29 ± 2, 28 showed cellular enlargement. Despite their changes in morphology, ± 4, and 28 ± 2 ml), diameter ofrubules (2 17 ± 6, 202 ± 6, and204 ± 3µm), no reduction in Sertoli cell viability was seen at any of the number of Sertoli cells/g parenchyma (26 ± I, 29 ± 3, and 25 ± I x 106), concentrations or time points studied for either toxicant. Using Sertoli cell number/man (1.2 ± 0. 1, 1.2 ± 0.1, and I.I ± 0.1 x 10"), and numberofpachytene primary spennatocytes (1.3 0. 1, 1.3 ± 0.3, and 1.6 ± mRNA differential display, a number of novel cDNAs were = detected when cells were cultured either with cadmium or PCBs 0.1) or spherical spennatids (3.9 ± 0.4, 3.2 ± 0.6, and 4.8 ± 0.5) accommodated by individual Sertoli cellsindicate no patternof diminished showing that changesin gene expression accompany the changes Sertoli cell number or function from 1980 to 1996. Based on directcounts in Sertoli cell structure. We propose that Sertoli cells in culture and of testicular cells in these North American men, there appearsto have been mRi'\J A differential display provide a sensitive morphological and no decline in number of Sertolicells or Sertoli cell function men of the biochemical assay system to detect early direct effects of low doses iu Texas region of North America during the last 16-year period. NIH K04 oftoxicants on mammalian testicular cells. AG00464-05

30 A MORPHOLOGICAL STUDY OF TI-IE EP!DID!MYS ANDTESTIS OF RATS 3 2 NO DECLINE IN SPERMATOGENIC POTENTIAL IN A UChA AND UChB SUB.\-OTTED TO EXPERIMENTAL CHRONIC GROUP OF NORTH AMERICAN MEN OVER THE LAST 16 YEARS. ALCOHOLISM. E. Bustos-Obregon, F.E. �oz*. M- �oz*. V.H.A. Ggnon •, L. Johnson, J.J. Barnard,and H. Meguerditchian. Dept. of Vet. Anatomy & P.J. Garci.a•, CR. Padovaru• and H.R. Con=• Dept. of Cd! Biology and Gencocs. Public Hlth., Texas Veterinary Medical Center, Texas A&M University, Faculty of Medicme. U. of Chile, Santi.igo. Chile. College Station, TX. Dept. of Pathology, University of Texas Southwestern Medical Center, Dallas, TX. An aninulmodd has bttn devdoped which describes the adveae effects of chronic ethanol ingestion on reproducovc function. Morphologial changes of the epidid1m1des Reports of world-wide diminishedspenn counts in recent years have sparked and testes of rats subrrutted to expcrunental chronic alcoholism were analyzed through interest in, and raised concerns for, the spennatogenic potential in humans. morphometric and microscopic teduuqucs. Male nrs o f the saains devdoped by genetic Further, geographic variations in human spenn counts require srudyof subjects sdection in our I.hontory for high (UChB) and low (UChA) ethanol conswnpuon in specific areas. Spennatogenic potential of men estimated as daily sperm were used. R= of each stnin were divided into four groups : (a) UChA nrs. forced production is determinedby countingpachytene primary spenn atocytes (PDSP) ethanol intake. receiving only 10% v v ol a< drinking m= conswnpcion of / ethan fluid. or spennatids with spherical nuclei (DSP) inthe process of spennatogenesis. UChB rats, I 0% 25g ethanol per leg body weight per day. (b) provided ethanol ad libowm The objective was to detennineif PDSP andDSP have declined duringa 16- and water, m= conswnpcion of g, c UChA r.ats, provided with water, and (d) Wuur 7 ( ) yearperiod between 1980 and 1996 in a of North American men. Testes, r.ats with water(Controls). The results indicated histologic:J altcr:ll:ions of the liver of the group obtained at autopsy in Dallas, were fixedwith 2% glutaraldehyde, further fixed and of auda epididymal epithelium and of the epithd.ium. UChA group scminifcrous with 1% osmium, embedded in Epon, and enluated by stereology for volume absence of spcci& germinal dcrnents, vacuobcion and increased lipid droplets in the density of testicular structures. PDSP and DSP were estimated from the Sertoli cdls. There was an increased nuclear area of the Settoli cdls and of the nwnbcr of number of spennatocytes or spennatids in homogenates of fixed testicular thesecdls per rubu4r cross-section. There wereno changes in the epididymal head nor in parenchyma divided by the corresponding life spansof these cells. For years Lcydig cdls Testoscerone production wassignificantly reduced. and lcvds did not LH FSH '80, '86, and '96, respectively, fairly large numbersof men (13, 8, and 21) of differ bctWttn the ;uwnal.,srudicd. There was a reduced epithelial cdl size in cauda when similar (P>0.05) ages (31 ± 2, 31 ± 3, and 28 ± I) were Estimated paired compared to controls due to reduction of cytoplasm of the epithelial cdls. The rcsuks used. testicularparenchymal weight (46 ± 3, 43 7, and 42 ± 2g), percentage of suggest that depressed testosterone lcvcds may have • role in the etiology of cthanol­ = seminiferous rubules (66 ± 2, 71± 3, and68 2 %), PDSP/man (4 19 ± 65, 379 induced male reproductive dysfunction. However, the present data do not exclude the = ± 100, and390 ± 35 x 106), PDSP/g (8.6 ± 1.0, 8.6 ± 1.2, and9.2 ± 0.6 x 106), possibility that ethanol or its m=holii:es can also act directly on the epithelial cdls of the DSP/man (304 ± 42, 223 ± 53, and 299 30 x 1()6), andDSP/g parenchyma auda epididymal region and on germinal dcrnents. (CNPq 200940/95-3; PLACIRH ± PLC 123/95; CEE01 "-CT92-0022) (6.3 ± 0.7, 5.3 ± 0.8, and7.1 ± 0.6 x 106) indicatedno pattern of diminished spennatogenic potential from 1980 to 1996. Based on direct measures of spennatogenic potential in these men, there appears to have been no decline in spennatogenic potential inthe tested region of North America during the 16- year periodtested. NIH K04 AG00464-05

P-32 Journal of Andrology •January/February 1997

AGE - RELATED ALTERATIONS IN PLASMA NITRITE 33 DECLINE IN LEYDIG CELL VOLUME IN A GROUP OF 35 AND NITRATE LEVELS IN ll\IPOTENT �IEN NORTH AMERICAN MEN OVER THE LAST 16 YEARS. L. Johnson, M. S. W. Brown, and 1.1. Barnard. Dept.ofYel Anatomy & Public Hlth., Texas Rajasekaran , W.J. Hellstrom, S.C. Sikka, Tulane University School of Veterinary Medical Center, Texas A&M University, College Station, TX. Medicine, New Orl"81l.5, LA. Dept. of Pathology, Universityof Texas SouthwesternMedical Center, Dallas, TX. Nitric oxide (NO). synthesized by nitric oxide synthase (NOS) is a non adrenergic, non

34 PROADRENOl\lEDULLIN N-TERl\IINAL 20 PEPTIDE (PAl\IP), 36 PENILE ERECTION INDUCED BY TRANSURETHRAL A NOVEL PRODUCT OF THE ADRENOl\IEDULLIN GENE, INDUCES ADMINISTRATION OF NOVEL NITRIC OXIDE DONORS. PENILE ERECTION IN THE CAT Wayre J. G. Hellstrom, Run Wang. Hunter C. Champion*, Suresh C. Sikka, H. C. Champion•, R. Wang, W. E. Fitzgerald•, W. A. Murphy•, D. H. Coy•, Larry K. Keeffer*, Paul Doherty, Tulane University School of Medicine, P. J. Kadowitz•, and W. J. G. Hellstrom, Tulane Univesity School of Medicine New Orleans, LA, National Cancer Institute, Frederick, MD, VIVUS, Inc. New Orleans, Louisiana. Menlo Park, CA

Proadrenomedullin N-terminal 20 peptide (PAMP) is a novel 20 amino acid earlier studies have demonstrated that intracavenosal injection of nitric peptide encoded by the adrenomedullin gene. PAMP bas been shown to have Our oxide (NO) donors can induce penile erection. This study investigates the direct, cAMP-mediated vasodilator activity in the cat. The present study was undertaken to investigate the ability of intracavemosal inj ections of PAMP to effect on penile erection in an in-vivo fe line model by the transurethral useof induce penile erection in the cat. the novel NO donors: Proli/NO, (CH,)3 CCoo·Na • N [N (0) NO] · Na• and PAMPwas delivered by a 30-gauge needleinlracavemosally. The response was piperazine, (CH,),NH2 • N ((0) NO]'. characterized by changes in intracavemosal pressure, duration of the maximal NO donors were administered with a 20-gauge Jelco™ angiocatherter pressure, total duralion of the drug effect, change in penile length, and alterations transurethrally. The response wascharacterized by changes in intracavernosal to the systemic arterial blood pressure. The reference drug combination (J.65 mg pressure and penile length. A standard drug combination of papaverine, papaverine, 25µg phentolarnine 0.5µg PGE1) was injected intracavemosally and phentolamine, and PGE1 injected intracavernosally served as control. at the end of each experiment for control comparison. N 0 donors caused penile erection in a dose-dependent manner. When Intracavemosal injection of PAMP caused penile erection in a dose.erectile dysfunction. penile erection via intracavemous injection.

P-33 TWENTY-SECOND ANNUAL MEETING

9 GALAi'11N INDUCES PENILE ERECTION CAT 37 ll1 -llLOCKERS ENHANCE l'E�ILE ERECTION EFFECT OF PGE, 3 1N THE IN CATS H. C. Champion•, R. Wang, W. A. Murphy•, D. H. Coy•, P. J. Kadowitz•, Run Wang, Hunler Champion• , Suresh C. Sikka, Paul Doherty, Wayne J, G. and W. ]. G. Hellsirom, Tulane Univers11y School of Medicine, New Hellslrom, Tulane University School of Medicine,New Orleans, LA, VIVUS, loc. Orleans, LA. Menlo Park, CA Galanin is a 29 amino acid peptide is widely

38 ATPyS, A POTENT PURI:-.t:RGIC AGONIST, INDUCES 40 ANDROGENIC REGULATION OF RAT PENILE ERECTION PENILE ERECTION IN THE CAT C M Reilly• V S Stopper'. and T M Mills. Depanment of Physiology ano H. C. Champion•, R. Wang, D. G. Lamben•, P. J. Kadowitz•, and W. J. G. Endocrinology, Medical College of Georgia, Augusta GA 3091 2-3000 Hellslrom, Tulane University School of Medicine, New Orleans, LA. Previous studies from this laboratory using untreated-castrated rats A

P-34 Journal of Andrology •January/February 1997

41 ASSESSMENT OF THE '.\IALE ACCESSORY 43 ISOLATION AND PURIFICATION OF BASAL AND SECRETORY GLANDS OF INFERTILE ME.'i WITH KALLIKREIN EPITHELIAL CELLS FROM THE RAT VENTRAL PROSTATE N. hK2 IN SEMINAL PLASMA. Ravindranath• and M. Dym, Department of Cell Biolo� . Georgetown R.R. Tremblay, E. Coulombe*, G. Frenene* and J.Y. Dube*, Lab University Medical Center, Washington, DC. of Andrology, CHUL Research Center, Quebec, Canada. The epithelium of the prostate consists of basal, secretory, and endocrine· paracrine cells. It is hypothesized that the basal cells are the stem cells which The human prostate expresses the 3 knO\\n kallikreins namely hK I. can either renew themselves to form new basal cells, or differentiate into hK2 and hK3 (Prostate Specific Antigen). While the roles and/or secretory and endocrine-paracrine cells. Our objective was to isolate and variations of hKl and hK3 in seminal plasma have been previously characterize a pure population of basal cells. Minced pieces of ventral prostates described, those of hK2 are totally unknown. Thus, the availability from four adult rats were suspended in DMEM/FI2 medium containing of the purified kallikrem hK2 and rhe development of highly collageoase (I mg/ml) and DNAse (I µg/ml), and incubated at 3i'C for 20 min specific antibodies against the protein (no cross-reactivity with other in a shaking water bath operated at 100 cycles/min. Large sheets of glandular prostatic proteins) allowed us to set up an ELISA assay for the epithelium, which separated from stromal cells, were sedimented and measurement of hK2 in biological fluids. This assay was used to resuspended in DMEM/FI2 containing collagenase (I mg/ml), hyaluronidase (I add complementary information or to ascertain observations, based mg/ml), and DNAse (I µg/ml), and incubated for 30 min at 37°C. The on Western analysis, indicating impressive vari ations in the profile dispersed clumps of epithelial cells were plated in DMEM/Fi2 plus 10% FBS. of free or truncated hK2 (secreted by the prostate) and the hK2 After incubation overnight at 37°C, the floating epithelial ceU clumps were bound to Protein C Inhibitor (PCI) in seminal plasma (SP). PCI is a removed and subjected to a 0.05% trypsin·EDTA solution for 5 minutes. The o specific product of the seminal vesicles. To achieve this goal, f ur dispersed cells were then filtered through SO·µM and 40·µ�! nylon mesh groups of subjects were selected: fe rtile men (FM), vasectomized successively, and separated by sedimentation velocity at unit gravity at 4 °C with subjects (V), oligoasthenoteratozoospennic patients (OAT) and use of a 2-4% BSA gradient in DMEM/FI2. After 2.5 hrs, 50 fractions of IO azoospennic men (A). hK2 was expressed in µg/ml of SP. In FM ml volume were collected at sec intervals. From several isolations, we have (N=32), V (N=36), OAT (N=34) and A (N=53), hK2 was 90 observed that fractions 16-20 contained large single cells and fractions 26-30 respectively 2.2±0.9 (SD), 1.8± 1.0 (SD), 1.8± 1.2 (SD) and contained predominantly small single cells. The large cells contained a centrally 1.7±1.4 (SD). No significant difference was observed between FM located nucleus with numerous granules in an abundant cytoplasm. The small and infertile subjects. In conclusion, the study of the secretory cells were polyhedral in shape and had a dark nucleus with a very rim of function of either seminal vesicles or prostate appears much more thin cytoplasm. The pools of fractions containing large single cells and small single discriminatory when using a Western blotting technique to cells were characterized as secretory and basal cells by immunosraining for appreciate PCI and/or hK2 variations. Our ELISA assay provides cytokeratin 8 and 5, respectively. The principal contaminating cells in the basal only valuable information on the sum of hK2 and hK2-PCI in SP. cell pool were mesenchymal cells whereas no contaminating cells were observed By contrast with hK3 Androl. 16, .'.'lo 6, 1995), hK2 is not (1. the secretory cell pool . In conclusion. our results indicate that highly purified decreased infertile men. in in populations of secretory and basal cells could be isolated from the ventral Supported by MRC grant. prostatic epithelium by the technique of sedimentation velocity at unit gravity.

:2 THE EFFECTS OF SPrNAL CORD 11'.JL"RY ON TRP'.\-1 AND 44 INTRAPROSTATIC INJECTION OF ZINC SALT AS A ANDROGEN RECEPTOR mRNA IN THE PROST.HE OF THE RAT TREATMENT FOR BENIGN PROST A TIC HYPERPLASIA. H.F.S. Huang•. M-T Li. T.A. Linsenmeyer. J.E. Ottenweller, L.M. Min Wang•I, Jian Cao• 2 , Mostafa S. Fahim 1. Center of Reproductive Pogach and R. J. Irwm, Departments of Surgery, Physical Science andTechnology 1 , School of Medicine, University of Missouri, Medicine/Rehabilitation, Neuroscience and Medicine, UMD-New Jersey Columbia, MO 65212. Peking Union Medical College Hospital 2 , Medical School, Newark; Kessler Institute of Rehabilitation. West Beijing, People's Republic of China. Orange; and V. A. Medical Center, East Orange. \"ewJersey Fifty percent of men over the age of 50 years have some degree of In men, sperm motility is usually abnormal after spinal cord injury (SCI). hyperplastic enlargement of the prostate while 95% of men will This could resuh from abnormal spermatogenests, or due to changes in experience symptoms related to prostate enlargement by the time they biochemicalpr operties of the seminal fluid. In this study, we examined the reach the age of 85 years. Twenty-five percent of all patients seen by expression ofmRNA fortestosterone repressed prostate message (TRPM- urologists sufferfrom benign prostatic hyperplasia (BPH). Therefore, we 2) and androgen receptor (AR) in the prostate of rats after surgical studiedintraprostatic injection of zmc salt as a treatment forBPH (U.S. transection of the spinal cord at the levels of T9 or LI vertebra. SCI Patent Nos. 4,946,688; 5,071,658; 5,234,698; Andrologia 25:369, resulted in an acute decrease inpro state weight and an increase (P<0.05) I 993). A pilot clinical study was conducted in 34 patients who had been diagnosed I 00 in steady state le vel of prostate TRPM-2 mRNA that concurred wrth a with BPH. One milliliter of mg zinc salt was injected into each decrease in serum testosterone. In contrary, there was a transient but lateral lobe of the prostate of patients with BPH. All of the patients 1-4 significant decrease (P<0.05) in the steady state level of AR mRNA. experienceda sensation of swelling at the area of injection for days . Ahhough normal serum testosterone concentrations and prostate AR No other side effects were observed. The results of the pilot study indicated in t ms truct n, an mRNA levels have subsequently been restored, prostate weights remained significant decrease symp o of obs io increase in m persistently lower in SCI rats than sham controls for at least 3 months. urinary flow, and a decrease rostattc volume. The average of the Concomitantly, the level of TRPM-2 mRNA remained elevated as late as Amencan rology ssoctatlon (AUA ymptom Index of all injected 27.8 to I 1.5 6 months after the injury. Morphometric analysis revealed a decrease in patients decreased from fourweeks after injection. Future clinical investigations bedesigned andinclude a longer post·injection the height of prostate epithelial cells in the SCI rats, and evidences of will abnormal epithelial morphology and possibly cell deathwere also noted in follow-up period . some animals. These resuhs demonstrate a prolonged effect of SCI on prostate fimction. These findings suggest that male accessory sex glands may be compromised after SCI. These changes may affect biochemical properties of the seminal plasma and may provide some explanation for abnormal sperm motilityseen inthe semen of SCI men.

P-35 TWENTY-SECOND ANNUAL MEETING

45 OSTEOPENIA IN MEN WITH PROSTATE CANCER !+7 ANDROGEN ABLATION 11\DUCED CELL KINETIC TREATED WITH ANDROGEN DEPRIVATION CHANGES IN A RAT PROSTATE Tn!OR M.L. Meistrich, R.A. D.A. Cook', M. Eisenberger•, C.R. Schneyer·, D. Hoover·, A.S. White, C. Sikes, D. Lim Joan, C.S. Wu. �- Hasegawa, N.H.A. Terry. Dabs, The Johns Hopkins University School of Medicine, G.K. Zagarsand A. Pollack, M.D. Anderson Cancer Center, Houston, TX Baltimore, MD. The decrease in growth rate of androgen-sensitive tumors fo llowing Current treatment for advanced prostate cancer uses androgen androgen ablation could be caused by altered cell cycle kinetics, induced deprivation therapy (ADT) to induce a hypogonadic state. We cell quiescence, or increased cell loss by apoptosis or necrosis. We hypothesized that this therapy would cause a decrease in bone investigated the contribution of these various factors to castration-induced mineral density (BMD), leading to accelerated bone loss and an changes in growth of R3327-G Dunning rat prostate tumors in vivo. increased risk of fracture. Osteopenia, a condition in which BMD Histological determination of cell numbers, TUNEL-staining for apoptosis, is between 1 and 2.5 standard deviations below the young adult and flow cytometry of thymidine-analog labeled cells were applied to a new mean, is considered prognostic of future fracture risk. In the model for cell population growth in which cell loss through apoptosis is present cross-sectional study we examined the femoral neck and included. Cell cycle progression was hardly changed by castration as the lumbar spine BMD of 13 men (age S7 to 80 yrs., mean 72 +I·6 duration of S-phase was only increased from 19 to 23 hours . In contrast, yrs.) with prostate cancer who had undergone ADT for at least 18 the potential doubling time (if cell loss were not occurring), calculated from months. The spinal BMD values for 6 of those men were unusable cell kinetic measurements , increased from 6.2 days to 42 days. This was due to reactive sclerosis. The mean spinal BMD of patients the result of a decrease in the fraction of cells that were proliferating from without sclerotic changes (mean ± SD 0.888 ± 0.123 g/cm2, n=7) 65 3 to 8 3 . The apoptotic index of this rumor was low, increasing from was significantly below that of age-matched normals (p < 0.02). 0.143 in intact rats to 0.43 in castrates . In intact rats the cell production Five of these 7 men (71 %) had osteopenia in the spine while 13 of rate was calculated at 11.2 %/day and cell loss by apoptosis at 0.3 %/day. 13 (100%) were osteopenic in the femoral neck. The mean BMD In contrast, cell production was reduced tO 1.2 %/day in castrates but the in the femoral neck (0.720 0.082 g/cm2) was slightly lower than +I· cell loss rate was only slightly increased to 0. 7 % . The differences between age-matched normals (p < 0.1 ). Deoxypyridinoline (DPD) and production and apoptotic loss rates agree well with changes in tumor cell osteocalcin, markers of bone resorption and formation numbers detennined by measuring rumor volumes and calculating the respectively, have been used clinically to determine the status of volume density of rumor cells from histological slides. Thus, the dramatic bone turnover. In this study the mean DPD concentration was decrease in R3327-G rumor growth after androgen ablation is mainly due 12.85 ± 4.58 nmol DPD/nmol creatinine (normal range 3.0 - 10.7) to a decrease in cell production because of entry into quiescence. Induction while the mean osteocalcin concentration was 9.3 ± 2.3 ng/ml of quiescence could make cells more radiation resistant and suggests that (normal range 5.1 • 16.9). These results suggest that androgen deprivation therapy in patients with prostate cancer may augment in the combined modality treatment of prostate cancer, unless there are the rate of bone loss and increase the risk of fracture, resulting in other factors that compensate, administration of the irradiation during a significant adverse effects on qualityof life and morbidity. period of androgen ablation might result in a poorer tumor response.

46 THE REGULATION OF PROLACTl:-.1 AND EGF 48 THE CYNOMOLGUS MONKEY PROSTATE UNDER PHYSIOLOGICAL RECEPTOR mRNAs IN A HUMAN PROSTATE CANCER AND HYPOGONADAL CONDlTIONS A.\; ULTRASONOGRAPHIC STIJDY CELL LINE (LNCaP) BY LHRH A. Kamischke, H.M. Behre, G.F. Weinbauer and E. Nieschlag, lnsUtute of J-Y, Liang*, J.N. Rao, J.F. Wilber*, and P. Feng*, Division of Reproductive Medicine of the University, Miinster, Germany Endocrinology, University of Maryland School of Medicine, Objective: Although the prostate of the cynomolgus monkey has been shown to Baltimore, MD 21201. be a particularly suitable model for the human, in vivo data on this organ are hardly available I _ In the present work we apply transrectal ultrasonography for Prolactin receptors (PRL-R), members of C)'tokine-hematopoietin monitoring prostate size longitudinally and mvestigate the dynamics of prostate receptor superfamily in which EGF-R is also included, mediate size under physiological and hypogonadal conditions. Materials and Methods: PRL biological functions, including cell prol iferation. LHRH Five adult intact and 7 long-term castrated monkeys were analysed repeatedly for analogs, known as anti-prostate cancer drugs, suppress androgen assessment of the fe asibility and variabilit) of the method. In addition, cross­ levels through the pituitary-testicular axis. Howe\"er, LHRH sectional evaluation was performed on 30 adult monkeys of various ages and on 7 agonists were also shown to have direct anti-proliferative effects long-term castrated monkeys. Finally, the vehicle (n=5) controlled effects of on prostate cancer cell lines. To ascertain whether LHRH can androgen deprivation treatment with the GnRH antagonist cetrorelix (n=4) for 25 directly regulate mRNA levels of PRL-R and EGF-R in days followed by semicastration at day 16 and full castration on day 25 were androgen-dependent LNCaP, cells were treated with LHRH for 3, examined and prostate size was followed thereafter. Results: The monkey prostate 6, 12, 24, and 48 hr, respectively, and mRNA levels were shows an ellipsoid shape and ultrasonic appearance similar to the h11man prostate. determined by using semi-quantitative RT -PCR. LHRH-R Variability in 5 intact monkeys (mean±SE= 4,67±0,55 ml) and 7 long-term mRNA was also confirmed by RT-PCR. Results: PRL-R mRNA castrated monkeys (0,88±0,08 ml) was 8,5% and 5,6% respectively. In intact levels were increased by 65% at 3 hr incubation with 10-7M animals a linear correlation between age and prostate size was found (p= 0,0004) LHRH, and by 50% at 6 hr with 10·9M LHRH, compared to Prostate size in castrated animals was reduced (p< 0,005) compared to intact controls (p<0.05). This up-regulation was mainly attributed to the animals. In the experimental study, GnRH antagonist led to a significant reduction of prostate volume and testosterone levels compared to the vehicle increase of the short form PRL-R since there was no significant change in the long form PRL-R mRNA. In contrast, EGF-R group, while semicastration had no influence. Following removal of the second mRNA was down-regulated by 49% (p<0.05) after 12 hr testis a further decrease of prostate volume was seen (p< 0,0001 ). When compared, castration and GnRH antagonist treatment induced a similar decrease incubation with LHRH (1Q-7M). Conclusions: 1. PRL-R mRNA of prostate volume over time. Conclusion: Transrectal ultrasonography provides a was shown for the first time to be expressed in LNCaP cells, of fe asible and reproducible approach for detennination of prostate size in which the short form was up-regulated by LHRH. These results cynomolgus monkeys. Castration and GnRH antagonist treaunent are equally encourage studies on the role of PRL in prostate cancer. 2. The effective in reducing prostate size. References: Habenicht U.F., M.F el Elreby. down-regulation of EGF-R mRNA by LHRH may renect its anti­ 1 .) Rationale ror using aromalase inhlbilors lo manage benign proslalic hyperplasia_ Experimenlal proliferativeaction on prostatic tumor cells. studies. J. Andrei. 12: 356, 1991

P-36 Journal of Andrology • January/February 1997

49 DEVELOPMENTAL SHIFT OF l\HIBIN OR INHIBIN RELA'fED S I EFFECTS OF EXERCISE ON TESTOSTERONE AND NITRIC OXIDE PROTEINS PRODUCING SITE Il'iFETAL THROUGH INFANTILE PRODUCTION IN TIIERAT TESTIS VJ Meskams•. F.S Hannan•, J.S Volek•. BC Nindl0, W.J. Kra=or", WelllSlock• and R.Deavc:r Departments ofDauy andAnimal RAT TESTES D. D Science and Veterinal)·Science and the Center for Sports Medicine, The PenJ1>1·Ivania State J_Noguch11• H.Hikono�·. the late S Sato. K Kikuchi'", A.Shimada1", H.Kaneko1 -_ Unh·ers1ty, Uni\'=ity Parl. PA Y Hasegaw a3", G.Watanabe • and K.Ta�a"· 1Natl Inst. Agrobiological Resources. Tsuk-uba. lbaraki. �at! Inst. .-\rumal Health, Tsukuba, lbarak1, Effects of exen:ise ontestostorone secretion varies among species and \\ith the type of exorciseprogram. rats inhibition of nitnc oxide production re-suits a marked incTease 3 Lab. of Amma. Sci . Kitasato Umv , Aomon. 'Lab. of Vet. PhysioL Tol..:o In in in testosterone pro

50 LOCAL TESTICULAR CONTROL MECHANISM ENABLED )2 PATERNAL AGE AFFECTS PROGENY OUTCOME IN THE THE TOTAL NUMBEROF SERTOLI CELLS FOUND AN ACT IN INT BROWN NORWAY RAT. V. Serre•, B. Robaire. Department of RAT TO BE EXCEEDED. Larry Johnson, Kevin L. Kunde, Genevieve E. Pharmacology and Therapeutics. McGill University, Montreal, Canada Keillor, and Angela J. Kaschmitter. Dept. of Vet. Anatomy & Public Hlth., Aging of the male is accompanied by functional and structural changes Texas Veterinary Medical Center, Texas A&M University, College Station, in the reproductive tract. In the testis, there is a progressive loss of germ TX. cells, atrophy of the seminiferous tubules and diminished capacity of Leydig cells to produce testosterone. In the epididymis, a major If the total numberof Sertoli cells in a rat is to be augmented by intervention, accumulation of lipofuscin and the formation of large vacuoles are seen in the number of Sertoli cells achievable in an intact rat (46.4 ± 1.0 x JO') must the epithelium; an increased incidence of abnormal spermatozoa is found be exceeded. Furthermore, if the testis itself contributes locally to the in the lumen. Consequently, we investigated the effect of increasing age regulation of total Sertoli cell number, transplantation of more than twotestes on progeny outcome. For fertility studies, each male was mated overnight should augmentthe total numberof Sertoli cells achievable per rat. Four, six, with 2 females in proestrus. On day 20 of gestation the fe males were andeight neonatal testiculargrafts were transplanted into young, adult, inbred, Cesarian sectioned, the ovaries were removed and the number of corpora castrated, Fischer rats. At 20 days posttransplantation, the hosts were lutea counted; the uteri were opened and the numbers of implantation terminated, and the testicular grafts were recovered, fixed, and embedded in sites, resorptions and live fetuses were counted. Fetuses were weighed Epon. Toluidine blue stained 0.5 µm sections were evaluated by stereology and examined for external malformations. Matings between male and to determine the volume density of Sertoli cell nuclei. Unstained 20 µm female Brown Norway (BN) rats resulted in a low litter size (5.0±0.7 sections were observed by Nomarski opticsto determine the height and width fetuses/litter). To determine whether this low litter size was due to the of individual Senoli cell nuclei to calculate a correct nuclear volume. When male or female, we cross bred BN with Sprague Dawley (SD) rats. Litter four, six, and eight testes were transplanted into five, four, and three rats size was significantly higher in the SD female (14.6±0.3) compared to the respectively, there was an increase (p< 0.05) in total testicular parenchymal BN fe male (5.2±0.7); paternal strain had no influence. Thus the reduced weight (38.8 ± 22.2, 72.9 ± 26.5, and 106.5 ± 41.2 mg) andan increase (p< fertility in the BN strain was due to a fe male factor. To assess whether 0.05) in the total number (IO') of Sertoli cells ( 36.0 ± 22.7, 61.6 ± 20.2, and aging affected the fertility of male SN rats, animals aged 3, 12, 18 or 24 85.9 ± 24.6). Hence, rats with six or eight transplanted testis had a total mo. were mated to fe male SD rats. There was no significantchange in the number of Sertoli cells/rat that exceeded the number in intact rats. However number of resorptions or live offspring or in the incidence of external on a per testis basis, there was no change (p>0.05) in the number (IO') of malformations. However, there was a significant increase in the pre­ • Sertoli cellsper testis (10.2 ± 6.9, 1 1.2 ± 2.8, and 1 1.6 ± 2.8) or intesticular implantation loss in older males (24 mo., 23.0±9.6%) compared to young parenchymal weight per testis (10.7 ± 7.0, 12.I ± 4.4, and 13 .3 ± 5.2 mg). males (3 mo., 4.6±1.3%). A dramatic decrease in the average fetal weight Since the total Sertoli cell number can be increased proportionally by was found. Fetuses fathered by 3 month old males were 3.97±0.07g increasing the number of testes transplanted, a component of regulation of while those fathered by 12 mo. old males were 3.62±0.07g. This decrease Sertoli cell number arid testicular size is at the level of the testis. In was sustained for males aged 18 and 24 mo. Together those results show conclusion, the total number of Sertoli cells is amendable to experimental that paternal age has a significant effect on both pre-implantation embryo intervention, and a local testicular controlling component of total Sertoli cell survival and fetal weight, thus indicating that the quality of spermatozoa number must be involved. NIBR OI AG 10093-10 may decrease as males age. Supported by NIA AG08321.

P-37 TWENTY-SECOND ANNUAL MEETING

53 STAGE-SPECIFIC LOSS OF GERM CELLS IN THE RAT AFTER 55 TPX-1 GENE IS SPEClFICALLY TRA.'iSCR!BED BY NORMAL A SINGLE EXPOSURE TO HEAT OCCURS BY APOPTOSIS HUMA.J.'1 GERM CELLS DL1UNG THE LATE STAGE OF Lue*, A.P. Sinha Hikim, P. Im*, K.S. Tiang*, T. Bui*, A. Leung•, YH. R. SPERMATOGENESIS. SwerdlolT, and C. Wang, Department of Medicine, Harbor-UCLA Medical NoriakiTok uda*, Shigeru Matsuzaki•, Kikuo Nomoto• andJoichi Ku mazawa , Center., Torrance, CA. • Department of Urology and Immunology, Kyushu University, Fukuoka, Japan Short termexposure of the testis to heat causes degeneration of germcells but Background : TPX-1 gene was found to map adjacent to ffiA on chromosome 6 has not distinguished necrosis from apoptosis. We have previously andhave 77.8% nucleotide 70'"• amino acid sequences imilarity with mouse Tpx-1 demonstrated that stage-specific loss of germcells afterhormonal deprivation gene(Kasaharaet al. Genomics 5:527,1989). TPX-1 gene are considered to be the occurs exclusively by apoptosis. The objectives of this study were to establish human counterpart of mouse Tpx-1 gene. Mouse Tpx-1 is a testis-specific gene whether heat also causesdamage to the rat testis by apoptosis; if so to document its stage-specific kinetics. Testes of adult male rats (5 rats per group) were and transcribed by mouse haploid male genn cells. However, the specificity and immersed in a water bath at room temperature 22° C (control) or at 43°C for 15 the function of Human TPX-1 gene remained unknown. min(heat treated groups). Animalswere sacrificed on day I, 2, and 9 afterheat Objectives : To study the activationsite of the TPX-1 gene and to investigate exposure. Apoptosis was characterized by a modified in situ procedure which the expression of this gene product in various tissue samples. specificallydetects apoptotic germ cells in the testis and quantitated as number Methods : Total cellular RNA from tissue samples was extracted by the ofapoptotic germ cell Sertolicell (apoptotic index, Al). Germcell apoptosis per guanidinium-isothiocyanate method After transferri ng RNA to a Gene Screen was markedly increased (p<0.05) on day I and 2 after exposure to heat but not 32 on day 9 when compared to control. On day 9, almost all apoptotic cells Plus(NEM, Boston, MA), hybridization was mare to the P-labeled disappeared and an active proliferation process was noted by presence of nick-translated TPX-1 probe. Approximate sizes of the hybridizing RNA were

increasing nwnbcrs ofpreleptotene and lcptotene spermatocytcs. On day I, the estimated using mouse ribosomal RNA as molecular size marker. mean Al was highest at stages I-IV, IX-XI, XII-XIV (3.4 to 5.9), and lowest at Results : Here we show that the messenger RNA derived from this gene(l.4 and stages V-VI (0.34) whereas stages VII-VIII (2. I) were intermediate. On day 2, 2.0kb) is specifically andabundantly transcribed in noanal adult human testis but AI remained highest between stages I-IV, IX-XI, XII-XIV (3. I to 5.5), but the not in epididymis, prostate, or ovazy. Moreover, we could not this gene lowest AI occurred at stages VII-VIII (I. I). The increase in AI in stages V-VI detect (2. 7) at day 2 may reflect progression of apoptotic cells from previous stages. expression in a testis biopsy sample ofinfertile patient with maturation arrest, an The results indicate that I) exposure of the adult rat testis to heat results in the infant undescended testis biopsy sample, or various testicular tumors. These stage-specific loss of germ cells via apoptosis, 2) The early and late stages of results suggest TPX-1 gene is a testis-specific gene and transcribed by nonnal spcrmatogcnesis arc most sensitive to the effects of heat on apoptosis. The mid human male germ cells during the late stage of spermatogenesis, raising the stages V-VI and VII-VIII (hormone response stages) are relatively protected from programmed cell death. We conclude that transient exposure to heat possibility that TPX-1 may play an important role in the human spermatogenesis damages the testis by stage specific activation of apoptosis. and thus fe rtilization.

s,t 54 BIOCHEMICAL CHARACTERIZATION OF SERTOLI CELL (SC) 56 SERTOLI CELL CYTOSKELETAL At\D RELATED PROTEINS. MEMBRANE PROTEINS THAT ARE LIKELY TO BE INVOLVED IN AND co-MACROGLOBULIN, ARE NOT IN\'OLVED IN INHIBITED SPECIALIZED JUNCTIONAL COMPLEX (JC) FORMATION. Dolores SPERM ATION (LS.). R.N. Wine* and R.E. Chapin. Reproductive Mruk"' 2, Will M. Lee2. and C. Yan Cheng'. 'The Population Council, 1230 I Toxicology Group, NIEHS, RTP, NC York Avenue. New York, NY 10021; and 2oepartmentof Zoology, Universily of Hong Kong, Hong Kong. A common feature of the testicular response to many toxicants is inhibited sperm release. We used imrnunohistochemistry to evaluate Throughout spermatogenesis, primitive germ cells Iraverse from the basal IJ cytoskeletal proteins (t b lin vimentin, actin. \lnculin, and a-actinin) lamina to !he adluminal compartmentof the seminiferous epithelium where u u , because several Sertoli cell cytoskeletal proteins are closely apposed to malure spermalozoa are released into the tubular lumen. It is conceivable germ cells, and have been related to movement of germ cells wit n the that inter-Sertoliand Sertoli-germ cell specialized junctions are continuously � seminiferous epithelium, 2) B4 integrin, a member of a fa1TIJly of disrupled and regenerated. Thus, JC component proteins are expected to intercellular adhesion molecules , and 3) the protease inhibitor az­ turnover rapidly. This study sought Io identify SC membrane proteins !hat macroglobulin (a2-M), found to be not present specifically at are likely to be involved in these events utilizing (35S]-Met incorporalion. spenniation. SD rats, 3/group, were treated with doses of compounds Pure SC isolated from immature rats were cultured athigh cell density (0.7 x known to produce I.S. (boric acid, dibromoacetic acid. ,5-he anedione or tri­ 1 cells/cm2) with (35S]-Met on Matrigel-coated dishes in Ham's 2/DMEM 2 x , os Fl -cresyl phosphate). Testes were snap frozen or fixed in paraformaldehyde in order Io allow the formalion of specialized junctions. Thereafter, SC were o prior to cryostat sectioning, and staining. Antibodies were obtained processed for membrane extraction by cell lysis under hypotonic conditions, conunercially, except a1-M antibody, kindly provided by Yan Cheng homogenization and detergent solubilization. The solubilized [35S]-labeled (Pop'n. Council, NY . Bound antibody was visualized with Zyme kits. proleins were fractionated by sequential CB and C4 reversed-phase HPLC ) � The distribution of staining was the same for both control and tox1cant­ and SOS-PAGE. Actively synthesized proteins of 45, 38, 28, 18.5, 18, 17.5 treated testes. Tubulin was prominent in the apical Sertoli process, was and 13 kDa were readily identified by fluorography. The 45, 38, 18.5, 18, not associated with normal step 1 9 spermatids, and was not localized 17.5 and 13 kDa proteins were furtherpurified to apparent homogeneity by adjacent to aberrently-retained spermatids. Similarly, actin was successive HPLCs. PartialN-terminal and internal amino acid sequence prominent around the apical Sertoli process, was not near ste 19 analysis revealed thal some of these proteins are indeed unique. Similar i: spermatids at normal spenniation, and was not localized near retamed protein patterns were seen using testicular membrane extracts except that spermatids. Vimentin, vinculin, and a-actinin, clearly visi le arou d the 45, 38, 18.5, and 18 kDa proteins were more prominent in the SC � � Sertoli nuclei and extending luminally, were never associated with membrane extracl. When the same amount of protein obtained from SC retained spermatids. az-M was distributed as preYiously described (Zhu membrane extract derived from cells cultured at low cell density (5 x 1 04 et al., J. Andr 15:575, '94), and showed no alteration with 1.S. B4 cells/cm2) in the absence of specialized junctions was examined, the ei, integrin staining was found in late spermatid cytoplasm and residual abundancy of !he 45, 38, and 18 kDa proteins was significantly reduced bodies, but was not near the heads of retained spermatids. Proteins suggesting !hat these proteins may be related Io JC formation. In summary, a comprising, and related to, the Sertoli cell c toskelet n appear not to unique set of proteins are identified in the SC membrane when JC are � ? mediate I.S.; current efforts focus on changes m adlurrunal proteases. formed in vitro suggesting that these proteins may be important for JC formation and are novel markers in studyinQ cell-cell interactions in the testis.

P-38 Joumal of Andrology • January/February 1997

57 5-AZACYTIDINE TREATMENT INITIATED DURING MEIOTIC 59 EFFECT OF TffYROID HOR�IO'."E ON TESTICCL..\R OEVELOPMENT OF MALE GERM CELLS RESULTS IN ABNORMAL DEVELOPMENT A:-.JD mRNA LEVELS OF THYROID HOR.\-10!\:E EMBRYO DEVELOPMENT. T.E. Doerksen• and J.M. Traster. Depts. of RECEPTORS IN THE TESTES OF l�l�!..\TURE AND ADULT Pediatrics, Human Genetics and Pharmacology & Therapeutics, McGill RATS University and the Montreal Children's Hospital Research Institute. Montreal. J.N. Rao, J-Y. Liang*. J.F. Wilber* and P. Feng*. Division of Quebec. Canada. Endocrinology, University of Maryland School of Medicine. DNA methylation patterns are established during gametogenes1s and Baltimore, MD 2120 1. embryogenesis and play an important rolein gene regulation and embryonic development. In previous work, administration to male rats of 5-azacytidine Thyroid hormone (T3) together with its receptors is critical for (5-aza). a cytidine analogue that leads to decreases in DNA methytation, normal growth, development and metabolism in mammals. To explore resulted ;n abnormal embryo development when germ cells were exposed the effects of T3 on the regulation of testicular function and its own during mitotic, meiotic and post-meiotic development but not if they were receptors, we im•estigatcd testicular development, and mRNA levels of only exposed post-meiotically. To determine whether germ cell exposure TR-a and � in the hyper- and hypothyroid testes of immature and initiated during meiotic development could also alter sperm function, male adult rats by semi-quantitative RT-PCR. Sprague-Dawley male rats Sprague-Dawley rats (n=61group) were treated with saline or 2.5(L) and were treated either with T3 or PTU from day 1 to day 21. One half 4.0(H) mglkg of 5-aza, 3 times a week for 6 weeks. To assess progeny of each group of rats, including control, T3 and PTUtreatment, were outcome, males were mated with normal females and cesarean sections sacrificed at day 21. The remaining half of each group was allowed performed at 20 days of gestation. Paternal 5-aza treatment resulted in a to recover until day 50. Significant decreases of body and testis dose-related increase in preimplantation loss (saline-5.0%, 5-azaL-9.5%. 5- weight were observed in both hyper- and hypothyroid rats of 21- and azaH-24.2%) with no increase in post-implantation loss or fetal 50-day-old compared to controls. Higher serum T3 levels found in abnormalities. Embryos were also examined at 17 hours (pronucle1 present) hyperthyroid rats of 21-day-old and lower levels in hypothyroid were and 2 days (2-cell stage) of age to determine whether the early embryo loss all restored to normal in adulthood. Histological examination was due to lack of fe rtilization or embryo death. At 17 hours, embryos m all revealed reduced size of the tubules and less germcells in the 21-day­ groups showed a pronucleus and sperm tail. indicating that fertilization had old hypothyroid testis. At 2I days of age, lower testicular TR-a 3 occurred. In contrast, on day 2 of gestation, mating with the 5-aza treated mRNA levels were detected in the hyperthyroid group (0. 8±0. l; P<0.05), and higher levels were observed in hypothyroid rats males resulted in an increase in the number of morphologically abnormal (0.98±0.04; P<0.05) compared to the controls (0.79±0.0-+). No embryos (saline-7.9%, 5-azaL-16.4%, 5-azaH-31.3%). The results provide significant changes were detected in TR-� mRJ'1A levels between evidence that the preimplantation loss seen at 20 days results from death experimental groups. At 50 days of age. neither testicular TR-a nor of embryos early in their development, at around the 2-cell stage We TR-� mRNA levels were altered significantl y. Conclusions: Our suggest that 5-aza leads to decreases in DNA methylation in meiotic germ results demonstrated that prepuberal hyper- and/or hypothyroidism cells that subsequently affect embryo development. (Supported by MRC) can directly influence 1) testicular development 2) mRJ'IA levels of TR-a but not of TR-� in the immature testis, and 3) the altered levels of testicular TR-a mRJ"\/A could be restored to normal at adulthood.

58 EXPRESSION OF CYCLIN H AND CYCLIN­ 60 IDENTIFICATION OF TWO KINETICALLY DISTINCTACTIVITIES OF DEPENDENT KINASE IN THE MOUSE TESTIS 7 I IP-HYDROXYSTEROID DEHYDROGENASE IN RAT LEYDIG CELLS. D t & J.T. McGaughy* and S.E. Raw1ik, ep . of Cell Biol. Biochem. R.-S. Ge•, H.-B. Gao•, V. Lakshmi•, G.L. Gunsalus• and M.P. Hardy, Population Texas Tech University Health Sciences Center, Lubbock. TX 79430. Council, New York, NY Cyclin/Cyclin-dependent kinase (Cdk) complexes are key regulators of the cell cycle. We have previously shown that cyclin A 1 and cyclin A2 Leydig cells are susceptible to the direct glucocorticoid-mediated inhtbitton of and their Cdk partners, Cdc2 and Cdk2, are differentially expressed in testosterone biosynthesis. This inhibition can be counteracted by I I P-hydroxysteroid the mouse testis during spermatogenesis. Recently, cyclin NCdk2 dehydrogenase (I l fl-HSD). which oxidatively inactivates glucocorticoids. Of the two complexes in cell lines were shown to be activated by another isoforms of 11P-HSD that have been identified. type I is an NADP(H)-dependent cyclin/Cdkcomplex, cyclin H/Cdk7 or CAK C.Cdk Acti vating Kinase). oxidoreductase with reductase activity predominating. In contrast, type [! 1s NAD­ To begin to test the hypothesis that CAK regulates the cyclin A­ dependent and acts exclusively as an oxidase. The identity of the I I P-HSD isoform in associatedkinases during spermatogenesis, we have begun to examine Leydigcells is unceruiin, since the protein in this cell is recognized by an anti-type I I IP· the expression of cyclin H and Cdk7 in the mouse testis. Analysis of HSD antibody but the activity is primarily oxidative, more closely resembling type Il. RNA in several adult tissues (heart, kidney, liver, lungs, ovary, and The goal ofthe presentstudy was to detennine whether the kinetic properties of I I P-HSD testis) showed that eye/in Hand are most abundantly expressed Cdk7 in Leydig cells areconsistent with type type [!or neither isoform. Leydig cells were in the testis. We detected a major cycli11 H transcript of -1.5 kb. A I, purified from male Sprague Dawley rats (250 g), and IIP-HSD oxidative and reductive ntinortranscript of -:!.5 kb was present only in the testis. Cdk7 was activities.types I mRNA levels, and type protein were evaluated in Leydig cells. expressed as a single transcript of-1.5 kb. To begin to determine the andIl 1 LeydigceU I IP-HSD had bidirectional catalytic activity that was NADP(H)-dependent. possible cell types which may express eye/in H and Cdk7 , we examined RNA from day 7, day 17, and adult testis. Interestingly, This corroborated the hypothesis that type I I I P-HSD is present i n rat Leydig cells. both cyclin H and were most abundant in day 7 and adult testis However, unlike the type I I I P-HSD in liver parenchymal cells that is primarily Cdk7 17 · RNAs, with lower levels in day testis. The 2.5 kb cycli11 H reductive, oxidation predominates in Leydig cells. Moreover, analysis of kinetics transcript was detected only in the adult samples. These results are the revealed two components, the firstbeing a low K. NADP-dependent oxidative activity first data indicating that cycli11 H and are developmentally with K. of 41.5 ± 9.3 nM and V,,,., of 7. 1 ± pmol/min cells. The second Cdk 7 a 1.2 • IO' regulated in the testis. Furthermore, these data suggest that eye/in H componentconsisted of high K. oxidoreductase activities that were consistent with type and may function in both mitotic and meiotic cell cycles. /11 sill/ Cdk7 I: NADP.

P-39 TWENTY-SECOND ANNUAL MEETING

61 N!TIUC OXIDEAS A FACTOR GRO\\/TII OF TIIE CANINE PROSTATE IN 63 LOSS OF TESTICULAR FACTORS CAUSES CHANGES IN THE r n S, L. T.S.K. .D AL J.K. C o e1, Chnmness1 , Chwig1 (d=sed),J . Strandberg"2 wid. STABILITY AND TRANSCRIPTION RATE OF GAMMA-GLUTAMYL Burnett"' Department of Urology, The Johns Hopkins Uruversity, Baltimore, MD 1 21287 TRANSPEPTIDASE mRNA-IV IN THE INITIAL SEGMENT OF THE RAT Division of ComparativeMedicine, The Johns Hopkins University, Baltimore, MD 120 2 2 5 & EPIDIDYMIS. D.B. Rudolph B. T. Hinton. Department of Cell Biology, University of Virginia Health Sciences Center. Charlottesville, VA 22908. INTRODUCTION ANDOBJECTIVES : Recent studies have suggested that nitric oxide synthase (NOS) is present in the prostate ofvnriousspecies including man. Nitnc oxide (NO), the biochemical productof the NOS pathwa� . may function in prostalic smooth Gamma-glutamyl transpeptidase (GGT) is an enzyme believed to play a role in the muscle relaxation and/orapoptosi s. To fu rther in\'estigate the role of NO in the prostate, protection of maturing spermatozoa in the epidtdymis. Our previous studies have we examined NOS expression in the canine prostate under different hormonal conditions shown that the fourGGT mRNAs (I-IV) transcnbed fromthe single copy rat GGT and correlated these results with histopathological fi ndings. gene are differentially expressed and regulated in the rat epididymis. In particular, METHODS: Using the beagle as a model for BPH. 3 treatment groups were established: the expression of GGT mRNA-IV in the epididymal initial segment is dependent intact control, subcutaneously implwiled with blank silastic capsules at years ofage 2 upon the presence of testicular factors. Efferent duct ligation (EDL), which (trtI); castrate al days of age and implwited "ith te ost ne (250 ug/kg/day) and 7 st ero estradiol ug/kg/day) silasticcapsules at years of age castrate years of age prevents testicular flow to the epididymis. results in an -50% decrease in the (0.4 2 (trt2); at 2 expression of GGT mRNA-rv in the initial segment after 12 hours. The objective and inunediately implanted with testosterone wid estradiol silastic capsules as in trt2 The implwits in all groups were replaced at yearly intervals until years of age, at of this study was to test the hypothesis that the decreased expression of GGT (trtJ). 6 which time the prostates were removed wid dissected. For all specimens, a transverse mRNA-rv in the initial segment following the removal of testicular factors is due segmentwas taken from the mid-apical portion includingcentral wid peripheral regions. to a decrease in transcription rate and/or an increased rate of decay. Changes in Part of the tissue was inunediately placed in protease inhibitor buffer and homogenized, transcription rate were evaluated by examining changes in the amount of newly the remainder snap frozen wid cryostat sectioned. The homogenate was processed for transcribed mRNA produced at a given time. The expre ssion level of an intron Western blot wialysis using a well..:haracterized rabbit anti-rat neuronal NOS (nNOS) present in the 5' untranslated region specific to GGT mRNA-rv was assayed using witibody. Cryostat sections were processed using routine histopathological techniques. RNase protection analysis and servedas a measure of newly transcribed mRNA-IV. RESULTS: Intrtl (n=7), complex BPH with a prominent glwidular component was Results of this analysis revealed that the transcription rate of GGT mRNA-rv in observed in all prostates. nNOS immunoreactivitywas absent in all specimens. In trt2 (n=IO), moderate to severe atrophy was observed in prostates, normal histology with the initial segment fell by -70% followinga hour EDL. Decay rates, a measure 7 12 small areas of hyperplasia in prostates, wid mild BPH in I prostate. with of the stability of an mRNA, were determined by evaluatin mRNA levels 2 Five of the 7 g atrophy exhibited nNOS expression. In trl3 (n=9), moderate to severe glandular atrophy following incubation of tissue in a transcription inhibitor for varyingamounts of was observed in prostates, normal histology with small areas of hyperplasia in I, wid 7 times. These stability studies showed that immediate removal of testicular factors moderate BPH in I. Six of the showing atrophy exhibited nNOS expression. 7 from the initial segment induced a rapid initial decay of GGT mRNA-rv (t 112 = CONCLUSIONS: These data describing the absence of nNOS expression in BPH tissue 0.86 (t 10.5 hours; 1-24 hours) wid, conversely, itsdistinct expression in atrophic prostatic tissue following hormone hours; 0-1 hours) which slowed at later times 112 = alterations (79%) suggest that NO is inversely relatedto growthwid development of the to a decay rate paralleling mRNAs-II,!II. Thus, the decreased expression level of cwiine prostate wid may be widrogen-regulated. Whether nNOS deficiency contributes to GGT mRNA-rv in the initial segment following the loss of testicular factors is a the pathogenesis ofBPH or occurs as a consequence of this disease process remains to be function of both a decreased transcriptionrate and an increased rateof decay. determined. Work supported by NIH grwit DK -1.�JOOi .! l � le c.J" Supported by N.l.H. grant HD32979 (BTH), N.1.H. PJO-HD28934, and training ML i,,. { �,Jo s �,,� �� grant N.l.H. T32-DK07642 (DBR).

62 SYNERGISM OF ESTRADIOL AND TESTOSTERONE ON BROWN DUCTULI EFFERENTES I� ESTROGEN RECEPTOR NORWAY RAT PROSTATE GROWTH P.P. B:u1erjee•, S. Bwicrjee•. B.R. Zirkio K."IOCKOUT MICE DO NOT REABSORB LUMINAL FLUID wid T.R. Brown, Division of Reproductive Binlngy. Johns Hopkins University. Rex A. Hess'. David Bunickl,Janice Bahr2",Dennt> B. LubahnJ· tDep! of Baltimore,MD. & IL & Physiologic to pharmacnlogic doses of exogenous tes1os1emne cause age- and lnbc­ Vee Biosci 2Dept of An Sci, U of Illinois. Crbana, 30epcs of & specific overgrowLl1 in llruwn Norway rats. H)'[>ertrnphy occurs in the ventral (VP), Biochem Child Healch, An Sci Res Center, U.of :\lissouri , Columbia. dorsal (DP) and lateral (LP) prustatic lobes of young and old rats, whereas DNA contenL a measure of cell number, increases in the DP wid LP, as a function or Although estrogen receptors have been localized in the epithelium and the VP. testosterone dose and age. but not in We subsequently discovered that age­ connective tissue of ductuli efferentes (efferent ductules), the function dependent wid lobe-specific overgrowth of the prostate occurs spontaneously in the of estrogen in the male reproductive system is unknown. The recent absence of exogenous hormone treaunent. Westernblot alyses revealeda decrease establishment of !he estrogen receP.!or knock ouc mouse (ERKO) has wi 10 in androgen receptor protein level in all three lobes of old rats. By contrast. a lobe· allowed us investigate th e functional role of estrogen m the male. specific (DP wid LP) increase in estrogen receptor protein level was observed with Histological examination revealed an expanded lumen in ERKO efferent ductules 10 that of normal mice In the present study. we age These results prompted us to investigate the effects of estrogen on the age­ compared (+I+). . hypo!hesizeo that estrogen may contribute 10 the regulation of fluid dependent and lobe-specific growth of the Brown Norway rat pros te. Young ta (4 resorption in !he efferent ductules. Thus, wichom a functional estrogen month) rats were implanted with t tosterone cm month)and old (22 2.5 cm es en. 0. 1 receptor, fluid would not be reabsorbed by the duc!al epithelium, estradiol (E), or a combination of T filled Sihi.

P-40 Journal of Andrology • January/February 1997

EFFECT OF TESTICULAR BIOPSY AND TRI ETHYLENEMELAM INE 65 6 7 SEASONAL COMPARISON OF SE:.IE:\." P .\RA:-.IETERS IN CAPTIVE ON TESTIS FUNCTION AND SEMEN QUAL ITY IN THE DOG R.H. CHEETAHS (AC/NONYX /UBATL/5). BS. Durrant J K. Yamada', SE Millard" Foote, Department of Animal Sci ence, Cornel l Un iversity, C.D. Burch" and D.M. Zimmerman·. Center for Reproductton of Endangered Specie>, Ithaca, NY . Zoological Society of San Diego. San Diego. CA Many zoos in North America pair cheetahs for brccjm:; m January and July. m the Semen was col l ected 3X per week for 5 weeks from 27 belief that the species experiences bimodal seJsonal reproduction. A re\'1ew of sexual ly mature kennel - reared Beagl e dogs . After 5 weeks birth dates reported in the International Cheetah Studbook reveals captt\'e births in every month of the year with a peak during the spnng. This apparent seasonal 20 of the dogs wi th good libido and trai ned for semen trend may be the result of winter pairings, or could reflect physiological responses col l ection were divided into four groups of fi ve dogs to environmental cues. It is of interest to detennine the role semen production mi'.!.y each . Semen col lect ion was conti nued 3X per week (Monday, play in annual reproductive rhythms. Semen was collected weekly by artificial vagina from three male cheetahs over a three year enod to determine the effects ot Wednesday and Friday) for 20 weeks . Group I was a control season on semen quality. Males #1 and #2 (6 and j\'ears of age. respecth·ely at the wi th no treatment until uni l ateral ly castrated at week 16. beginning of the study) were hand-reared indi\•iduals with no opportunity to breed. Male #3 (4 years of age) was a mother-reared prm·en breeder. Semen analysis Group II had three testicul ar biopsies on one testis on included volume (V, ml), concentration (C, x106), motility (MOT) and speed of weeks 1, 5 and 9, with th is testis-epididymi s removed on progression (SOP, scale of 1 to 5, 5 being fastest), and percent normal morphology week 16. Groups III and IV recei ved, respectively, 0.2 (%N}. Motility scores (MS) were calculated as \IOT x SOP2. To facilitate mg/kg and 0.4 mg/kg of bodywe ight, of tri ethyl enemel ami ne comparison of males and seasons, semen qualitv indexes (SQI) were calculated as (\' %N)/100. (TEM) intravenously at the outset of the 20-week test x C x MS x Means, ranges and number oi e1aculates for each measured semen p rametei for_ l males during the t dy '.''ere� \', 1.68, .11-5.10 n= 9 C, _a ? ! ::_� '.. 3 2; period. These dogs al so had testicul ar biopsies on weeks 27.74, 0· 132.0, n-27:>,:__ Yo N, 36.08, 5 _69, n--01, MS, 066.91, 0-1424, n-270, SQI, 1, 5 and 9 and a unilateral castrat ion on week 16. Al l 120.98, 0-791.01, n=173. Within males, volume of ejaculate was highest for each #1 dogs were castrated on week 20 . The average sperm output male during the summer, but was significant only for :-.tales and #2 (p=.04). Between males, Male #3 had a hi gher (p=.05) mean volume during the winter season per week during the fi rst four experimental weeks in compared to both Males #1 ana #2. Male #3 exhibited greater (p < .05) sperm Groups I and II was 760 and 876 mi llion, respectively, and concentration during the summer and fall compared to bofh Males #1 and #2. The duri ng the last four weeks before unil ateral castration percentage of morphologically normal sperm aid not differ between males during any season. However, Male #1 had more normal sperm (p=.05) during the winter (weeks 13-16) was 685 and 811 mi llion, respectively. Thus, than at any other time. Male #3 had a higher (p=.05) mean MS during the winter the control s decreased 9.9% and the biopsied group 7.4%, than Male #!. When all parameters were factored into the SQI, there were no indicating a possibl e seasonal effect , but no detectabl e significant differences between or within males dunng any of the four seasons, although there was a tendency for each male to have higher SQis in the winter than biopsy effect . Un ilateral castrat ion reduced sperm output in other seasons. by 47 and 46% in groups I and II, respectively. In the Mean SQ!+sem- Surrurer TEM-treated groups III and IV, weekly sperm output during Male Winter Sj)l'i11g_ Fall within 1 130.7 92.5 ±2 29.8 ns weeks 1-4 averaged 933 and 1094 mi llion, respectively. By ± 28.9 4.9 94.4 ±192 64.1 ± 2 188.5 ± 702_ 112.9 ±2 8� 87.5 ± 16 s 82.3 ± 27.3 ns weeks 13-16 corresponding average weekly sperm output was 3 180.8 ± 41.9 121.8 ± 22.4 174.3 ± 50.4 156.7 ± 36.9 ns markedly depressed to 375 and 124 mi llion. The percentage between ns ns ns ns of moti le sperm was not affected. These data indicate that there are no seasonal differences in sperm quality in captive male cheetahs.

66 SENSITIVITY OF DOMESTIC CAT SPERM TO 68 EJACULATE CHARACTERISTICS OF THE BLACK-HANDED COLD-INDUCED ACROSOMAL DAMAGE. SPIDER, SOUTHERN BLACK HOWLER AND DIANA MONKEY. B.S. Pukazhenthi, K.M. Pell·, D.E. Wildt•, and J.G. Howard. National JA Long, N Lamberski* and AH Shoemaker.* Conservation Research Zoological Park, Conservation & Research Center, Smithsonian Program, Riverbanks Zoological Park and Botanical Garaen, Columbia, SC. Institution, Washington, DC 20008. Considering the large number of primate species, basic knowledge Previous sperm cryopreservation studiesin lelids reveal loss in about reproductive physiology is available for only a disproportionate few. sperm motility and extensive acrosomal damage post-thaw. To evaluate In light of the current biodiversity crisis, it is essential to begin accumulating factors that affect sperm structure and viability during freezing, we data for little-studied species. Objectives here were to examine seminal analyzed the effects of cooling on sperm from normospermic (N; >60% traits of the black-handed spider ( /e black nowler normal sperm/ejaculate; n=3 males) and teratospermic (T; <40% normal Ate s geoffroy1) , (Alouatra and the endangered Diana (Cercopithecus monkey. Elec­ sperm/ejaculate; n=3 males) domestic cats. Ejaculates were divided into caraya) diana) troejaculates were collected from anesthetized males ejaculates/male) either raw or washed (Ham's F1 0+5% fetal calf serum; FCS) aliquots. (2 Washed pellets were resuspended in: 1) Ham's F1 0+5% FCS; 2) PDVF using either a 1.3 cm (spider monkey) or 1.6 cm (how1er, Diana monkey) (20% egg yolk, 11% lactose); and 3) Refrigeration Medium/Test Yolk rectal probe. Electrical stimuli were delivered in serially-increasing incre­ Buffer ®(TYB). Sperm aliquots were maintained at 25°C (control) or ments (2-6 volts; 10 stimuli/voltage) until semen was obtained. Following an initial assessment of spermmotility, ejaculates were diluted 1 :1 with exposed to: 1) 0°c for 1 O min; and 2) 4°C for 30 min. Following wanming to 25°C, samples were evaluated at 0, 5, 60, 180 and 240 min for Ham's F10 (+ 25 mM HEPES; 10% fetal bovine serum). Ej aculate volume % sperm motility and % intact acrosomes using FITC-PNA. In all varied extensively among the 3 species (spider monkey, 3 mi; howler mon­ treatments, sperm motility was not affected (P>0.05) by cooling. key, 0.12 ml; Diana monkey, 0.02 ml). Howler monkey ej aculates exhibited However, % intact acrosomes was reduced (P<0.05) in raw, Ham's F1 O no coagulum; however, Diana monkey ejaculates rdquired 30 min (37°C) to and PDVF aliquots in both cat populations following exposure to 0°C complete liquifaction. In contrast, spider monkey ejaculates coagulated (N, 39-63%; T, 19-34%) or 4°C (N, 54-68%; T, 23-28%) compared to within 2 min of collection and did not dissolve completely, even aftera 2 h 25°C (N, 75-84%, T, 71-73%). In contrast, TYB maintained (P>0.05) incubation (37°C) in Ham's F10. Although the coagulum remained intact, 3 acrosomal integrity in nonmospermic ejaculates exposed to 4°C (71%), x106 spenm/ml were present in medium aspirated after30 min of incubation. but provided less (P<0.01) protection at 0°C (67%) compared to 25°C Fewer sperm (0.4 - 1 x106 sperm/ml) were recovered after 1, 1.5 and 2 h of (81%). Furthermore, % intact acrosomes in TYBwas reduced (P<0.01) incubation. Spenm concentrations in howler and Diana monkey ejaculates in teratospermic ejaculates at both 0°C (24%) and 4°C (37%). These were 0.8 and 104 x106 sperm/ml, respectively. High numbers of morpholo­ results indicate that: 1) cooling induces acrosomal damage in domestic gically nonmal sperm were observed in howler (52.4%) and spider (41 .8%) cat sperm without altering sperm motility; 2) PDVF fails to protect cat monkey ejaculates; whereas Diana monkey ejaculates contained few struc­ sperm acrosomes during cooling, while TYB is only protective for turally nonmal spenm (1 5.5%). Diana monkey sperm anomalies included normospermic ejaculates cooled to 4°C; and 3) teratospermic ejaculates flagellar (36.2%) and multiple (cephalic + flagellar, 22.4%) defects. Initial are highly susceptible to acrosomal damage during cooling compared to spenm motility was poor (20%) in Diana monkey ejaculates compared to normospermic ejaculates. (NIH grant HD 23853; Smithsonian Institution howler (80%) and spider (80%) monkey ej aculates. Collectively, these data Scholarly Studies Program). demonstrate the unique ej aculate characteristicsof these primate species and will help to establish species nonms, including ejaculate traits, seasonal variations and reproductive soundness.

P-4 1 TWENTY-SECOND ANNUAL MEETING

71 INCREASED FERTlLf'rY TURKEYS RESlJLTING FROM a:::PEATED FOLLICUUR STIMULJ..TIOtl IH' THE ::::::::.;uc;:::R::D LION-TAILED JN MAC;.QUE69 (� �) WITH aECOMBIN.\flT HUH.AN G::�:ADCTROPHINS SEMEN DONOR SELECTION A.M. Donoghue• Germplasm and

2 3 M. Cran!ieldl", U. Berger * , J. Liu2 ;; . SmiJ.rt. , :J. Su=ers� Gamete Physiolog)l-Laboratory, ARS, USDA, Beltsville, MD 20705 2 lThe Ba.i.tiriore Zoo, Bal't.inore, MD; Greater Bal'::.:-:.:::ire Med!.cal Center , Balt.i:::io:-e, !10: lJ'ohns Hopkins University, School c: :1ed !.cir:e, Ba lt. i:Jore, and D.P Froman• Department of Animal Sciences, Oregon State HO; 4 ocper Cent r for !VF, Marl c a tan, NJ University, Corvallis OR, 9733 1 The urinary preparat.i:ins of non-pri:i.at.e hu:tan ;:::nad:::it::-:::iphins used to Commercial turkey production depends exclusively upon artificial sti::i.ulat.e follicular developt'lent in r:iacaques cause =ap.!.::! develop::i.ent of neu:.ral!.z.inq an"C.ibodies and ovarian r !:- to ne s !ut.ura e ac ri s -:.:i s't.ir.tulat.ions insemination of pooled semen. The objective of this study was to pre­ in t.he :-hesus ::ionkey and l!.on-:.ailed macaque but �O't. in the pig--:ailed :i.acaque. Thi s s:.udy exa=ined the follicular ::e·.·elcpcen-: following screen individual males using an objective motility assay. Semen was repea:ec! st:.ic.ulat:.!.ons with recor:ib,inant hucan gonac!:-:=:::phins in ':he licn­ tailed ::i.acaque . Two naive lion-tailed cacaques we=e ?=e-t:-eated !'or six then pooled by motility ranking and fe rtility evaluated. The motility assay days a GnRH antagonist [An"C.ide; Ares Ser no ; 0.5::q/kq body weigh:. I:-! wi':h e differs from conventional motility assessment for poultry in that the . BID] , wl"u.chcon;:inued through :he adr.i.inist.ration o!" =e:::::=-:>inant hu:an FSH [R-hFSH; Ares Serano; J C IU IM BID] for six days !::::10-,..ed by R-hF'SH plus. sperm must overcome a physical barrier to forward movement. Semen I1R-h!.H (:.res Sereno; J O IU each IM BID !or one to -=.:-:.:-ee :!aysJ fo= ':.hr!IH sti::iula;:ion cycles (Cl, C2 , CJ J. R-hHCG {Ares Se:-:�o; 1000 IU IM] was fromeach ej aculate (n=75 toms) was diluted to lxl09 sperm/ml in pre­ 19i•1en -:he day !allowing the appearance of follicles ;:-eat.er than �:m on ult:-asound. surgeey ':o aspirate follicles occurre:! :? O t.c 35 hours !":':er. warmed TES based motility buffer and placed over 3 ml of a 2% With bo�'i. c.onkeys , during Cl and C2 , E2 peaks ;.;e=e si::;.i:ar at R-hHC:i \ ad::linist.ration, but in both cases had lower E2 eaks and spon;:aneous Accudenzsolution (Accurate Chemical, Westbury, NY) in a polystyrene drops of E2 levels ;:: pre:-R-hHc::; adc.inis;::-ation during CJ . It "Jas fel-:. :.:-.at. ":hi s drop in =:2 cuvette at 41° C. After a 5 min incubation the cuvette was placed in a levels das not:. due -:.o a spon-:aneous UI surge d1.:e :.o unc!er:los:.n; with "An-c:.de bu"t:. neu"t:.::"alJ.zing antibodies because the=e ·o1 as :io =!.se in ?4 levels densimeter and % transmission recorded after l min. Semen from toms a!"':e.r :.h.a E2 d!:"op . ranked in the highest and lowest !0% after3 screenings were pooled by Wi -:h bot.h ::ion}:eys, the nu?:tbe!:"s ot oocy-:es c::::e -:.c:! f=oc. Cl �:id C2 •o1ere s!.:ular (16 to 19J . The num.ber of M2 oo=�.. :.es = at -:.he -:.:..:::.e o! group and used to inseminate hens weekly (n=20 hens/group) for IO collec-:.!.on increased significantly in both monkeys !!:"::l;! Cl :o C2 bu-:. was :felt. -:.o be influenced by an increase in the tir:ie i::-.":-!:-"Jal be"C.·o1een il-hHCG weeks. Fertility from hens inseminated with semen from the HIGH ad:iinist::"ation and surgery than by the reac-:.ion of :.�e repeat.ed ho=::onal E2 the:-apy. Neither monkey reached the patt.e=:-. O!:" !olli:::ula:- siz11e MOTILE males averaged 95.8 ± 1.3% over the l 0 weeks and was higher c:-i-:.e:-:!..a during CJ that a l lc·,..ed su!:"gery . Al':.h.ough -:.he :-ecoc..binan':. ;oncido:.:-ophins did n::::-: sc.:::cess!:J!ly p::-o:::!:uce than fertility from the LOW MOTILE tom group 90.4 ± 2.2% (P<.05). a thi!:"d si:Jula-:ion, bu-:. the product.ion o! t'.o ::: s-::.�ula::..?.ons is a sign!.!"i.c.ant event:. wit!l these hig ly endangered pri=?.-:.es. Hatchability of fe rtilized eggs did not differ between groups. Weekly h motility evaluations on pooled semen were consistently higher for the HIGH MOTILE males than the LOW MOTILE males throughout the study. Motility differencesbetween males influenced fertility outcome. By mimickingthe hens vagina, this assay mayidentify males whose sperm have a greater ability to reach the sperm storage tubules and subsequently fertilize oocytes.

70 '!'HE USE OF I!1':'ERC'!TO?LASMIC SPERM !llJEC:":c:: ::::s :) TO Etn:..;..:rc:: :'HE. 7 2 CILHACTERIZATION OF Hill!AN AND TURKEY SPER.'IIOPHAGES CO?ISEil'/.\TION OF '!'HE. EUDA1lGErtED LION-TAILED liAC!..·;U:: \:;::·I ) : u,;.c.;.c;, c:H�nt;S) AND IDE�TIFICATION OF TURKEY SPERMIOPHAGE PRECURSOR CELLS

ran!" el l J. Liu: , D. Sut:u:iersJ J, Smar:..; 2 USING A �IONOCLONAL ANTIBODY . ti · c i d •, , ::.. Se=:;e:- • B MD; 2 "'The 3al timore Zoo . al ticore ' Greater aal-:.:.. ::ore �edical Cent.er I Fle tcher C. Derrick, Jr. , Benilda P. Pooser* , Thomas R. Sa l -: i=ore, MD; JJohns Hop}:ins Uni·1ersity, School o!. !o!eC.i::::.ne , Bal-:.ieore , . XD : ;Cooper Cen-:.er for r:F , Marl -:.on, NJ Scott* , Ronald J. Thu rston* and Nancy Korn* , Dept. of Poultry Science , Clemson University , Clemson, SC and The When IVF technia1.:es were applied tc u:::::!.e!:"::"e:::::-csent:.eC non­ reproduc-:.ive LTM individuals as a ::ieans t.o inc:-ease ;ene-:.:..c dive=sit.y in Dept . of crology , MUSC, Chas. , SC population, ter-:.iliza"C!.on ra es ...,ere 3;:;::=ox!.::at.ely 10\. The -:he cap-:ive t Sper:iiophages , immunoresponsive cells in semen of proble� was poor sper::i sa::ples collec-:.ed fro::i -:nese :-:on-:i:-eedim; :.ales by rec':.al probe elec'!:.roej aculation. Fewa1es -we!'e s-:.i:iu: a-:ed u-:.!.li:::inq vertebrates , have been shown to phagocy tize sperm and !:"ecor.o.binant c;cnadotrophins (Ares Sereno ). R-t-:.H ::; fAres Serono; !ODO :u c bacteria . In man, marked increases in spermiophages have IM) was administ.e!'ed when follicles great.er -:.han ..:.� were obse=ved :iy ".llt.::-asound and follicles "Je:-e surgi:::ally aspira-:.ed JO -:.o ::!� hours post ?.­ been proposed as being pathognomonic of chronic non­ �HCG ad?:iinis-:.ra::ion. Spern · was rec-:.al p:'obe col!.e:::-:.e.=. :iy p elec-:.::-oejacula-:ion and then e1ther !"rozen ut.ili::: ir:; �e::-.:.ge:-a-:ion :-tedil.!:::­ specific e ididymal infection. Turkeys with yellow semen (':'"!BJ 10\ -:.es-: yoH: bu!fer {!:-vine Scien-:.ific) 1: l !Jy ·..r c:.;::ie �:--:d q:ycer:>:. syndrome and reduced fertilizing capacity , also have as a c::yoprot.ec-:.an;: o= pu-: -:.hrouqr. a percoll doub le g.:-adie:r:. 'PerCept.ior:; Fer-:.ilit y Technologiesl , sl!;!;.pended in TALP-H:E.?::s a:-.:: us�::! fresh. increased numb ers of spermiophages . These factors yte i c c suggest that the domestic turkey may be a good animal er c s3;� ;..� i::;�c�7�; :ediu�� Whe5n ;���r ��� =�� o�s!�1e��n��� ���;���:1�� � ; � de!i as; t!"le :::-ese�=a o! ·o1 as pe:=!or:ned. Fer-:iliza-:.ion ·..,r as ned .e. sec::::::! model for studying spermiophages. Little information pola!' body -:·.;.::i-ce:! s-:.age bi' and -:-...o pronuclei and t.he ad·:ance::i.en':. -:: "::?.e exists on the origin of spermiophages . Thus the obj ect­ 2� hou.:-s pos:.-::::csI. ives of this study were to 1) compare the morphology of ;.�: �i���:���\��F::�Jh�:�<,�i<:li�:;�fai� human and turkey spermiophages and 2 ) determine the pre­ The second procedl.!re produced imna-:ure qernw���'d�}yoinal 'les:.::::e �=!c:::;�}�:����·�;=��cy':es tha-:. were cursor cells of seminal spermiophages . ::a-cured in v it:-c and wnen MI: eggs developed i:-1je=:.e:! -...i-:.:-.. spe==i. ':ha<:. had been held in '!'AL?-HEPES a-:. :roor.'I -ce::::pe.:-at.u:-e !o:- :;e h::':.!:-s. This !'"es':.llt.ed Sper:iiophages from semen of human and domestic turkey !.n the produc-:ion of two e::.::i:-yos ro !-:::::: las-:. o: f c:!ive eg;s . ':';:e the were prepared for transmission electron microscopy . -:.hree ?rocedures produced six H!I eggs at ':he -:..:.::-.e :: : re-::-ieval. Three "Ji!:!'e i:-ij ect.od. -....it!". !!'eshl;· collec-:.ec! spe=r.:. and tn:-ee "J !.":� !:-o:en- -:.na"Jed Mouse monoclonal antibodies to turkey spermiophages were s::ie:-:i. Three o!" ':.hree t.he fresh spe.:-= be:::::i.::e e:-:.!Jryos ar:d U"t.!.l.!.;:: ing one­ also prepared . After to 24 h in culture , turkey sperm­ -:.hi=:! ·.- i -:.h the trozen-tha\Jed spe=::i became er:U::i.:-yos . 1 12 res: In su:r.ma:-y, of 2 !. (57\J M!I eggs inse:-.!::.e.-:eC. =: oe::::a=ie iophages developed motile granules within the cytoplasm e::ib.=ycs - a c;:-ea-:. i.n::::-ease over ::egular !VF inse::i:.:;a-:!cr.. Ir: ':he !.dea: sit.ua-:..i.on ot MII eggs a-:. re-:.=ieval and fres�ly c:: :!..le=:.2d spe==., -:he which have been shown to be mitochondria. Three mono­ per-::e�;:age is 69\: (9/13) . The fac': -chat. frozer?-:.ha... ·ec spe=:: ....as a!:lla -:.::: clonal antibodies (MAb) bound to antigens of turkey ;:=odu::::e 30\ fer--:.i :!.. i::: a-:ior: !.s also e:icouraging. !": ;;culC. .e.;::pear -:.ha-:. -:he spermiophages demons trated that only the epithelial :;:.:s:;: -:.ec!mique gre�':.ly er:han:::es ':he chance o!" i! s� :::cess c:onse:-:a�ior. ;irog:a= u-:.ili::ing re rodu::::-:.::.vely s"';.!b-op-:. ical indi·.':.:!-.;als anc! a=-:.:!":.cia: cells of the ductuli efferentes of the epididymal region ve e ;J ab u e th :::: h had antigenic determinants which bound the third MAb . ���������! � �������i it.i'! �f i����� u��n; -:�i�� ;�;:;;�-:i����� gene-:..ics into a cap':.ive conservation prograr.t "Ji-:.!i ::: ::!.:?..i a:!. dis::-up-:.ion t:::: Human and turkey spermiophages are morphologically -:..he wild popula-:.ion. similar. Epithelial cells of the ductuli efferentes may be the source of spermiophages in turkey semen .

P-42 Journal of Andrology • January/February 1997

POWERCALCULATIONS OF VIAB!LITYESTIMATES 73 SPE!Uvl-B E\Ti l"NG ..\SSAY TO DETECT MALES UK.ELYTO 7 5 VARlABll.ITY . ... 'ID HAVE RELATIU:LY LOW FERTILITY IN DUTCH BELTED RABBIT SPERM S.M. SCHR.\DER S.D. SIMON", NATIONAL R.H. Hammerstedt. S.P. Gill*, R.P. Amann, P.C. Cramer*, and G.F. MICHAELJ. BREITISSTEJ);", AND lNSTITUrE FOR OcCUPATIO�AL CINCINNATI, Barbato*, BioPore Inc, State College PA and The Pennsylvania State S.>.FEITAND HEAL1H, OH. University, Uni\·mity Park PA 16802 The use of\ ital stainsfor assessing sperm viability is a subjective measure of In animal agriculture. elimination of males likely to be subfertile is of the nwnber of live cells (not stained) vs dead cells (absorbing stain). It can be economic importance. An in vitro sperm-binding assay utilizing difficult to distinguish ifthe cell has absorbed the dye. Oftenthe distribution . segments of hen's egg membrane (BOR 50 Suppl I: 128, I 994) was of the live/dead ratio is not unifonn across the slide Inthis studyan objective modified to incorporate: (I) pre-labeling of extract containing viability method was assessed using a flow cytometer. A Coulter Epics Elite perivitelline membrane pro tein \\ ith Ai\·!C..\; (2) drying 50 µg flow cytometer \\ith an air cooled argon laser was used. When cells are mixed protein/well in a microwell plate. ( 3) incubation of replicate sperm with the LIVE/DEAD FERTILIGHT"' spcnn viability kit reagents (Molecular suspensions (buffer control or 0 :5-10 x I 06 sperm/well) for30 or 60 Probes, Eugene, OR) and excited Y.ithvisible-wavelength light the cells (4) 14 min at 37C, \\ashing 3X to remove unbound sperm; (5) drying at fluoresce. The two components of the kit SYBR and propidiumiodine (Pl) 60C for 18 hr; (6 1 ;raining DNA with a YOPRO and protease bind to the DNA of thesperm. SYBR 14 is a mcmbrane-penneant nucleic wcktail, (7) reading fluorescence at 485/530 to quantify DNA and acid stain and PI is the conventional dead-0:ll stain. Three different 360:'460 to quantif\· binding-protein, and (8) expression of data as populations canbe distinguish on the C)1ogra.m, live cells, dead cells and dual sperm bound per 50 µg protein and % bound. The assay likely is stained cells. Semen samples were collected from 70 Dutch Belted rabbits for applicable to mammalian sperm as the perivitelline membrane of a 5 weeks. The semen was collected byej aculation with four ej aculates per hen's egg is s1m1lar to the zona pellucida. For bull, horse, human, rabbit, 10 µl of the firstejac ulate was diluted with 500 µl of Ham's F-10 and mouse, rooster or turkey sperm, binding was negligible for sperm used to measure viability. The average percent of dead cells over the five rendered immotile by blocking energy transduction or snap freezing, week period was I 7 .9%. The intraclass correlation, a measure of relative whereas motile fre;h or frozen-thawed sperm bound in a dose­ consistency within a rabbit, is 18.4o/o. This low value implies that a single respons1ve manner % bound sperm was maicimal at 0.25-2 xl06 measurement on a rabbit is not representative of the rabbit's overall mean sperm/well Capability of the assay to detect males likely to be level. This measure also hasfairly large relative variability, both within subfertile e\'aluated with chickens. Data for2 of 5 studies are \\ as rabbits and acrossrabb its. The within rabbit coefficient of variance (CV), a presented [A[ Sperm in ej acula from each of28 roosters of 35.4%. 3 measure of relative variability foran individualrabbit, equals The unknown fertility \\ere evaluated by the sperm-binding assay and, total CV, a mea5\llc of relative variability across all rabbits, equals 46.9%. applying a rule of--:; bad roosters were identified; semen In out of 3", 7 spite of this variability, thismeasure good powerfor detecting a shift a from each then \\as used to inseminate 5 hens (3 4 d 80- 125 has in X apart; longitudinal design. For example, an experimentwith 15 rabbits, with 5 pre­ sperm) Femlity of the 7 bad roosters averaged 70% vs 76% for xl06 exposure and 5 post�xposure measurements, there is a 94% chance of the other 2 I males. there was I of false positives. [B] 57 roosters of be 7 detecting an average increase from 18% dead cells pre�xposurc to 22% dead a different generic line were used in a similar study but with 6-7 cells post �xposure. hens/male, forthe bad quartile fe rtilitywas 69% vs 88% forthe top quartile. USDA-92-202-073, USDA.-95-00178 and USDA 96-03683

. ROLE OF REGULATION OF 74 TEST!CL1. .\R MORPHOLOGY AND GONADOTROPIN 76 Il\HIBI� IN THE RECEPTORS REFLECT AGE-RE LA TED ALTERA TIONS IN FSH SECRETIO� Ii\ I:\I:\IATURE BULLS. H. Kaneko1-, J. FUNCTIOl\ Noguchi', K. Kikuchi'", ..\. Shimada'·. G. Watanabe2, K. Taya2·, Y. MA Ottmger•. N. Thompson•. M R. Bakst••, K. Kubakawa*#, Hasegawa3·, K. Okuda''. ?\atl. Inst. of Agrobiological Resources, \I. Kikuchi+", and S Ishu+•, Arumal and Avian Sciences, Ibaraki 3051, Tokyo Uni\'. of Agr. and Tech., Tok.')'o 1832, Kitasato Univ of \Iru: land, College Park. �ID 29742; •tiermplasmand Garn� Univ Aomori 0343• Okayama University, Okayama 700' , Japan Pllysiology Lab. ARS, USDA, BeltsviJle, MD 20705; #Ocean Research Our previous findings provide evidence that inhibin is a major Institute, Utm ersity of Tok.·yo. Nakano ku,Tok. 1·0 I 64, Japan: +Department factorcontrolling FSH secretion in 6-month-old bulls (J Endocrinol, 169-50 ofBiology, \\'aseda Universi� . Tok.1·0 Japan. 1993, 15-19). However, the regulation ofFSH secretion in infant bulls has not been clarified yet. In the present study, we furtherinvestigated Our stud.tes ha\ e fo cused on reproductive aging in male Japanese quail. the physiological significance of testicular inhibin in the regulation of During agmg. the male shows a loss in fe rtility and cessation of sexual FSH secretion duringthe early life of bulls, by I) immunoneutralization behavior. both of which precede measurable changes in plasma levels of of endogenous inhibin ancl 2) immunohistochemical detection of gonadotropms o� gonadal steroids. Further, in vitro Leydi g cell function testicular inhibin. Animals were given a single i.v. injection of 40 ml revealed that these cells retain function in spite of re ession of the testes In inhibin antiserum at 7 (n=7), 21 (n=IO), 60 (n=6) and 120 (n=6) days ese expenments we mvesugate morphological changes that accompany ofage. The antiserum used in this study, raised against bovine 32 kDa age-related tesucular regression and alterations in the nwnber and response inhibin in a castrated male goat, recognizes the a -subunit of bovine (LH of the gonadotropm and FSH) receptors. Male quail were grouped inhibin. Treatment of control serum caused no significant changes in according to age and sexual activi�·; additional groups of young plasma FSH levels during 48 h post injection at all ages. Concentrations photocastrated males and old testosterone implanted males were studied. of plasma FSH showed a marked increase (p

P-43 TWENTY-SECOND ANNUAL MEETING

77 CO!lt!PARISON OF MO\"OPERCOLL SPERM SEPARATION WITH 79 ISOLATE "' AS A NEW METHOD FOR SPERM SEPARATION: A TWO-LA YER PERCOLL METilOD K Sc1fanh*. D. Garlak*, L Cordck•. C PRELIMINARY STUDY. S.M.Tarchala and R.G.Rawlins. Rush Medical Fitzhugh*, A.J. Thomas. Jr. and A. Agamal. Andrology Research & Chmcal Center, Dept. Ob/Gyn, Chicago, IL Laboratories, Department of Urology. Clc\'eland Clinic Foundauon. Cle\'cland. OH 44 195 Objective: !So late"' (Irvine Scientific), is a colloidal suspension of silica particles stabilized with covalently bound hydrophilic si!ane used for the separation of human spermatozoa from seminal plasma prior to use in Previous studies have shown that sperm rcco\'ery by MonoPercoll procedure is significantly better than the S\lim-up technique for male factor patients and in assisted reproductive technologies. The purpose of this study was to test patients with normal sperm characteristics. The cost and labor of conducting a the efficacy of lSolate"' compared to currently used methods of sperm separation in respect to yield, overall motility, forward progres sion, two-layer Percoll procedure vs single layer sperm washing procedure are viability, acrosin activity and % normal sperm morphology. significantly more. Therefore. the purpose of this study was to compare the Design: A prospective study of patients reporting to our clinic for eITect of MonoPer�oll procedure and tl1e two-layer Percoll on .perm diagnostic andrology procedures. characteristics of normozoospennic men. Semen specimens from IO normal Materillla and Methods: A total of nine human ej aculates obtained for donors were allowed to lique�· prior to processing. Semen specimens were diagnostic semen analysis were each processed into three test groups. divided into two equal aliquots. One aliquot was processed by layering I of ml Group 1 was processed by gradient separation using ISolate"', Group 2 80% (Pcrwash) Percoll (MonoPercoll), and the other on a two-layer Percoll was processed using polyvinylpyrillodone (PVPI coated beads (Percell "', 90% gradient (lower phase and 4 7% upper phase). Both aliquots were Pharmacia) and Group 3 using traditional swim up. The neat ej aculate 1600 20 centrifuged at rpm for nun at room temperature. The supernatant was served as the control group. The resulting pellets were resuspended in removed and pellet resuspended m 2 ml of sperm washing medium (human Modified Ham's F-10 with HSA and assessed according to WHO guidelines tubal fluid, HTF) and centrifuged for7 min at 1600 rpm. The final sperm pellet for sperm count, overall motility, forward progression, viability and 1 was resuspended in ml of H1F. Routine computer-assisted semen analysis morphology. Acrosin activity was measured colorimetrically according to and sperm function tests were done to examine the following parameters: total the assay established by Kennedy et al, 1989. motile sperm, percentage recovery of motile cells, motility (MOT), curvilinear Results : While statistically significant differences (p< 0.05) were seen velocity (VCL), linearity (LIN),amplitude of lateral head displacement (ALH), between the control and test groups, no significant differences were sperm motility after 60 minutes of separation. hypo-osmotic swelling (HOS) observed among the 3 test groups for any of the semen parameters test, bovine cervical mucus penetration teSl percentage viability by eosin­ tested. nigrosin staining test and sperm morphology differentiation by WHO and Conclusion : The results of this preliminary study show that !Soiate "' is Kruger's method. MOT, LIN, ALH, percentage recovery, HOS and viability an equally effective method of sperm separation compared to currently were significantly better in specunens processed by Perwash than MonoPercoll used methods. (P <0.05). Results of sperm morphology by WHO and Kruger's criteria were similar between the two methods. In summary, discontinuous two-layer Percoll procedure provides significantlybetter semen characteristics than MonoPercoll sperm separation technique in ncrmozoospermic men.

78 MICROSCOPIC SPERM PREPARATION MIGHT BE A NOVEL 80 CHANGES IN SPERM MORPHOLOGY AFTERPREPARATION APPROACH FOR INTRACYTOPLASMIC SPERM INJECTION (lCSI). A. FOR IN VITRO FERTILIZATION (!VF). OS Karabinus, C Racowsky", LA Hossain', S. Barik2", B. S L)llll Jr'" and I. Tborneycroft' " , Univ of South Rizk'", Rudzitis", and TJGelety". Department of OB/GYN, The University of Arizona Alabama, Departments of Ob/G}n 1 and Biochemistry '. Mobile, AL. College of Medicine, Tucson, AZ.

Intracytoplasmic sperm injection procedure requires optimization of sperm and egg Sperm head sizeand shape are major determinants of normal sperm morphology preparations before injection of a single into egg. Notable sophistications have sperm which, when assessed using strict criteria, can reliably p�ict IVF success. We in egg preparation, microsurgery, injection equipment andin jection strategy. occured investigated whether these sperm characteristics are affected following The preparation,sperm however,has not undergoneany significant improvement In !CS! procedure, the sperm are prepared by just aboutany method that yields a clean preparation and subsequent incubation. Raw semen for IVF (n = 72 specimens; preparation. In this study we have developed a microscopic sperm preparation 60 patients) wasprepared using discontinuousPercoll columns followedby wash teclmique thatis speciallysuited for !CSL Sperm separation column is designed to be and swim-up. The prepared specimens were incubated for24 and 48 hr at 37°C construcl!:d on a 60 mmpetri dish that is usually usedin !CS! so an ideal number of in 5 % C02 in humidified air. Sperm motility wasdetermined by CASA. Sperm capacitated and hyperactivated sperm are ava ilable at the time of!CSI procedure. A morphology was evaluated using strict criteria; specific abnormalities were 15 uJ volume microgradient column was constructed in the shape of asquare with an recorded forthose sperm judgedabnormal . Motility for raw, prepared, 24 and open corneron a petridish. One o� end of the square contained semen, while the 48 hr specimens was 69.0±2.1 %, 96.5±2.2%, 75. 1±2.2% and 37.2±2.2%. arms contained 3 variable concentrations of albumin to maintain the three used Theoveral l fertilization rate was 60.9± 2. 1 %. Compared theraw semen, gradient The microgradient column, covered with mineral oil, was observed under with an inverted microscope. When sufficient number of sperm reached the harvest zone the proportionof normal formsincreased followingpreparation (22. 0 ± 0. 6 % vs. (column-end), it was disconnected from the rest of the column by mild scratching at 17.3±0.6%, P<.001), then decreased after 24 and 48 hr (15.9±0.6% and the specific locationwith a pipettip. Twelve semen samples.,.; thdifferent male fa ctor 15.3 ±0.6 % , respectively). The incidence of abnormal midpieces, droplets and etiologies were processed with the column and the quality of the sperm yield was tails decreased after preparation, while the overall incidence of head 2112 evaluated. Spermpreparation was successful fromI (I00%) semen samples. The abnormalities remained unchanged. The prevalent abnormal head shapes in raw motility of the prepared all samples were I with grade a quality sperm in 00±0"/a semen were large oval (>5µ long and/or >3µ wi de), asymmetric, pyriform, (WHO criteria). 96±6% sperm in the }ield exhibited hypo-osmotic swelling. The tapered, andamorpho us. Although the incidence of most of these prevalent head percentage of spermatozoa with normal morphology (Kruger's strict criteria) was 53±7%. The number of sperm reaching the harvest zone correlated with thetime in abnormalities were unaffected by preparation, that of large ovals was increased (34.9 ±1.2 % 44.4±1.3 % ; P< .001),but not furtherchanged by incubation. all samples. Five sperm injected into hamster OOC)1es exhibited swelling head vs. (indication of decondensation). Microscopic sperm preparation does not induce any In contrast, incubation resulted in an increased incidence of asymmetric heads gross and ultrastructural physical damage to the sperm since theprocedure doesnot (prepared:13.9±0.7% vs. 24 hr: 19. l±0.6% and 48 hr: 19.2±0.6%; P<.001 require anyprelreatment such ascentrifugation of the semen. Furthermore, since the for both). Therefore, following preparation andduring subsequent incubation, teclmique is based on self migration of motile sperm in horizontal column, it always there was a reduction in the incidence of normal forms concomitant with an yields morphologically superior with I 00% motilityfrom semen irrespective sperm increasein the incidence of asymmetric heads. These changes appearedunrelated of its male factor quality. The microscopic approach of sperm preparation therefore, to sperm viability or fertilization. The present observations suggest that levels might be an improved way of controlling the quality of spermto be usedin !CS! and thus improving the !CS! fe rtilization rate. of large oval and asymmetric heads and the shift from normal forms to asymmetric headsmay reflect normal, fertility-related eventsthat occur i.!!vit ro, and possibly i!! vivo.

P-44 Journal of Andrology •January/February 1997

METHOIJ FROLEN [XJNOR WITH 8 I PROCESSING OF SPERM CORREL.;�s FINAL o3 POST-THAW PLAS�IA �IE.i'v!RANE INTEGRITY (P�lll OF SPE!Uvl PARAMETERS ANDINTRA!ITERINE INSEM!NATION PREGN.>SCY OUTCOME Ceni:-: �IOUSE SPERM CRYOPRESERVED IN tvtEDIA TAl:-.llNG co:-. S.K. Jindal• nnd K.J Ltpetz. UMDNJ-Ne" Jersey Medical School. for Ferulity and Ri:pro:riod. 102 used Re production and Women's Health, Uni ,·ersity of Pennsylrnnia, frozen/thawed donor sperm. The purpose of this study was to retrospcc:-ely compare the Philadelphia, PA 19104 etfecti,·enessof threeprotocols rounnely used to prepare frozen/tha\\ ed sperm. ..::Jto evaluate thru signifiC3Ilcc on pregnancy outcome Previous studies of the sensitivity of mouse spenn plasma membranes 41 patients"ith v�ing diagnosesof infertility underwent eithernarura1 cycl< lUl or controlled to cryodamage (Noiles et al.,C ryobiol. 32 [1995] 220-38) indicated a ovarianh�perstimulanon "ith bMG Once lead follicles reached 16 mm cli!!ceicr, panents were 2: phase transition around leading to membrane embrinlement at given hCG (5.000 lU) nnd Ms were performed approximately 35 h later "ith donor spcmL 4oC, pre\iously matched and obtained from one offive commercial banks. Semen"JS thawed at 2-l'C lower temperatures. Glycerol (GL) abolished this transition. This led fo r30 minmid at37'C for 5 min. and the concentration of motile spermmid inm3I percent motili� to the hypothesis that GL could act as cryoprotectant by 'plasticizing' CASA were measurodby nnpreparations were pcrtbrmed by Swim-Up (SUJ. P«eoll (P) or Sperm the membrane as well as by inhibiting intracellular ice fonnation. Wash(SW) . SU samples were diluted I·I with Modified SpermWash Medilll:l1 Irvine Scicnti.tic. !nine,CA) mid centrifuged at300xg for JO min. The pellet was ovcrlmd"ith 0.5 :nL fr esh medium Trehalose (TH) plus GL was used with success in cropre servation of and allowed 10"'' im-up for 60 min at 37'C. The supernatant was collected_ =::ifuged and sperm murine embryos (Honadel and Killian, Cryobiol 25 (1988] 33 1-7). were resuspended. P samples (I ml aliquot) were placed undtluted on a ch3..=tinuous pcrcoll This suggested that TH, rather than raffinose (Nakagata, J. 95% (RF) gradient of mid 75%. centnfuged for 20 min at -l50xg and pellets """' combined and Reprod. Fertil. 99 (1993] 77-80), plus GL combined with another resuspended(0.5 ml). SW involved ccntnfugationof the whole sample for 5 min 350xg.11 removal of the supcmal8ntand resuspension of the pellet as the finalpl sam e. Final sperm:oncentrations and polyol as specific inhibitor of intracellular ice fonnation might be a percent motility were recordedby CASA Patients returnedin 2 \\Ksfor a bloc-.J pregnancy lest superior cryoprotectanL The following polyols were tested for Statistical significancewas deter:mmedusing the Students' I-test. preservation of sperm PMI as assayed by retention of calcein loaded ester. ethylene glycol (EG); I,:!- There was no significant difference ininitial sperm concentrations (motile spct::iml) per or imnal as the acetox�methyl propane- and percent motility prior to SU (n=IO), P (n=2 1) or SW (n=7 1). Nor \\et: the final sample 1,3-diols (PD l2, PD13), 1,1,1-tris-(hydrox}methyl)-ethane concentrations significantly different, regardless of protocol or sperm bonk U30\I. However, the (THME), 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHPD), with percent motility of the final sample was significantly different (p<.001) bemeen SU and SW. and without GL, in combination with TH or EG and EHPD were whereby SU samples bad the highest motility (73.3% 20.3), which was DOI different from P RF. :!: the least effective, with post-thaw PMI below 20%. None of the samples (60.6% :!:23.2). and SW samples badthe lowest motili�· (33.3% :!:l i' 7). SW samples }ielded a pregnancy rateof 44%(1 4132), SU samples gave a mte of 38% (3/Sj. :?Ild P preparations other polyols enhanced the cryoprotective effect of GL. The best �elded a 7% pregnancy rate (1/15). giving a total of 18 pregnancies for 41 panc:nts (44%). post-thaw PM! was achieved with 7.5% (w/v) TH plus 69' GL al 48±6% (SEM, n=S), with cryoprotectant added in 8 separate aliquots. It isgen c:rallybelieved thatsperm most compromisedby the freeze/thaw proccs;�i elds acceptable samples \\ith SW, less compromised sperm improves greatly after P, and spct::i \\ith the higbt:St With 7.5% (w/v) RF plus 6% GL, with 8 aliquot addition, the value parameters give the best results after SU The resultsof tlusstudy mdicate thm SU, P or SW !!l'·e was36±9% (SEM, n=S). These values differ. P < 0.05. The TH/GL similmconcentrations final of sperm_ but SU or P give higherpercent motili�·.�ess of initial system thus provides a well defined medium for mouse spenn measurements. However, when correlated to pregnancy outcome. SU prepar:monof the sperm is cryopreservation withreasonable recovery post-thaw. tiirsuperior to P preparation, asis SW, despite the lower motility of the final =pie. Therefore. based on the results of this study, swim-up and sperm wash procedures are :te0mmended for Supported by NICHD through grant HD-3 1757. preparation of frozen/thawed sperm.

8 2 FROZEN HUMAN SPE!Uvl MAINTAINS MOTILITY FOLLOWING 8 � TREATMENT OF HUMAN OR BULL SPERM WITH A PROCESSING AND REFREEZING. Pl. Jones. L.Thanh'.J.Schulm:u:' Jnd E_ Fugger �- SYNTHETIC PEPTIDE INCREASES BINDING TO EGG­ Gi:m:tics & !VF Institute, Fairfax. VA. and the Mcdic:al College of Virginia. Ri.:-'".:nond. VA In general, human semen survives cryopreservation with enough dficiency to be MEMBRANE SUBSTRATE used for 11..:1/IVF cycles. In some cases it may be necessary to rerreeze the processed R.P. Amann, R.H. Hammerstedt and R.B. Shabanowitz, BioPore Inc, frozen sample for use in future cycles. In an effort to evaluate the 50% for3 of 10 samples treated with increased the sample motility by 35 (35.7) percentage points to 62.9 = (2.0 I 31), and 6 160 pM peptide. For fresh human spenn (I sample from each of 8 refreezing this specimen (zf) decreased the motility by an average of 20 ( 18.5) percentage points to 44.3 :!: 11 (2.0 I 22). men), 0.8-5.9% of untreated spenn bound and for 5 of 8 samples In conclusion, processed frozen spenn may be refrozen. There are quantitative binding was increased by �80% at optimum peptide doses. Averaged losses of total spenn cells during each processing step, and additional lo;ses of motility across all 8 samples, binding was increased 59% at 320 pM peptide. occur following rerreezeof these specimens. Yet, enriching the motili� of the sample For frozen-thawed human spenn (1 sample from each of7 men), 1.7- by processing the frozen specimen through glass wool prior to refre

To1. C1.1IO" '" JU J6J "' "' " similar in vitro treatment with UPSEBP or FertPlus™ peptide is knownto increase fertilityof spenn fromcertai n roosters, it is MeanMoL ...., 50.0 J6A 27.1 12.1 ':.' 4.&.J assumed that in vitro treatment of spenn from somehumans or bulls Pros.Grade 2.5 2.5 2.0 2.0 2.0 �· u also might increase fertility. Donation samples of frozen semen by California Cryobank Inc., Methodist DIC dO" 2JJ 112 IJ2 " " " 2! of Hospital of Indiana, & Colorado State University is gratefully acknowledged.

P-45 TWENTY-SECOND ANNUAL MEETING

85 GENDER PRESELECTION IN MAJ\!MALS: UPDATE ON 8 7 EVALUATION OF MEN WITH VARlCOCELES FOR IVF, ICSI OR OTHER OPTIONS AN 0 Glaz1ar JL Marmer, M Gibbs, SL Corson, Division of Urology, Robt. Wood Medical FLOW CYTOMETRIC SPERM SORTING L.A.Johnson: G.R. •. X/Y School et Camden, NJ and FertlJity Tes1ing Leboratory, Phila. PA WelCh" and"W. Rens" Germplasm & Gamete Physiology Laboratory, Following varicoceleotomy, about 50% of the infertile patients achieve pregnancy with ARS, USDA, Beltsville, J\ID 20705 USA coitus end/or IUI. However, many remain infertile and are candidates for IVF. ICSI or other options. In the past, these patients were evaluated by semen parameters alone. For example, ICSI has been recommended for patients with less than 50-100,000 Flow cytometric sperm sortingbased on DNA is now a standardized method motile sperm per oocyte in e prepared specimen or In cases with oligoasthenospermia for selling sperm. DNA is 3 to 4 % greater in the X chromosome that in the (OAT) in whole semen because fertilization with IVF has been only 7-10% for these Chromosome. Gender preselection in man and animals can be predicted men. Recently, acrosome induction studies have been used to predict fertilization with Y IVF. In addition, these acrosome studies have evaluated sperm function of men with by virtue of the fact that the DNA method can be validated by measuring the vericoceles. At our institution, we developed a novel acrosome induction test which DNA of the sperm after separation. Since DNA is the only know difference included hypoosmotic swelling and a double stein !Bismark Brown and Rose Bengal) to between, X and Y sperm it constitutes the marker for the separation. determine the percent viable acrosome reaction in a capacitated specimen (% VARI. In this report, we utilized semen parameters and the % VAR test to avaluate 106 men Hoechst 33342 a DNA binding stain is added to intact viable sperm to wtio wara potential candidatas for !VF, ICSI or othar options. Each man failed 2 years differentiate X from Y sperm. Prior to passing the sperm past the laser of of coitus and a minimum of 5 IUl's, Control studies were carried out on 42 donor the flow cytometer/cell sorter, sperm are incubated at 3 2 C for 40 min to specimens. The mean % VAR was 14.5% with no values less than 10%. These specim&ns yielded greater than 50% egg f&rtilization with JVF. The study group attain uniform stain penetration. Procedures are effectively validated using consisted of 32 infertile malll!ls without varicoceles (VNI, 30 with varicoceles not reanalysis of the separated sperm forDNA content and sex at birth. Sexed repaired (VNRI and 44 with varicocll!lles repaired (VAi. The percentage of OAT was ·semen is sorted into X and Y sperm populations and used forfe rtilization 12.5% (VN), 11.1 % (VNRI end 31.8% (VR). This group was recommended for ICSI through: in vitro fe rtilization (IVF), intratubal (IUI) or by deep-uterine because none with OAT had greater than 10% VAR. The remainder of the study group was classified by the pll!lrcentage with graster than 10% VAR as follows: 27.3% (VNJ, insemination (DUI) in the cow Intra cytoplasmic sperm injection has also 56.6% IVNRI, 36.3% (VRI. These patients were recommended for IVF. However, been reported by others to produce offspring of the predicted sex. Skewing among the VNR patients there were 44.4% with less than 10% VAR. Despite normal of the sex ratio from the standard 50:50 to 85 to 90% in either the X or Y semen parameters, this group was recommended for a varicocelectomy as another option to ICSL We anticipated that 1 /3 of the VNR group would improve their direction is effective in cattle, pigs, sheep and rabbits. Purities recorded for acrosome reaction following surgery based upon our experience among other sorted sperm from the human, average 75 to 80% of X or Y sperm. varicocelectomy patients. Thus, we believe that classification by semen parameters Depending on species, sex ratio can be skewed from 75 to 94%. To date IUI and a modified acrosome induction study (%VAR) may help determine IVF versus ICSI versus other options among infertile men with varicoceles. and IVF have proven to be the most effective for utilizing small numbers of sperm to gain fe rtilization. DUI has proven effective fordelivering about 2 xl01 sperm to the tip of the uterine horn in cows to produce sexed calves. The sexing technology is an effective means for shifting the sex ratio in .o.ffspriog in sever�! mammalian _s pecies

88 xIICRO�TCLEI OTHER '.L\LITIES .S6 Vasectomy, vasovasostomy and MESA I ICSI: is it the ..\.'-. lJ '.'.TCLEAR ..\E:.:OP ... L' ; PEOPLE EXPOSED I'ERO'.'."'E future triad ofvasectomised man who regrets vasectomy? ' BT:CC -\L '.\·IT:cos.i. l:'i TO ESTOS OR .:..DOTROPHf.\"ES. ·:VI. DuJ.L. M.T.W.T. Lock, H. Roshani� Dept. Urology and P. Kastrop Dept. iGOX. 0. Torm-Bug:irm ' . E Varga,;�,:\ ·l.P. � •R.lnura-:\-Iuiioz•, S.i.ncha..Coro!ll1'. OBGYN, University Hospital Utrecht, Utrecht, The Netherlands. J G. Zm!!�a· ·'S cr.1c10 de: '"-\JidrnlL•gm dd Ho,;pi1:1l de: Gincc(l-;.'bstelricia dd ·:· '.\L\cJ y Lat>. de la :\.Jolccular 1.s.s We studied the pregnancy and patency rate of our micro-surgical and our :.\Iutal!enes1s de Di\'1sion .i e '.\1erticina .. i.:.. .. Guadalai:· ua.. MESAIICSI treatments to evaluate the future roll of the micro­ .raJ.i;co.:.\Iex1rn . 1 manipulation andvasovasostomy in the case of vasectomized man. The ill vivo ruicrnnuclei test detect; the effects ot':nul.'.! geuic agems b; A number of 84 men underwent vasovasostomy. The hormonal state of the 1demific:mon of 1cenuic fragment.'> :tnd 'N chr.'n1<);;0me� that :itter FSH, LH and testosterone were studied. Two layer technique as described b.:ing left uo t of the nudeous. give rise to thc::>t: strucrures: it is dire<�! by Silber was used. Testis biopsies were taken peroperatively. Semen an.J there is no need !O nmke tissue culture�. '.l.l:c:c·uudeogenesis 1 in analysis occurred per 3 months post-operatively during one year. Six bUIP.a.n buccal mu,cosa·1 ret1ects the genotoxic e\e:Jh th.at could ha•:e months afterthe operation the data were refreshed. taken place m the previous thret! weeks. It is well known that the Nine men didn't cooperate to examine the semen. The patency of the vas hormonal therapies used to mJuce hyperl''1.!ia11on Je\·elupe was reversed at 75% (56175) of the cases. Six patients lost their child wish. chromosome alterauons in h= and other :ij:ec:es. Some human The number of 69 patients were divided in three groups: group A; (fertile fe male steroids resuli in mice, liver and m:uurual rumors. spem1 head group) n=2 l (30%) with average spermatozoa counts (ASC) of 37±19 ( 12- abnormalities and chromosomal nberrauo!l.5. be,1Jts. it is known that 82) XJ06/ml, group B; (subfertile group) n=29 (42%) with ASC of 20±2 1 the androgen tluoxi.w.esteroue, uorwally 1u rreauuenL> for erectil (1-79) XJ06/ml and an azoospermic group C, n= 19 (28%). Ten couples u,t:J dis functiou and oligos a microunucleogenic from group C were treated by MESAIICSI. Seven healthy children have perm.i is in mice and sister·chromatid exchange l:IBpbcc;·1es. Howe\'er. it been already born. Since 1-4-1996 MESA-treatments were stopped in the causes in human are Netherlands. There was a significant difference between group A and B has been reponed that female sterio score. The interval between the vasectomy and the vasovasostomy was to whom Testosterone or Gonadou-ophins were adruinisu-ed 10.8±4.1 and 6.7±2.5 years in azoospermic fertile group (p=0.001), iden�ing the preseuce of micronuclei. picuosis. cariorrexi;_ respectively. canolisis, condensed chonuatine, broken egg

P-46 Journal of Andrology •January/February 1997

89 SPER.'\.I MOTION PARA.t'\.lETERS INFLUENCED BY RHEU:lI 91 ARE J. R�latioo:shipwith sexual hormones ARICOCELE LIGATION? V Irvin H. Hirsch, Mohamed T. Ismail. R pia.. Scrra. ,o; FI Jimt!ocz·Balc!:r3...<*; M E Fonseca•:C Lara•; A aeltrl!!•: Ta John Sedor, Depanment of Urology. Jeffe rson Medical College, A F�•' ·Rhreumotolo�· department and Andrology scctior. t• f The Hospital de Philadelphia, PA J:'speci:tlidadcs. CentroMedico Nacioaal Siglo XXL ln.nituto Mexicano del Scguro Social , Me>..ico D.F. Although there is no pathognomonic semen pattern induced by OBJETJVE: To domminc if th• mal e hypogonodism ha,·e more :'requcnc) �r rhci.-:1ack varicoceles, sperm motility has been shown to correlate most commonly innammatory uid/or autoi.:n.'?lune di$C'ase depending on their i:tiology a::d gor.;tdal (RT-Al with this clinical entity. In addition, of all semen parameters. spenn steroid� l�o,,·el:i. MATERIAL AND METHODS: Tn irtoen men "ith hype>gonndism (Group Ii. b� motility is reponed to improve most commonly following varicocele ncurotnd•Jcrine and s�i'!t!t!c �:-ittri3.S were r.ia.t.ch:::d by age. sex and bud�· ma:is!ndex l BMl I liga!ion. To objectively evaluate this hypothesis, computer-aided semen tn the control group 10 mer. ..i.6XY tGrnup Ji) . Rhrcumatologic and a.r:drologic climcal analysis (CASA) was utilized to assess spenn motion parameters in studi� were perfonn� on the groups'. subfenile men before and after varicocele liga!ion. The group I was formed oy 9 pacicnr:; "ith hypcrgonadot:opic hypogt•n•u1sm fSub�oup This study includes 34 men with varicocele as the primary cause of A}. six of thmi had anormal carion·� and 4 paticn:s wich hypogonadotrc•pic hypogonadi>mSubgroup B);Two had Kallmam Syndrume and the :est had ldi'1p•:ic infenility who underwent physical examination, honnonal profile. hypogvnodotropic :uid cryptf)n:hidism.group B). Doppler ultrasound. CASA was perfonned before and after either RESULTS ' The findingswere: subinguinal or laparoscopic varicocele ligation. Patients were followed Subgroup A. -Five pMients hadRl-A: t"'o ;ystem;c erythemotosus l p s one had u u (SE! ), of between 3 and 18 months postoperatively and average CASA values chczrt antiphost'oiipid syndrome: cwo had Anqu±!osis Sp n l es i A� 1. B 2 i and with o u1 it - were obtained pre and postoperatively. Using the paired t-test CASA one with Juvenilerheumatoid anhritis {JRAJ. values were analyzed with special reference to motility and spenn motion Sub111oup B • 'l'hrec pa:t:nt> hod R!·..\: two had AS B27 + and one had Juvenile dermathnmyosicis. parameters. d1ft'erenc< In the group II Rl·A wa; not fo und andthere v;as a signjlic.uit Postoperatively patients demonstrated increases· in mean spenndensity CJ.01 with up I. p= gro and in the overall distribution of sperm with rapid velocity. although not The levels of Tcstoscerone \1) and Estradiol (E2) were lower (p< 0 07) in panencs "'ilh Rl-A than in patients without RI-A and controls. Two patients v.i!hout RI-A had high to statistically significant proponions. Increased mean values of Prola<:!ine (PR!.) levels. straightness, linearity, and track speed were observed postoperatively, CONCLUSIONS: Mak hypogonadism had more frequency of Rl·A independent of th eir but only progressive sperm velocity increased to statistically significant eciolngy. Thepresence of Rl·A was fouod l e withlow lc\'cls of n <.t steroids corro at d go a al levels (P < 0.008) postoperatively. Since progressive velocity has been but chromosopathy not with a primary CASA parameter predictive of male fe llility (Mathur. I 986) and successful assisted reproductive techniques (Holt, I 985), we conclude that varicocele ligation results in improvement in most semen parameters and, significantly, in the key parameter of progressive sperm velocity.

TESTICULAR �IICROLITHIASIS (TML) Il' �lEN WITH ON 90 9 2 EFFECTS OF MULLERIAN INHIBITING SUBSTANCE AZOOSPER.l\IlA SEVERE OLIGOSPER.\IlA. HUMAN A.t"ID SPERMSUR VIV AL E. Falla!,Falguni A Amin*,Sheryl C. Yoffe and Arnold Irvin H. Hirsch, Rick I. Feld. Leonard G. Gomella, Peter T. McCue. Yong Siow, Maiy M. Belkcr. Department of Surgery, University of Louisville and Louisville Depanments of Urology, Radiology. Pathology_ Jefferson Medical Andrology Laboratory, Jewish Hospital, Louisville, Kentucky. College, Philadelphia. PA Purpose: The use of cryopreserved sperm as a treatment for infertility is a Testicular malignancy has been associated with both the clinical spectrum maj or medicalapplication of sperm banking. Upon thawing, however, spenn of reproductive dysfunction and the ultrasound finding of testicular survival (\iability and motility) is poor. This project investigates the effects microlithiasis (TML). Yet few studies have investigated the association ofMullcrianinhibiting substance (MIS) on sperm survival in freshsp ecimens after cryopreservation. of TML in the infenile male population. This study's goal is to establish and Methods: Sperm fresh semen (n=6) was assessed using Cell-Vu the incidence of TML in the severely iofenile male population. motility in microscope coWlting slides after 0.5, 1, 3, 5 and 22 h incubation with and A group of 15 men presenting with azoospennia or severe oligospermia without (Coolrol) MIS (0.5 µg/ml). Viability was assessed by eosin-nigrosin ( < 5 x 106/ml) was scanned by high resolution scrotal ultrasound and 8 exclusion test. The same specimens cryopreserved in TES-tris-glycerol-egg underwent testicular biopsy with PLAP stain for carcinoma in situ. yolk buffer +14% glycerol (TYB) with or without (Control) MIS for 2 weeks Sonographic characterization of microliths included their number, size. were then thawed at 37°C, andsurvival assessed at similar time points. The specific effect of MIS was examined by co-incubation with anti-MIS laterality, and pattern of distribution. Based on clinical and laboratory monoclonal antibody (6E 11) in freshspecimens (n=6). Statistical analysis parameters patients were characterized with a diagnosis of either genital was by Signtest, and a p value of < 0.05 was considered significant. tract obstruction or spermatogenic failure. Ultrasound detection of TML Results: was present in 2 patients (spermatogenic failure and genital tract FRESHI CRYOPRESERVED obstruction). Io the former, ultrasound characterization of TML was Oh Sh h Oh Sh h multifocal with 6 microliths ranging in size from mm. Histologic 22 22 2-3 Motility correlation showed early spennatogenic arrest with tubular wall ·MIS 71 :I:4 41 :1:9 23 :I: 7 67 :!: 12 43 :1:9 15:I: 2 thickening and myoepithelial proliferation. Io the latter patient a single echogenic focus of TML measuring 1 -2 mm was noted. All of the +MIS 71 :I:4 53 :I: 7t 38 :1: St 67 :!: 13 52 :1: lOt 29 :1: St specimens were negative for carcinoma in siru by PLAP stain. Viability The association of TML and azoospennia/oligospennia remains unclear -MIS 69 :I: 5 43 :I: 8 28 :!: 7 67 :!: 13 44 ±9 15 :I: 2 at this early phase of study. Yet because of the detection of TML in this small initial coholl, periodic clinical andultrasound monitoring is +MIS 70 ±4 54 :1: 6t 40 :1: 7t 74 :1: 13 53 :1: lOt 29 :1: St in recommended subfenile men with TML. 22-76xl 62-78%; 'Sperm concentration (range): O'/ml; motility: nonnalmorphology %. (WHO criteria):15 -51 tp <0.03 compared with Control (-MIS). Co-incubation with 6El l eliminated these effects of MIS. MIS Conclusions: improves spermmotility and viability, and thus may have potential fo r use in assisted reproductive technology.

P-47 TWENTY-SECOND ANNUAL MEETING

93 INTRAUTERIKE INSEMINATION FOR CERVICAL OR MALE 95 IMPROVEMENT IN SEMEN QUALITY :\... '\D FREQUE'iCY FACTOR PROBLEMS IN WOMEN >40 HAVE VERY POOR VIABLE OF SPONTANEOUS ACROSOME REACTION AFTER PENTOXl­ PREGNANCY RATES J.H. Check, D. Lurie•, M. Peymer•, D. Katsoff and FYLLINE ADDITION IN NORMOZOOSPERM!C MEN. S.C Esteves. R. Long•, UMDNJ, Robert Wood Johnson Med. School at Camden, Cooper R.KShanna, A. J. Thomas, and A. Agarwal, Andrology Research & Hosp /Univ. Med. Cntr., Dept. OB/GYN, Div. Repro. Endo. & Infertility, . Clinical Laboratories, Department of Urolo� . Cleveland Chruc Camden, NJ. Foundauon, Cleveland OH 44 I 95 t The obj ec ive of this study was to compare the efficacy of intrauterine Incubation of highly selected motile sperm population mth pentoxif:Hine insemination (IUI) for male and/or cervical factor by age of female partner. A retrospective review of all patients who underwent IUI at a private practice improves sperm motion parameters and optimizes induced acrosome t of a universi y based infertility center during an eleven month period in 1995 reaction. The aim of this study was to evaluate 1f pentoxifylline \\hen was undertaken. 281 patients who underwent IUI therapy for cervical and/or added directly to the seminal plasma can l!Tiprove sperm motion and male factor were classified into two groups by age at time of their first IUI: acrosome reaction. Sperm specirnerts from 15 volunteers with nonnal younger than 40years (n =232), 40 years old or older (n=49). Cervical factor semen volwne, sperm count, and motility were divided into two aliquots was diagnosed based on a post-coital test that failed to show sperm with good one aliquot was treated by directly adding pentoxif:lime (5 mM) and the forwardp o e ion at time of mature follicle. Male factor wasdiagnosed the r gr ss if second without pentoxifylline (control). Both aliquots were incubated for semen analysis demonstrated either low count, low motility, antisperm 30 rrunutes. Percent motility and motion characterirucs were evaluated by antibodies or subnorm al hypo-osmotic swelling test. About half of the patients underwent IUI in natural cycles. The remaining patients underwent IUI computer assisted semen analyzer. Spontaneous acrosome reaction (AR) following ovarian stimulation with either clomiphene citrate or low dose was assessed by fluoroscein conjugated peanut lectin Viability was gonadotropins for the treatment of anovulation or follicular maturation defects. assessed by Hoechst-33258 stain. Pentoxifylline treatment increased The main outcome measure compared was the cumulative probability of percent motility, amplitude of lateral head displacement and ongoing pregnancy (\iable at end of first trimester) following 3 cycles of IUI. hyperactivation status of sperm (P < 0.05). No change was seen in o!fter Based on life table analysis, the cumulative probability of ongoing pregnancy motion characteristics. The frequency of AR in viable sperm was higher following 3 cycles of IUI was 28.2% for the younger group and for the 0.0% in the pentoxifylline treated group ( 19 2 ± 8-4%) compared to control older group (p< .05). The age groups did not differ in terms of infertility (10.7 ± 5.9; P = 0.005). The effect of pentoxifylline on sperm mouon history, indication for IUI, use of ovarian stimulation, number of follicles characteristics varies in normozoosperrnic men. In selected cases it may released and baseline semen parameters. The treatment of male and/or enhance the fertilizing ability of the sperm by increasmg the number of cervical factor by IUI is ineffective for women 40years old or older. Thisstudy acrosome reacted spermatozoa. reaches the same conclusion as did a previous reported investigation of JUI following superovulation. The study presented herein did not use u s perovulation techniques. Furthermore, all patients in the study presented herein had male or cervical fa ctor whereas the majority of cases in the afo rementioned study had unexplained infertility in the majority of cases.

01' 94 ABSENCE GLUCOSE DECRE.\SES HU\IAN FERTILIZATION 96 EFFECT OF PERITONEAL FLUID ON SPER.i\11 MOT!O\ AND SPERM MOVE�IENT CHARACTERISTICS IN VITRO CHARACTERISTICS A.i'\ 'D ACROSOME REACTION IN WO:O.IE\ M.M. Mahadevan, M.M. Miller•. D.M. Moutos•, Department of \\lTH ENDOMETRIOSIS Y. Wang, R.K Sharma, 1T. Falcone• and OB/GYN, University of Arkansas for\!edical Sciences A Agarwal. Andrology Research & Clinical Laboratories, Department Culturing human zy gotes in modified Ham's FI 0 medium lacking ofCrology, 10bstetrics and Gy necology, Cleveland Clinic Foundation. glucose, hypoxanthine, phosphate and transition metals (MM) resulted in Cle\·eland, OH 44195 better pregnancy rate compared to normal Ham's FIO (Mahadevan et al, in press). The effect of glucose in MM on human fertilization and sperm Endometriosis is common in women with infertility, but the etiolog: of survival in vitro was determined. Mature human oocytes from !VF patients or Percoll washed human sperm were randomly allocated to one infertility in these women is unclear. The adhesion and concomitant of the treatment groups. The treatment groups were as follows: Normal distortion of the pelvic anatomy may account for the infertility in Hams FIO, MM, MM with 5 mM glucose (HGMM) and MM with 0.5 ad\·anced endometriotic patients. However, infertility is difficult to mM glucose (LGMM). Oocytes were inseminated in one of four media explain in patients with minimal or mild endometriosis There are for 12-20 hours and checked for fe nilization. Sperm were incubated conflicting reports in the literature that peritoneal fluid (PF) of patients likewise for 4 and 24 hours and sperm motility and sperm movement characteristics such as average path 'elocity (VAP), curvilinear velocity with endometriosis could adversely affect the spermatozoa! motion (VCL), straight line velocity (VSL), amplitude of lateral head characteristics. The aim of the study was to I) determine the effect of displacement (ALH), beat cross frequency (BCF), straightness (STR), PF on sperm motion parameters, and 2) assess its effect on sperm and linearity (LIN), determined using computer-assisted semen analysis. acrosome reaction (AR). PF was aspirated from endometriotic and Fertilization rates were significantly lower in oocytes cultured in MM tubal ligation patients (serving as controls) during laparoscopy. Sperm (23.8%) compared to LGMM (75.5%), HGMM (73.6%) or Ham's FIO from normal healthy men were incubated for 3, S, and 24 hours with (7 1.1%). Sperm survival after four hours of incubation in all fourmedia were similar except VAP, VSL, VCL and ALH were significantly lower PF from endometriotic (n = 13) and tubal ligation (n = 14) patients to in MM (no glucose) compared to the ocher three media. At four hours, assess sperm motion parameters by a computer-assisted semen VAP, VSL, VCL and ALH were significantly higher in LGMM analyzer. Sperm AR was evaluated by a monoclonal antibody kit compared to MM. At 24 hours VAP, VSL, VCL, ALH, LIN and % "Acrobead test" in endometriotic (n = 20) and tubal ligation (n = I.+) rap id sperm were significantly higher in sperm incubated in HGMM or patients. No significant differences were seen in sperm motion Ham's FIO compared to MM or LGMM. At 24 hours % Rapid sperm was higher in HGMM than in Ham·s FIO. Absence of glucose in a parameters and percentage of AR between endometriotic and tubal modified Ham's FIO medium significantly lowered fe rtilization rates and ligation patients. These results suggest that PF from patients with sperm movement characteristics in vitro. endometriosis may not directly affect the sperm function. ItCould haYe a deleterious effect on the egg, sperm-oocyte interaction or early embryo development.

P-48 Journal of Andrology • January/February 1997

97 TOXICITYOF LL::�.CANTS ON S?ERM 99 PROGRESSrYEMOTILITY IN COMPUTER-ASSISTED SPERM K.O. Pomeroy, W. Sa-. ;,5e. M. Zimmer. D.V. Moffitt and J.H. Mattox. Samaritan ANALYSIS (CASA). V.L. Slott', S.D. Perreault. Universityof North Institute of Reproduc:;,;;'1edicine. The Samaritan Health System. Phoeniz. AZ.. Carolina, Chapel Hill, NC, U.S. EPA, Research Triangle Park, NC.

Lubricants have been c;:: to overcome vaginal dryness, to assist in the collection of semen and as a tron;:.c1ng gel fo r ultrasound. We examined the toxicity of There is a general opinion that the percentage of progressively motile sperm several lubricants (inclc: -g one ultrasound gel) on Percell-isolated sperm. Sperm (PMOT) may be a more appropriate measure than the percentage of motile in an assessment of sperm motion, but there is no standarddefinition of (200 ul with a minimur :i 50 million sperm/ml) were placed into tubes previously spenn

coated with approxima:: v 20 mg of lubricant(KY- Jelly, Astroglide, Vaseline. Clear PMOT. With CASA, PMOT is generally defined as sperm exceedinguser­ a Image. and a control vr..- no lubricant). Ten fresh semen specimens, in duplicate. definedthr esholdpath velocity(V AP) and/or straightness (STR). To examine were allocated to each :-eatrnentand a computer assisted semen analysis theinfluence of different VAP an d STR thresholds on PMOT, cauda epididymal (Hamilton Thome) was :erformed after I and 24 hoursexposure to the toxicants. spenncollected were from an untreated rat and allowedto disperse in Medium- Clear Image. an ultrasc_-d gel. was extremelytoxic to sperm and in all but one 199 at 36 °C. One aliquot was diluted and digital images collected on an specimen there were -: -notJle sperm after I hour exposure. Lubricants 1n order optical disk using an HTM-rYOS (Hamilton ThorneResearch, Beverly, MA) of to'"city were Clear ·-"ge. KY-jelly, Astroglide and Vaseline. Interestingly. sperm with the sample chamber at 36 °C. A second aliquot was "treated" by cooling exposed to Vaseline ha: ;.gnificantly higher parameters (percent progressive roomtemp., and at 27 °C. Two hundred spermimages from motility, straight line ve lateral head displacement and straightness) than at diluted recorded :::cy, sample analyzed at 60 for 0.5 sec. The distributions of YAP and controls, sperm not ex:-:sed to lubricants (paired t-test; p<0.05). KY-jelly and each were Hz Astroglide were toxic ir ::i1s assay, but the clinical significance of this needs to be STR in each sample were compared, and cut-off values for defining PMOT examined. Clear Image ; toxic and should not be used as a transducinggel pnor to were chosertcontrol The spermwere judged to be 95% progressivelymotile by Insemination. manual assessment. The median VAP and STR of these sperm were 13 8 µ/s Mean Values Mer 24. '--urs. - and68, respectively. The median VAP and STR of the treated spernt were 119 µ/s and 36. Using a YAP threshold of 50 µ/sec and a STR threshold of 30, Ccc:-ol KY-jelly Astroglide Vaseline Clear Image gated outtwitching spermso that %.0% ofthe controlsperm and 59.5% of the %Motile 59 22• 37' 64 2• treateddefined spennwere as progressively motile. Raising the STR threshold to50 yielded lowerPMOT values (85.0% and 18.5% forthe control and treated % Progressive 20 9' 14' 28' I' samples)than manual assessment, but provided a greaterdistinction between the VSL (u/sec) 27 15' 26 32' 2• treated and control sperm. Choosing the median V AP andSTR of the control spermto define PMOT selects for the fastest, straightest spernt. Using these 4' 0.2' ALH (u) 3.7 1.5' 3.4 cut-offs, 26.5% of the control and I% of the treated sperm were defined as progressive. Thus, different definitions of PMOT may be used in CASA to STR 68 44' 68 71' s· differentiate between controland treated spernt populations, depending on the different from Control ":<0.00I) : '(p

98 100 \'ASECTO\l\'-RELATED !:"FERTILITY: A COMMON AND CO:\DlTIONED MEDIA FROM CU�IULUS CELL CULTURES EXPE'."Sl\"E MEDICAL PROBLE:\I ST!Mt:LATE IN VITRO ACROSOME REACTION. G.M. Centola. S.P. Anne \I J•guier King Edward Memorial Hospital. Perth, Western Weisensel. V.L. Lewis •. Andrology Laboratory, Department of Obstetrics and Australia 6008, Austnlia. G;necolog; . University of Rochester Medial Center, Rochester, New York.

In the ,-ear 1995 in the State of Western Australia, some 1243 men underw;nt \'asectomy while another 117 men (9.4%) underwent The acrosome reaction (AR) is an exocytotic process necessary for successful vasectom; reversal. Between January 1992 and December 1993, a sperm-egg interaction. The AR can be facilitated in vitro by addition of calcium total of 860 couples were seen by the author in an infenility clinic. ionophore A23 l 87, progesterone. follicular fluid or even solubilized zona pellucida Of these couples, 80 (9.3%) were found to have infenility that was (Delonge CJ. Reprod Med Rev 3:159, 1994). The purpose of this study was to related to as vasectomy. Regret concerning vasectomy is thus a J p t determine the effects of conditioned medium from human cumulus cell cultures common problem. (CC) on the AR. Cumulus cells were acquired following !CS! from IO oocytes Of these 80 men. 73 were requesting treatment due to remarriage while the rc::maining 7 1• ascctomiscd men were in the same subjected to hyaluronidase digestion or from IO broken oocytes not subjected to the relationship but now wanted more children. The median age of the enzyme. CC were washed using human tubal fluid (HTF) supplemented with 0.5°,

80 vasectomised men was 42. 7 years (range = 26-58 years) while human serum albumin. Approximately 0.3 million viable (by eosin exclusion) cells

that of their present partners was 32.4 years (range = 22-47 years). were placed into each well of a 4 well culture plate, and held for 24 h at 37 ° C. 5° o The vasectomy interval in this group varied from 1-26 years with a CO 2• Sperm with normal parameters, obtained from a known fe rtile donor and a median "asectomy interval of 9.3 years. The pattern of seminal semen analysis patient, were prepared using a two-layer percoll (90%, 47%) abnormalities was very di,·erse in these men but either azoospermia gradiem centrifugation, followedby washing in HTF. They sperm were exposed to or oligoasthenozoospermia were the most common abnormalities I 37' seen. Among the female partners of these vasectomised men, 1 3 untreated conditioned media at a I: I (v/v) concentration, for hour at C, 5% ((16.3%) were found to have pathology that would have caused CO', then centrifuged, and resuspended in BWW with 3.5% HSA supplied with the infertility any semen problem in their partners. Acrobeads assay. The control was fresh, non-conditioned HTF., and conditioned Among these 80 men, a total of 79 vasectomy reversals had been medium from human granulosa cell cultures (GC). The acrosome status was performed. while 73 cycles of donor insemination had been carried assessed using the Acrobeads Test (Fertility Technologies, Inc.), which uses out, 3 of which were associated with ovulation induction. A further polyacrilamide beads, coated with monoclonal antibody MH6 l which binds to 18 cycle; of !VF and IVF/ICSI had also been completed. acrosome reacted sperm. Bead binding to sperm was assessed at 2, 4, 6 and 24 The cost of this treatment has clearly been very high. A plea is thus hours after exposure of the sperm to the beads. The higher the acrobeads score made for far more careful counselling of men undergoing vasectomy and for the cryopresen'ation of semen pre-operatively. The initial (scale of I to 4), the more acrosome reacted sperm present. Sperm incubated in application of epididymal aspiration of sperm and IVF/ICSI to this control or GC conditioned medium showed a score ofO at 2, 4, 6 h and a 3 (patient) problem may now be the more successful and cheaper treounent and 4 (donor) at 24 h. For the donor sperm, CC media resulted in a score of3 at 4 options. and 6 h, and 4 at 24 h. The patient sperm showed a score of2 by 4 and 6 h, and a score of 3 by 24 h. We conclude that cumulus cell conditioned medium is a source of stimulation of the acrosome reaction in both donor and patient sperm. This is the first demonstration of stimulation of the AR by a cumulus cell secretory product.

P-49 TWENTY-SECOND ANNUAL MEETING

101

MJTOCHOl'iDRlAL DNA, SDIES QUALITY AND CLINICAL 103 A NEW CATEGORY OF INFERTILITY DUE TO ASSESSMENT l 2 2 2 SPER'.'vl-SPECIFIC ABNORMALITY OF CD46 Anne M !equier , R. Martin , Denise Mehmet , J. Goldblatt & J.M. 3 I Cummins . King Edward �lemorial Hospital , Division of Genetic M.Kitamura, K.Matsumiya*, M. Yamanaka*, A.Oku�arna. Services, Princess Margaret HospitaI 2 & Murdoch University, School of Veterinary Studies3 , Perth. Western Australia. Department of Urology, Osaka University Medical School. Osaka. JAPAN and T.Seya*, Departrnent of Immunology, Center for Male infertility has been likened to accelerated testicular aging and our preliminary results on testicular biopsies from infertile men has Adult Diseases, Osaka, JAPAN indicated strong links with mitochondrial (mt) DNA deletions( l,2). These findings are consistent with models of aging and tissue degeneration based on progressive mtDNA dysfunction(3). Three infertile subjects fulfilling nonnal or subnonnal cri1eria on We have studied 66 semen samples from 64 men attending an infertility clinic and have used semi quantitative PCR to amplify routine semen analysis showed abnonnal spenn CD46 (membrane control regions of the mitochondrial genome as well as spanning the "common" 4977 basepair deletion. The deletion was classified as mild cofactor protein of complement) by SDS-PAGE/immunobloning (<0.03%), moderate (0.03-0.09%) or severe (>0.09%). analysis using a panel of monoclonal antibodies. These patients Deleted mitochondrial DNA was detected in 34 of the 66 (52%) were screened fr om male infertile patients with nonnal semen samples studied. or the 34 patients with deletions in the cells of their 63 seminal fluid, 8 were mild, IS were moderate and the remainin parameters, using Acrobeads test, which utilize agglutination were severe. Jn only I of these 34 men could pathology be det�cted clinically but of these men with evidence of deletions, 4 were reaction with anti-CD46 antibody. The spenn CD46 isofonn has oligozoospermic (W.H.O. Classification) and I was azoospermic. been reported to be associated with spenn-egg interaction. These or the men where no significant deletions could be found in their 3 semen, 5 were oligozoospermic and 2 were azoospermic due to patients expressed normal CD46 isofonns on lymphocytes and germinal aplasia. granulocytes. Thus, spenn-specific abnonnalities in these proteins It is clear that the presence of a mtDNA deletion in semen bears little relationship to the aetiology of the infertility and thus seminal fluid parallels male infertility, suggesting a new category of infertility, cannot be used to detect such lesions. These investigations must thus probably due to aberrations in the molecules related to spenn-egg be pursued in testicular tissue rather than semen. interaction. I. Cummins et al. (1994) in Bradley, M & Cummins, J.M.Seventh International Symposium on Spermatology, 7.5. 2. Cummins, J.M. et al. Mol Reprod Devel (1994) 37: 345-362. 3 Wallace, D.C. et al. Biochim Biophys Acta (1995) 1271: 141-151.

!O� CREAT!NE K!NASE(CK)-M ISOFORM !N HUMAN SPERM:ISOLAT!ON 102 CASA CHARACTERIZATION OF HYPERACTIVATED MOTILITY IN HAMSTER SPERM. S.D. Perreault*, S.C. Jeffav and D.F. OF CK-M AND DEVELOPMENT OF A CK-M SPECIFIC ANT!BODY.G Katz•, U.S. EPA, Res. TrianglePk, NC; Duke Univ., Durham, NC. HUSZARAND L. VIGUE, Dept. Ob/Gyn,Yale Sch.Med. New Haven CT The (CK)-M isoform in human spermatozoa, which is expressed in late Heretofore, computer-assistedsperm motion analysis(CASA) in rodent spermiogenesis simultaneously with cytoplasmic extrusion and reproductive toxicology studies has been conducted on freshly diluted remodeling of the plasma membrane, is a biochemical marker of sperm epididymal or vas (uncapacitated) To include more direct measures of spenn. maturity and predictive of sperm-zona binding and occurrence of sperm functionalcapacity, we are exploring the feasibility of using CASA pregnancies. We have now isolated the CK-M in human sperm and during capacitation to monitor the hyperactivated motilityassociated in vitro generated a CK-M specific polyclonal antibody. The key element was the with fe rtilizing ability. Hamster epididymal spermwere capacitated in vitro and sampled at intervals forCASA analysis using the HTM-IVOS (Hamilton development of a chromatography approach which separated the CK-B and CK-M isoforms, allowing us to monitor CK-M by its activity and not Thorne Research, Beverly, Spermwere imaged in 100 µ deep flat MA). by optical density, which is <5% of that of the whole sperm extract. cannulae usinga 4x objective and pseudo-dark field optics. hnageswere stored 9 Typically, we extracted 4x 10 sperm in lmid azol-DTI buffer, and the to optical disc andrecalled foranalysis at 60 Hz for0.5 sec. (minimum extract was passed through Blue Sepharose(CL-68) and Accel ion­ contrast = 40; minimwn size = 6). Afterverifying tracking accuracy during playback, spermtracks were printed and categorized visually as"progre ssive" exchangers. Tr� fractions with CK-M were further purified by Accel Prog, "transitional" (Trans) or "hyperactivated" (Hyper) in accordance with rechromatogra�r ! The overall recoveries of the protein and CK activity previous descriptions of hamstersperm motion (Suarezet al., 1984). Mean were about 9% a1.. :i 48%, respectively, with a 10-fold enhancement of CASA parameters of ten representative tracks for each category are listed the CK-M activity/0 .u. ratio. High activity peak fractions combined with below, along with the % acrosome reaction (AR) at the time of analysis. adjuvant were injecied into rabbits. The antiserum was used in studies toward the characteriz.atiun of CK-M. On western blots of sperm extracts Path VSL VAP VCL STR LIN ALH BCF AR the CK-M antibody s;a!ned a 64 kDa band, indicating a unique, sperm 173 238 434 74 ., Prog (O.Sh) 43 23 ,_ 0 specific CK, whereas on the same blots a CK-B antibody stained bands at the 32-35 kDa range, characteristic for CK-B and mitochondrial CK TraDJ 207 272 673 76 31 33 19 (1.lSh) There was no cross-staining of the CK species with the CK-M and CK-B TraDJ (Jb) 206 293 678 71 31 35 13 antibodies. In the Accel fractions, we observed the simultaneous presence of the 64 kDa band and CK-M activity. lmmunocytochemistry 158 348 783 45 20 43 29 13 Hyper(Jh) of high CK-M sperm fractions, showed enhanced staining in the tail­ area. In testicular tissue there was no CK-M staining of spermatids Hyper(4b) 138 380 861 37 17 40 35 65 i dica ng in agreement with our biochemical data, that the synthesis of These data document the dramatic increases in sperm velocity and \igor that n ti , accompany capacitation and the decrease in linearity associatedv.ith the CK-M occurs only in late spermiogenesis. The CK-M antibody and hyperactivation. Such data can now be analysed with multivariatemethods to the respective cDNA probes will be very important !cols in the develop integrated models for defining hyperactivated sperm tracks. Supported assessment of male fertility in both ejaculated spermatozoa and in by NIH ES03614. This abstract does not necess arily reflect EPA policy. testicular biopsies, in monitoring efficacy of male contraception and in the study of the Sertoli cell-germ cell interaction.{HD-19505 and 32902)

P-50 Journal of Andrology • January/February 1997

105 VAGINAL DELIVERY OF NEW FORMULATIONS OF NONOXYNOL-9 EFFECTS OF !:'1HIBITOR OF ADENYLATE CYCLASE (AC) ON CO­ I 0 7 AN PRECIPITATED WITH POLYVINYLPYRROLIDONE: ASSESS;\ll'.,YT OF TWO SPERll PROACROSIN ACTIVATION FORMULATION-DELIVERY SYSTEMS ON THE ONSET PREGNANCY or IN T. Toda' , Y. YaJDa110to ' , I. lliyagawa ' P. 11. Zavos, Sofiki tis, Dept. RABBITS. P.M. Zavos 311dJR . Cor:rc:i. Department of Animul 5,,ences. Uru,·c:mt) ofKcntucL' , l of Urology, Tot tori University Schoo l of lledicine, Yonago. Japan. Lexington. KY.

The objecliveof this study \\asto o.sscssthe efficacy of two deliverysystems cont.:muugc spermi id:il le have previous ly shown that 1) a high concentration of f3 r formulations, consistingnonoxynol-9 or (N-9) coprccipibtcd v.ith polyviny�=hdooe (PVP) or subunits of inhibitory GTP-binding protein {Gi) which is involved in PVP/N-9, andtheir ability toprevent the onset of pregnancy in rabbits. Semen >-pecun ens from 8 nulc receptor mediated inhibition of AC can almost total Jy inhibit sperm rabbits (New Zc::ilimd White) were collected, pooled. llSSCSSCd and utili:zeJ fot the ortificial acroso11e reactions induced by AC sti11ulators (Biol Reprod, Suppl. 46 mseminalions Thirty-two does were ullocntcd into 4 Groups. Spcrmictd:il formulations \\ere (Al). : 146. 1992) and 2) /3 r -subtmits of Gi have an inhibitory effect on delivered in tilefotm of Capsulc:sTahlds or (vaginal spermicidul vebicl.,). Docs m Group 1 (control) proacrosin (PRO) activation Rep Suppl, p94, 1992). In the r=ivcd plax:bo Tablet orC:ip!oule, vaginnlly.Docs in Groups 2 to 4 received an e'!"'rimenbl Tablet (8U11a11 rod, a present study, the effects of 2" -0-methyladenosine (llA ;inhibitor of or Capsule.Docs v:iginnlly. in Group I were inserninntcd al 0-h pol>1 -insertionof C:ipsulcs orTab lets AC) on sperm PRO activation was studied. containing no spermicides ( PVP only).Does in Groups 2, 3 and4were insc:mm3lcd (vagul.l!ly) at Semen sa11ples containing more than 100 x 10" sperm/111 were col lected 0, 0.5 and6-bpost-insertion of Capsules or Tablets containing spermicides(PVP;N-9), respectively. • fertile men and washed in medium containg 35mg/ml of The insemination dosage consisted of 0.5 mL containing 70-80 xl o sperm.llozoa with 70-80% from 13 BR I motility. The inseminateddoes were induced to ovulate 6-h (IMinjection: 200 nJ hCGJ prior to the hU1t811 serum al buai!l Two a 1 iquots of the same vo wie, sperm timeof insemination. The pn:Jl!lan"'· rat°' (PR 's) obtained from the matingsru.h · :ire sbown below· concentration. and percentage of moti le spermatozoa. were subsequently prepared frDll each mp e sample of each pair was Al time posl-inscrtion of delivery system end PR"s Cl'• ) sa l . One mixed with !IA {final concentration 100 m.ll ;Exp sample) dissolved in Delivery 0-time 0-time 0.5-h 0. 25 ml of BR 11edit11t. The other sample was mixed with 0. 25 ml of system p!.'lcebo e�enbl cxpcrimenbl Bn medium (contro l sa.1tple). All of the samples were incubated at 37 At 'C in an atllosphere of 51' carbon dioxide for 18 hours. the end of ·the incubation period each saaple was mixed with CaCI (final Capsule 75% 75% 25% 75% , I concentration: 1. 7 1111) and further incubated for 5 hours. Then al Tablet 75% 50% 0% 25% of the samples were centrifuged and the acrosin system (] Andrei 8: was assessed both in the pellet supernatant of each sample. The results thatdelivay both systems c:xbibi tedsome vari:lti O!lllin PR' s over the various time 267) and showed PRO intt:rvals (eocpecimai tal) as compmalthe to control vulues(placebo Ill0- time). When considering both The sperm content was significantly higher in Exp samples than in control sa.1tples vs m spent; li!coxon "s test deliverysystems, the PR's werereduced the most at 0.5-h post-insertion of the deli'·ety•system. Most (91±16 64±13 !U/10 7 intercstingly however, the Tablet seemed to be a more efficient delivery system th:Jnthe Capsule, for paired observations ; p

108 I 06 ASSESSMENT OF SPERMICIDAL EFFICACY OF NEW FOR.\lULA TIONS OF CYPROTERONEACETATE (CPA) PLUS TESTOSTERONE EN ANTHATE !Te) 100 mg/w,.k NDCCE:' NONO:\l�OIAI COPRECIPITATED WJTH POLYVINYLPYRROLIDO:-iE L'I RABBITS: A DOSE-DEPENDENT SUPPRESSION OF SPER."1ATOGENESIS IN �OR."1AL ME:->. MC COMPARISON BETWEEN TWO FORMULATION-DELIVERY SYSTE!\IS. P.M. Z.avos Meriggiola, C Flamigni, WJ Bremner. University of Bologna, Bologna, lt.1ly am!J.R. Correa. Department of AnimolSciences, Uciver>;ty of KentucL> . Le\'.ington. KY. and University of Washington, Seattle, WA. Previous studies of hormonal male contraception have resulted in We have Pf"'1ously sho\\11 bothin humansand rabbits. \\ith in vitro studies. th3l nono")nol-9 (N-� , inconsistent suppression of spermatogenesis and side effects such as coprccipit:l!ed v.ith polyvinylp)noliJone (PVP) couldoct as potent contr.ICCp!ivcs. We have also dcmonstr.Ued that thecoprecipibtion of N-9 and PVP (PVP/N-9) is a n�· step to alterthe decrease of HDL-cholesterol that would greatly hinder the acceptability of cbcmic31 st:de ofN-9 (liquid to powder) for spermicidaldelivery purpoocs. Therefore. the objective the contraceptive. Recently, we tested whether the combination of of this stud) was to assco. the in vitroctlicacy of PVP/N-9 '!'CIDlicidal agents delivered via f\\ o physiologic doses of TE with CPA could induce a more profound sperm different fonnulation-deliverv svstertlll.was Semen collected from NewZcol:md White male rabbits suppression and avoid androgen related side effects. Twenty-five normal (AV) (N=S) ,;a the ar1ilicinl v� method Ej aculates from all rabbits were collcctcU. poolcd men were divided into 5 groups of 5 men each and treated with TE 100 (oppra.3cly5.0 ml..) maintainedand al37'C prierto in vitro spermicidaltesting. Semen specimens mg/week alone or in combination with CPA at the dose of 100 mg/day were assessedcomxntr.ltion for (158.0±2.8 xl o• spermatozoa/ml..)and % motility (76.00.2%). The (CPA-100),50mg/da (CPA-50), 25 mg/da (CPA-25), and 12.5 mg/da (CPA- poobl \\<:respecimens split into 5 (1.0 mL):iliquru. Aliquot 1 consisted of fresh semen and wos used 12.5) The study consisted of a control phase, treatment phase lasting 16 as control Aliquots 2 to 5 were used for tc>ting of the placebo or e:3M/ml) iocubebcn.cithSUn:. In a previousstuJy. it was shownthat thispllltcna of sperm motility JccrenseJ CPA-25 4/5 (80%) 1/5 (20%) 0 spermpcndralion the in ccrvic.:dmucus penetrationtest. The dalo suggest tha!a) the form ulations CPA-12.5 3/5 (60%) 2/5 (40%) 0 tested :ire potent spermicido:s and b) both form ulation.Jclivery systems assessed ano capable of No change of any blood chemistry, including HDL-cholesterol, could be tile tested Further slUdic:sfor invivo assessment ofthe f\Vo fo rmulation­ dclivaini! spc:miicidcs. mating detected. A significant decrease of hemoglobinand hematocrit was found delivery· �stems will becarried out in rabbits, to test their efficacy as •ltj!inol delivery systems. (Supponedby NICHD Contmct No. NOl-HD-3-3 184). that was dependent on the dose of CPA and did not achieve statistical significance in the CPA-12.5 group. CONCLUSIONS: this hormonal combination provides a very effective, safe and therefore promising hormonalregimen to suppress fertility in men.

P-5 1 TWENTY-SECOND ANNUAL MEETING

I 09 TRIPTOLIDE: A POST-TESTICULAR ANTIFERTILITY AGENT : : l TEST KIT FOR THE DETERMINATION OF LEUKOCYTE •, RD Daqs• YH. Lue-, A.P. Sinha Hikim, A. Leung S. Baravarian•. C. Wang, and R.S. COUNT IN SEMEN. JG Alvarez. S Balgobin*. Powers and G Swerdloff, Dept. of Medicine, Harbor-UCLA Medical Center, Torrance, CA. Dep1 of Ob1 Gyn. Beth Israel Hospital. Boston !VF, Harvard :.Oledical School. Boston, �L.\.. The antifertility effect oftriptolide (TW), isolated from Triprerygium wilfordii, Leukocyte infiltration in semen is frequently associated with genitounnar: has been demonstrated in male rats and mice, as well as in men. TWhas been mfoction, antisperm antibodies and male infertility. A leukocyte count tn semen proposed to work at a post-testicular level but additional antispermatogenic greaterthan I million/ml is diagnostic of leukocytospermia by WHO criteria The effects remained possible. Our objective was to determine whether this obiecti\'e of this study was lo evaluate the performance of the novel le11k0Score""' compound selectedat dose levels that induce infertility have anti-spermatogenic ktlin the determination- of leukocyte count in semen, as compared lo brighlfield aucroscopy. A total of 128 semen samples were obtained from males effe cts. Groups of six adult male SD rats were given oral administration of 128 presenting for infertility diagnosis. Aliquots of 50µ1 of the liquefied semen were eithervehicle (control; C) or triptolide (50 or IOO µglkg B\\") dailyfor 35 or 70 mi�ed with 200µ1 of a solution of letramethylbenzidinelH,O, (TMBJ in days. Body weight gain was normal in all treated groups All six rats treated dtmeth) lformamide (Dialech Inc., Bos!on, MA).incubated al room temperature for2 with high dosage of triptolide were sterile during the second (days 63- 70) aunutes and the number of pero'.'tidase-positive cells exhibiting a blue color scored matingtrial (pregnancy rate \\ith exposure to 2 females per male was TW: 0/1 1 b: brighlfieldmicros copy. Leukocyte count in semen was also de!Crm.ined usin2 the Ler1k0Score"' a ­ C: 10/10). Lower dose (50 µg) ofTW gave intermedial.! values (pregnancy kit as follows: 50µ1 of the liquefied semen was added lo 3 mm vs chameler well in a plastic module and allowed lo through a filler (2.7 µm in TW: 8/12 C: 10/10). Plasma levels of LH, FSH, and T were drain vs pore size) sandwiched in the plastic module, followed by addition of 50µ1 or the indistinguishable from those of the C. No effects of triptolidewere observed on nm reagent. The absence of color on the filter fo llowing addition of the T:.OIB testis and accessory organs weight, volumes of tubular lumen, and the total reagent was considered a negative lest (s 1 million/ml) and the presence of blue Lcydigcells, tubule diameter, and homogenization-resistant advanced spermatid color was considered a positive test (> 1 million/ml). Ofthe 128 samples evaluated, 3 numbers. There was a modest decrease (p<0.05) in tubule volume. In situ 3'­ 31 hadleukocyte counts > 1 million/ml (I to million/ml) and 96 had counlS s I million/ml (0.02 to 0.85 million). semen samples with leukocyte counlS s I end labeling of apoptotic DNA fragmentation, which permits recognition of All nullionlml were correctlyidentified as negative by the leukoScoreTN kit. Of the d)ing cells at a very early stage of degeneration that could have escaped 32 samples with counts > 1 million/ml, 31 were correctly identified as positiveand I detectionin routine histological procedure, fu rther confirmed ourmor phological was incorrectly identified as negative. Leukocyte count in this sample was 0.90 findings of no effects of triptolide on spermatogenesis. In striking contrast. aullionlml. The sensitivity, specificity and positive and negative predictive \'alues cauda epididymal sperm concentrations were decreased by 60.0% and the of the l.eukoScore"" kit forleukocyte count at the I million/ml cutoff point were motility, which averaged 58.2% in the control rats, was reduced to almost nil. 100'1:,99%, 97%and 100%,respectively. Total test kit time was 1 minute. These r

110 TEST KIT FOR THE DETERMINATl01" OF SPERM l l 1 THE SPER;\I PENETR..\ TION ASSAY: STILL AN IMPORTANT VIABILITY. JG Ah'arez, S Balgobin*, RD Powers andG Da• is* Dept. ot TOOL I:\ THE AGE OF ICSI. J.D.Wininger, Ph.D. Abington Memorial Ob1 Gy11, Beth Israel Hospital,Boston l\'F, HarnrdMedtcal School. Boston, :.OL.\.. Hospital. Abington, PA.

Determination of sperm count and percent molih1: 10 semen b) The sperm penetration assay (SPA)is a common tool fo r measuring sperm microscopic analysis is a commonly used test for the diagnom of male factor nuclear decondensation in male infertility patients. Sperm nuclear infertility. In addition, the determination of sperm viability is often used as part of decondensarion is an integral step in re rtilization which leads to formation the rouune semen an alysis performed in the andrology laborato11 The obi ecth·e of of the malt pronucleus. With the advent of intracytoplasmic sperm this study was to evaluate the performance of the no•·ei Vita/Score™ kit in the injection 11CSI) , many in ,· itro fe rtilization(IVF) programs may be tempted determination of sperm count and viability in semen, as compared lo microscopic to bypass sperm fu nction tests in below normal semen specimens and analysis . ..\. lolal of 50 semen samples were obtained from 50 males presenting for proceed to ICSI. However, some men with below normal semen analysis infertiliryscreening. AliquolS of the liquefied semen were scored for sperm count ani parameters (count, motility,and/or morphology) are capable of fertilization percent motility, and percent viability by phase-contrast andbrigh dield microscop) in vitro "ithout ICSI. The sperm penetration assay, when performed Sperm count and sperm viability in semen were also decermined using correclly, c:in be a valuable tool in determining whether ICSI is necessary. the Vita/Score"' kit as follows: AliquolS of 50µ1 of the liquefied semen were added A critical component when performine the SPA is to minimize folse­ negative results. In our bonds. percoll washes, use of test-yolk buffer and lo wells # 1 and #2 in a 2-well plastic cartridge and allowed lo drain through a filler cold shock have been effective methods in minimizing fa lse-negative results. sandwichedin the cartridge. Then, 50µ1 of a0.1% solution of Triton X-100 in PBS Our laboratory considers a score of 6 penetrations per egg a positive result and 50µ1 of a propidium iodide solution in PBS ( 1 mg/ml) was added lo well and #1 and would perform routine rvF on these patients. A score of less than � 50µ1 of propidium iodide alone added lo well Both wells were nosed with 50µ1 #2. penetrations per egg is negative and ICSI would be performed on all of distilled water. The color produced on the wells was then scanned with a oocytes. A score between � aod penetrations per egg would receive ICSI densitometer and the integration areas used for quantitation. The color produced in 6 and rourioe IVF. Each laboratory should determine their own values fo r well was usedto obtain total sperm counl and thecolor produced Ill well was #1 #2 positi\'e aod neg3tive scores based on their own experiences. Our used to obtain the number of permeabilized sperm. The integration areas were laborato�··s experience with the SPA suggests a strongly positive comparedlo the results obtainedby phase-contrast andbrigh lfield microscopy using association between sperm nuclenr decondensation and the fertilizing regression analysis. Sperm count in semen ranged between 10 and 80 million/ml. ability oftbe sperm in routine rvF. percentmotility between 10 and 90 and percent viability between 12 and 95, as de1ermined by microscopic analysis. A significant correl ation was found between sperm countand propidium iodide-stainedsperm pretreated with Tnlon X-100 (r = 0.96). The number of spermatozoa stained withpropidium iodide m the absence of Tnton X- 100was highly correlated with the number of permeabthzed sperm (r =

0.98) and with the number of immotile sperm in semen (r = 0.96). Total test kit time was 2 minutes. These results indicate that the Vira/ScoreTM ktt could be used as a convenient an reliable lest for the determination of spermcount, sperm viability and sperm motility in semen.

P-52 Journal of Andrology • January/February 1997

t l 3 THE FERTILITY SCOREni TEST KIT DETECTS HIGH 115 ROLE OF ELECTRON :VllCROSCOPY IN THE EVALUATION OF SPERM DENSITY BUT IS NOT AS SENSITIVE FOR LOW MALEfNFERTILJTY DURING THE ER..\ OF !CSL D. Carbone.• J. McMahcn.• H Levin,• A. J. Thomas Jr. and A. Agam al, Andrology Research & Clinical DENSITY. Laboratones, Department of Urolo�" Cle\ eland Clinic Foundation. Cleveland E.V. Younglai, G. Tuke*, J.A.Collins* and A. Vawda OH 44 195 Department of Obstetrics and Gynaecology, McMaster University With the advent of !CS!, ultrastructural abnormalities of sperm pose less of a Medical Centre, Hamilton, Ontario, Canada LSN 3Z5. barrier to fertilization, and thus the use of electron microscopy (EM) has decreased. This study was conducted to determine if there are still indications for Home kits fordeter mination of male fertility potential has recently and value to EM of sperm during the era of JCS!. The history, semen analyses, and EM findings of 55 male-factor patients "ho underwent EM of their ejaculate been introduced commercially. between 1983 and 1996 were re\iewed Eleven men (20% of the sample) had Objective: To evaluate the FertilitySCORE Kitn1 with our regular normal sperm ultrastructure, while 44 men (80% of the sample) had abnormal EM semen analysis. results Abnormal EM findings included necrospermia (12/44, or 28%), tail Methods: One hundred and eighteen consecutive samples received for abnormalities (2 1/44, or 48%), acrosomal abnormalities (9/44, or 20%), neck routine semen analysis were included in the study. Sperm density was abnormalities (1/44, or 2%), and incomplete maturation (1/44, or 2%). The EM findings were then correlated with the semen analysis data to determine which assessed microscopically using a hemocytometer and expressed as parameters, if any, could predict the E�! results. Volume, sperm concentration, million/ml. FertilitySCOREn1 test results ranged from 1-6 for negative total sperm count, motility, and percentage of normal forms were compared tests and 7- 14 for positive tests. For comparison purposes the cutoff between the normal and abnormal groups Only normal morphology varied for sperm density was taken as 20 million/ml and the coresponding between the two groups. Patients \\ith a normal EM had significantly more normal cutofffor the FertilitySCOREni test result as 6. Nine samples could forms in their ej aculate (59%, range 48% to 65%) than patients with an abnormal not be tested with FertilitySCORErnbecause oflow volume. EM (8%, range 0% to 13) (P <0.0001). �e:-.1, EM findings were correlated with success \\ith in-vitro fe rtilization (JVF) Wlule patients normal EM's who Fifty nine samples had a negative FertilitySCOREn.1 test, of \\ith Results: underwent !VF were able to achieve a pregnancy, I I patients \\ith abnormal EM's which 13 were equal or lower than 20 million/ml. The remaining 50 who underwent JVF failed to achieve a pregnancy. Finally, the EM reports were samples had densities greater than 20 million/ml and all had positive examined for evidence of inheritable geneuc disorders. Five of the 44 patients FertilitySCORErn test scores. The sensitivity of the test was 22%, (I 1.4%) were diagnosed with immotile cilia syndrome directly as a result of their specificity 100%, PPV 100% and NPV 52%. EM testing. In summary, I) the best predmor of an abnormal EM is an abnormal sperm morphology ( 13% normal formsby light microscopic examination); 2) An positive FertilitySCORE1M test is indicative of 'good' < Conclusions:A abnormal EM identifies patients unlikely tobe successful \\ith !VF, and thus, EM semen sample, i.e. normal sperm density. The kit cannot be used for can be an effective screening test prior to !VF, and 3) EM can identify patients low volume semen samples and a negative test should be repeated. who may be at increased risk for passing on genetic derangements to their offspring, and therefore, it may be an imponant element in the proper counselling and therapy of patientsconsidering JCS!.

114 CORRELATION BEDVEEN MA;'\11\0SE LIGAND RECEPTOR 116 SPER-"•1 FUNCTION TESTS IN THE ASSESS:VIENT OF SEMEN EXPRESSION, ACROSOME REACTION, KRUGER'S MORPHOLOGY AND QUALITY. R.S. Sidhu.• R.K Sharma, Y. Wang. A.J. Thomas. Jr. and A. SPERM MOTILITY IN NOR.\!OZOOSPERlvllC MEN. R.K. Sharma, A.J. Agarwal. Andrology Research & Clinical Laboratories. Depanment of Urolo�" Thomas Jr. and A Agamal . ..\ndrology Research & Clinical Laboratories. The Cleveland Clinic Foundation, Cleveland. OH 44195 Cleveland Clinic Foundation. Cle,·eland, OH 44195. Sperm concentration and motility are insufficient in the diagnosis of male Spermconcentration and motility are not sufficient in the accurate diagnosis of the infertility. Sperm characteristics that can predict the fe rtilizing capacity can patient's fe rtility potential. Assays that probe the functional activity of the provide useful information in the management of infertile men. The aim of the spermatozoa are imponant. Occurrence of acrosome reaction and the recognition study was to evaluate if sperm motility. morpholo�·. le\'els of lipid peroxidation of zona pellucida by the head directed mannose receptors on the spermatozoa arc (LPO) and creatine kinase (CK) correlate \\ith the e:-.1ent of mannose ligand two imponant steps m fe rtilization. The aim of the study was to evaluate the receptor (MLR) binding and acrosome reaction (AR). Semen samples from 16 correlation between mannose ligand receptor (MLR) assay and acrosome reaction normal volunteers were analyzed by computer assisted semen analyzer. Seminal (AR) as well as between the above tests and sperm motility and morphology. smears were assessed by Kruger's method. Thiobarbituric assay was used to Semen samples from 14 normal rnlunteers were analyzed by computer assisted determine LPO levels in the presence of a fe rrous : ascorbate promoter system. semen analyzer and morphology smears by Kruger's method. Spermatozoa were Creatine kinase levels were measured using the CK-40 Sigma kit. Spermatozoa assayed after 6 and 24 h of capacitation at 3 7° C in Ham's F-10 medium containing were capacitated for6 hat 37° C in Ham's F-10 medium containing 3% human 3% human serum albumin (HSA). Hoechst-33258 (lµg/mL) was used to assess serumalbumin (HSA), and the viability assessed using Hoechst-33258 (lµg/mL) viability. Appearance of head directed surface mannose receptors were detected by staining. MLR binding (pattern III) were detected by the binding of lluorescein the binding of fluorescein isothiocynate labeled (FITC) mannosylated bovine serum isothiocynate labeled (FITC) mannosylated bovine serum albumin and AR albumin. Acrosomal status was simultaneously evaluated by rhodamine (RITC)­ (pattern III) was simultaneously evaluated by rhodamine (RITC)-labeled pisum labeled pisum sativum lectin. FITC and RITC Type III patterns were similar at 6 sativum lectin. Results were analyzed using the Pearson correlation test. A and 24 h of capacitation. A strong agreement was seen between these patterns at 6h negative but non-significantcorrelation was seen between sperm morphology and (r = 0,80, P < 0.0007) and at 24 h (r = 0.80, P < 0.0008). Sperm morphology the levels ofLPO and CK. Significant correlation was seen between FITC-pattern showed no significantcorrel ation nith MLR and AR binding patterns at both time III at 6 h and RITC-pattern III at 6 and 24 h (P <0.0006). A negative although periods. However, a good correlation was seen between motility and MLR (r = non-significant correlation was also seen between FITC-pattern III and CK. We conclude that I) sperm morphology is a good predictor of semen quality, 2) 0.65, P <0.01) at 6 h and between motility and AR (r = 0.66, P < 0.01) both at 6 and 24 h (0.5 1, P <0.06). Sperm morphology can not adequately predict the biochemical parameters such as LPO and CK are in\'ersely related to the sperm functional status of the spermatozoa. MLR assay is a reliable and inexpensive test, quality, and 3) MLRand AR can be used to assess the fe rtilizing potential of the it is easy to interpret and may be used effectively in the assessment of male fe rtility spermatozoa. These tests when performed together may provide relevant potential. information important in the treatment of infertile men.

P-53 TWENTY-SECOND ANNUAL MEETING

117 DIFFERE�CES P.\ THE '.\!Al\C..:.J. A.\";) .\5. C \ i'..E SL1..TS I:-. I 19 ASSESSMENTOF THE RELATIONSHIPOF SPERM MORPHOLOGY SE�1E!\ SPECll'v!E:\S BEFORE A.'-.U AITER FREEZC\G RS Stdhu.• R K WITI-1 SEMINAL ANDOTHER CLINICALCONDITIONS OF THE PATIENTS. A. Hossain', B R. Selukar'", D. Bhaumik ' , C. Huff '", J. Dyer-William '", Sharma. A J. Thomas and A Agarn a! Andro!o� R�search & Cilm:al Rizk1·, " R. Yeoman '" and I. Thomeycroft,. Dept of OB/GYN' and Dept of Mathematics Laboratories. Deparunent of L·ro !ogy, The C!e1 clan::! Chmc Foundatton. and Statistics '. University of South Alabama, Mobile, AL. CleYcland. OH .!.! 95I Theoccurence of abnormal forms of spennatozoa inhuman semen is quite common. Computer-assisted semen analysis (CASA) was mtroduced during the last decade According to V..110, a semen sample is considered nonnal even it contains 50% of to improve the accuracy of results and bring objectirn:y to the assessment of morphologically abnonnal spermatozoa. In this study we have assessed ifthe spenn characteristics such as concentration and motility The present study was morphological abnormality of thespennalD2D8 maintains any relation with the relevant carried out to e1·aluate: I) the accuracy and pre:tsion of CASA. 2) to determine tf clinical conditions of the semen donor. One hundred semen samples representing nonnal (nonnozoospermia) or different types of male factor etiologies differences in manual and CASA results were related to the type of CASA ( oligozoospermia, hyperspermia, teratozoospermia, aesthcnozoospermia, machine used or the type of semen specimen analyzed. �lultiple ej aculates from oligoaesthenol

120 115 LIPID PEROXIDATION CRYOPRESERVED SE:\!E\ CORRELATION OF HYPOOSMOT!C SWELLL-;G(HOS) TEST \\ ITH IN VITALITY STAINING, :l>IOTILITY A,.;D SPER.\IATIC MORPHOLOGY. FROM CANCER PATIENTS. Y Wang, R.K. Sharma, AJ Thoma; A ARREDONDO-PINEDA• ,C BRAVO-GATICA•,PT RONCOSO-TORRE7.0• and A. Agarwal. Andrology Research and Clinical Laboratories. R TAPIA· SERRANO.Aodrology section of tbe Hospital de E.specialidad••· Centro Department of Urology, The Cleveland Clinic Foundation, Cleveland. Medico N•cional Siglo XXI. Jnstiluto Muicanu dtl Seguru Soci•l.�hico D.F. OH 44 195. OBJETIVE.To assess tbe value of the hypoosmotic swelling test in relation with !iperm vitalitystainina1 motility a11d morpholo1y in Infe rtility patients. Preserving the fertility by cryopreserving semen specimen before MATERIAL AND ME!HODS. Fony two semen samples of infertility patients were rtudird in thl! labontory of the AndrololD' uction. Semen analyns were chemotherapy, radiation therapy or surgery is a realistic option for performed a"ordlng to WHO criteria (1992) . Sperm morpholoc wos e> aluated by cancer patients. Higher level of lipid peroxidation can adversely affect Sll'ict criterio ofKJ-upr and HOS test (Lomco and Giarnbcnio 1991) si�· percent or S Psed semen quality in men with male factor infertility. Although the pre­ HO tat reacted was as :a cut-oft'p oint . Statistical analyses w1re a'sn'5ed by t studenl and no correlatio freeze and post-thaw semen quality in cancer patients is poor, it is not - P .. a n. clear if lipid peroxidation (LPO) affects the semen quality The RESULTS. Tberm dings were sbowod on this Table, as fo llows: purpose of the study was I) to compare lipid peroxidation levels in I cryopreserved semen specimens from cancer patients and normal VARIABLE X:!:SD PEARSON t-STI'DE!\T •1. CORRELATION healthy men, and 2) to determine if sperm characteristics correlate TEST with vs. HOS •·s. HOS levels of LPO. Cryopreserved semen specimen from patients with HOS 59.7.l;J6.6 -- -

= = VIABILITY 0.496 testicular (n IS , non-testicular cancer (n 16) and normal men (n = 60.6:!:16.3 0.529 ) !110TlLITY 39.2 +1R.7 0.459 <0.00001 20) were thawed at room temperature for S minutes and 20 minutes in MORPHOLOGY 20.1::15.7 0.293 <0.00001 a C02 incubator at 37° C. Post-thaw sperm motility was assessed and The the sperm concentration adjusted to 20X106/mL. Thiobarbituric acid HOS was normal In 21 patients (52.J"t.), Viabiliry in 15 patients (35.7%), assay was used to determine LPO levels in the presence of a ferrous Motility in 1" patients (33.3%) and .\forpholol!Y In 21 patioat> (50.0%). ascorbate promoter system. No significant difference in LPO levels CONCLUSIONS: HOS test does not show any additional 1dv1nta2e through th• was seen between testicular (25.9 ± 3.9 nM MDN108 sperm/hr), non­ vitality staining test, so that It can be used indistinctly to ovalute the integrity of spermatlc membrane independent of the motilityand morphology observed. testicular cancer patients (24.S ± 6.6 nM MDN108 sperm/hr) and normal men (24.9 ± 6.4nM MDN108sperm/hr) . Post-thaw motility showed poor correlation with LPO levels. The poor post-thaw semen quality seen in testicular and non-testicular cancer patients may nor be related to LPO levels but may be the result of cell damage associated with cryopreservation and freeze-thaw.

P-54 Joumal of Andrology •January/February 1997

121 DIFFERENT TECHNIQUES TO IDENTIFY AND LABEL HUMAN 123 SUCCESSFUL INTERCONTINENTAL GENOME ROUND SPERMATIDS RESOURCE BANKING AND ARTIFICIAL INSEMINATION L.T. Culumbcru•, P. N. Schlc�d. M Fdicianu·. M.L. Papal<'. M. Goldstein, Z. WITH CRYOPRESERVED SPERM IN CHEETAHS. Rosenwaks•, G.D. Palermo. The Cenier for Reproductive Medicine and Infertility-Th< 1J.G. Howard, 1T_L. Roth', 1W.F. Swanson•, 1J.L. Buff", 1M. Bush', New York Hospitol-Cornell Medical Center. New York. NY, USA 2J.Grisham•, 3L. Maris to develop me>ns of identifying and labelling haploid Oklahoma City, OK 731 11; 3Cheetah Conservation Fund, Namibia, Africa. cells in testicular biopsies from azoospermic men. Their ploidy was confirmed by To determine the potential of_a genome resource bank for cheetah cytogenetic an>lysis of indiv1du>I cells. conservation, we assessedthe feasibility of collecting sperm from wild Material and Methods: Testis biopsies of -SOD mg were divided into small pieces individuals then transporting and infusing this material into the genetically and tubules were disrupted by homogenization, or m.l.nuaUy with fine forceps or needles. stagnant captive population. Our objectiveswere to: 1) collect and To assess the tissue for the presence of spermatids, S µI aliquots of the resulting cryopreserve spermfrom wild-caught cheetahsin Namibia; 2) assess suspensions were smeared on pre -stained slides and srudied by bright field microscopy at cheetah sperm survival post-thaw; 3) determine the efficacy of using lOOOx, on phase contnst microscopy at 400-600x. and also by acrosin immunolabeling laparoscopic intrauterineartificial insemination (Al) wtth cryopreserved using F!TC-anti boar acrosin antibodies. In order to isolate individual round spermatids. cheetah sperm for infusing wild genes into the North American cheetah cells were selected from 3 µI of the suspension placed under oil with a glass micropipeue breeding program. Electroejaculates from 11 Namibian cheetahs were of 7µ inner diameter controlled by a micromanipulator at 400x. Tue cells were then assessed for % sperm motility (M), % normal sperm (NS) and % non-intact analyzedon a microslide by fluorescence in situ hybridization (FISH) for chromosomes acrosomes (NA). Semen was washed in Ham's F1 O + 5% fetal ca� serum 18, X and Y. and pellets resuspended in POV cryodiluent (20% egg yolk, 11% lactose, Results: A total of 16 biopsies with different degrees of spermatogenic arrest were 4% glycerol). Samples were cooled (30 min, 5°C), pelleted on dry ice and processed by dissection with fine forceps or needles. In addition, tissues from three stored in liquid nitrogen for shipping. After thawing (37°C), aliquots were patients were homogenized. The most reliable method for disruption of wbules wbile assessed for M and NA. Ten Al procedures were conducted in 9 females maintaining the integrity of lhe putative spermatids proved to be manual dissection. treated wtth 200 IU eCG/100 IU hCG. Mean pre-freezeM, NS and NAwere Round spermatids were identified as cells of 6-7µ in diameter, with an acrosomal granule 69.4%, 20.7% and 12.8%, respectively, which were similar to semen tratts or cap and displaying an intact plasma membrane. Spermatids were isolated from 12 observed in other populations of free-ranging and captive cheetahs. The biopsies using light microscopy and the acrosin assay as a marker. and these were POV/pellet method provided cryoprotection for sperm motility (mean post­ det.ected in 3 of S patients with a complete absence of spermatozoa. The incidence of thaw M, 47.5%), however, a high incidence of acrosomal damage was round spermatids in the different biopsies ranged from 1-17% of the total cells scanned. detected after thawing (mean post-thaw NA, 59.5%; range, 44-68%). In order to confirm their ploidy, individual cells wen: isolated from four biopsies by Two females inseminated in utero wtth frozen-thawed sperm (6 and 15 micromanipulation, with a total of 7 slides being processed for FISH analysis. Due to a x1os motile sperm/Al) became pregnant and produced 1 and 3 cubs, higb rate of loss, only 19 cells wen: assessed but all displayed a signal, and 17 were respectively. These results demonstrate the: 1) feasibility of collecting and haploid. cryopreserving sperm from 'wild' cheetahs; 2) susceptibility of cheetah Conclusions: Spermatozoa or round spermatids were found in -70% of men with non­ sperm to acrosomal damage during freezing; 3) biologicalcompetence of obstructive azoospermia. ln the complete absence of spermatozoa. round spermatids were frozen-thawed cheetah sperm; and 4) potential of a genome resource isolated from 3 of S cases. Thus, cell diameter aod the presence of the precursor for bank and Al for gene infusion in captive cheetahs. (Funded by British acrosin •ppear to be useful markers througb whicb to identify round spermntids. Airwaysand a grant from 12 :zoological instttutions in North America)

122 CRYOPRESERV:\TION OF SPER.'v! COLLECTED BY TES E 124 INTERPRETATION OF CASA DATA USING DISTRIBUTION BASED AND MULTIVARIATE STATISTICAL METHODS. PROVIDES ADEQUATE POST-THAW VIABILin- AND SUCCESSFUL D Higdon1, M Lavine1, J Liu1, S Perreaulf2 , V Slott3 and D Kat:z"1, IVF-ICSI PREGNANCIES. R. Do!gina, G Wolf". P Studney•, B Kaplan•. 1 Duke University, Durham, NC, 2us EPA, Research Triangle Park, C Niederberger, L Ross, GS Pnns. Department Crology, Um,·ers1ty of NC, 3LJniversity of North Carolina, Chapel Hill, NC. Illinois, College of Medicine, Chicago, IL; Reproductive Genetics Insmute. Illmois Masonic Medical Center, Chicago, IL. This study begins development and application of multivariate methods that simultaneously assess sperm pattern and vigor, Testicular sperm extraction (TESE) combined with IVF-ICSI 1s a new revealed in the full set of per-sperm kinematic CASA variables. The approach focuses upon individual sperm as the unit of measure therapeutic modality for patients presenting with azoospermia due to vanous rather than, e.g., mean values per specimen. The goal is to enhance etiologies. TESE and IVF-ICSI procedures are commonly scheduled and two primary objectives of statistical analysis: (1) inference of performed concurrently to allow for the use of fresh sperm during !CSL biomedical outcomes from CASA data (e.g. fertility. toxic exposure); However, simultaneous scheduling presents several drawbacks particularly (2) delineation of changes in sperm motion from such outcomes, when there is inadequate sperm or egg extraction to allow for completion of leading to hypotheses about mechanisms of action. CASA data from the case. alternate approach is to perform TESE in advance and a study of air pollution in the C:zech Republic (2 cities X 2 seasons X An 2 years; Slott et al, 1995) have been reanalyzed with a set of cryopreserve the extracted sperm for later use in an "F-ICSIcycle. Herem I\ multivariate techniques: logistic regression and recursive partitioning we present data from 32 pal!ents who underwent TESE followed by sperm analysis - for (1); MANOVA, principal components analysis, and cryopreserval!on. The mean recovered sperm count per patient was 2. I 3 ± discriminant analysis - for (2). Initial univariate analysis had 0.53 million following unilateral or bilateral testicular extraction. While suggested that sperm vigor increased in spring vs. fall, reflected in motility was low or absent, eosin stain revealed 62 ± -1% viablecells. Sperm average VSL rising by 16%. The new analysis demonstrated that this average increase was due to increased straightness of individual were subsequently frozenin TEST-yolk buffer and stored at 96°C. A sample -l sperm trajectories (LIN rose on average by 19%). However, sperm aliquot was assessed for post-thaw viability which averaged 27 ± 2%. Only vigor actually tended to decline in the spring, particularly in the two patients had zero viable cells post-thaw and of those, one achieved second year, with simultaneous reductions per sperm in both VCL pregnancy following !CSL Thus we conclude that m most situations, enough and BCF x ALH. This effect was most pronounced for the viable sperm are available post-thaw for use in !CSL Seven patients subpopulation of the more straight swimming sperm. These latter results imply an increased symmetry of flagellar bending underwent IVF-ICSI using thawed TESE sperm and all cycles resulted m accompanied by a decline in sperm metabolic activity. The pattern of successful fertilization. Seventy-one oocytes were injected; 46 showed normal simultaneous changes in sperm motion could not be revealed in the fertilization (65%) and 33 exhibited normal cleavage divisions (72%). Four classical, average-based univariate analysis, and thus illustrates the viable pregnancies ensued (three singletons, one twin) with one normal potential power of the new methodology. Supported by NIH delivery and three ongoing. This 57% pregnancy rate using frozen-thawed ES03614. Does not necessarily reflect EPA policy. testicular sperm documents the efficacy of cryopreservation for sperm collected at TESE.

P-55 TWENTY-SECOND ANNUAL MEETING

125 FLUOROMETR1C ASSESSMENTS OF MITOCHONDRIAL FUNCTION 126 LOWER CONCENTRATION OF SPERM USED FOR OOCYTE AND VIABILITY IN CRYOPRESERVED BOVINE SPERM. D.L. Gamer' . INSEMINATION FOR IN VITRO FERTILIZATION (!VF) MAY C.A. Thomas•'. H.W. Jbrg•', J.M. DeJamene•' and C.E. Marshall'. 'School of RESULT IN HIGHER PREGNANCY RATES: A PRELIMINARY STUDY Veterinary Medicine. University of Nevada. Reno, NV. 'Swiss Federal Institute J.H. Check, C. Hourani', K Benfer• and A. Baker•, UMDNJ, Robert Wood of Technology, Zurich, Switzerlandand 'Select Sires. Inc .. Plain City, OH. Johnson Med. School at Camden, Cooper Hosp./Univ. Med. Cntr., Dept. OB/GYN, Div. Repro. Endo. & Infertility, Camden, NJ. The combination of fluorescent staining and flow C)1ometry provides for a rapid and precise assessment of the functional status of thousands ofindividual sperm. Recently data has been presented suggesting that a dissimilitude between We sought to quantify two seminal characteristics, mitochondrial function and fertilization rates and pregnancy rates (PRs) may exist in certain couples sperm viability, using fluorometric techniques and compare these with the undergoing !VF. The sperm has been proposed as the probable source of this classical microscopic measurement. sperm motility. Fluorescent staining of problem by releasing some toxic substance that may permeate the oocyte and sperm with active mitochondria was accomplished using rhodamine 123 (Rl23), remain throughout embryogenesis and thus impair implantation. Some 5,5' ,6,6' -tetrachloro-1, l ',3,3 '-tetraethylbenzimidazolyl.;:arbocyanine iodide (JC- researchers have presented data suggesting that a shorter incubation time of 1) and MitoTracker Green FM (Min. These metabolic measurements were sperm and oocyte may result in better implantation. Theoretically, reducing the compared to those found with microscopic assessments of sperm motility concentration of sperm used for oocyte insemination should reduce the (progressive spenn motility) initially (MOTO) and after3 hr at 37°C (MOTJ) and concentration of this hypothesized toxic sperm factor. Many !VF centers use with a fluorescent assessment of sperm viability (SYBR-P and propidium iodide 50,000 sperm per oocyte as the standard oocyte insemination concentration. [Pl] staining). Cryopreserved sperm samplesfrom eachof 12 bulls were thawed, The preliminary study presented herein evaluated the efficacy of using a 25,000 fluorescently stained and quantified by flow cytometry. Significant erencesdiff sperm per oocyte insemination concentration as compared to 50,000 sperm. (P<0.01) were found among bulls for each of the mtiochondrial function Couples undergoing !VF were randomized into 2 groups: oocytes inseminated measures, but not among the metabolic measurements. The mean percentages with 50,000 spenn per oocyte (standard concentration) versus the lower (n=3) of progressively motile sperm initially (MOTO) ranged from 27 to 83 for concentration of 25,000 sperm. All women used the same controlled ovarian 2 the 12 bulls. After 3 hr this range decreased to to 33% (MOT3). From 4 to hyperstimulation protocol (luteal phase leuprolide acetate-gonadotropin 70% of the sperm were viable (stained with SYBR 14). The proportions of sperm regimen) and all transfers were performed by one physician for this pilot study. 2 staining with Rl 3 ranged from 2 to 67% while MIT fluorescence was exhibited The fertilization rate for standard versus lower concentration was very similar in 4 to 71% of the sperm. Thepercentage of spenn staining with JC-I was also (62% versus 59%). The clinical PR (ultrasound evidence of pregnancy) was (355%) similar ranging from 4 to 68. The three fl uorescent measures of mitochondrial higher in the group using the lower sperm concentration than the function, RI23, JC-I and MIT, were correlated with �!OTO; r = 0.96, 0.87 and group using standard concentration (21.2% ); however the differences were not 0.96 and MOT3; r = 0.96, 0.86 and 0.96, respecti\ely. The mitochondrial significant (p=.204). The implantation rate was 12.3% for the lower sperm measurements also were correlated with SYBR-14 staining; r = 0.98, 0.93, 0.96 concentration and 9.9% for standard concentration (p= .564). Though these for Rl23, JC-I and MIT, respectively. These data indicate that the metabolic differences are not significant with this sample size, the trend is sufficient to activity of individual cryopreserved bovine spenncan be readily quantifiedusing warrant a larger prospective study. It would be especially interesting to evaluate a variety of mitochondral-specificfluorescent stains. (Supported by USDA grant cases where the insemination concentration is even higher than 50,000 when we 95-37203-2 186). adjust for low percentage of normal morphology by strict criteria.

P-56 We invite you to join the American Society of Andrology ! • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

Introduction

ounded in 1975, the American Society of credits (CMEs) are offered to qualified attendees of the F Andrology (ASA) is composed of clinicians and annual meeting and postgraduate course. Members basic research scientists involved with the physiology receive early notice and lower registration fees for the and pathology of male reproductive functions. The Annual Meeting and Postgraduate Course. Upcoming research interests of andrologists encompass several meeting locations include Baltimore ('97), Long Beach basic science and clinical disciplines including ('98) and Chicago ('99). biochemistry, urology, immunology, psychology, genetics, gynecology, pathology, pharmacology, internal medicine and animal science. The Society ASA On-Line: Androlog fosters a multidisciplinary approach to the study of male reproduction and sexual function. SA members have access to Androlog ...the on­ A line forum for discussing the latest issues and topics in andrology with colleagues from around the world. The Society also maintains a Home Page which can be used to find out the latest association news as Statement of Pur . ose - --- - p - well as an e-mail link to our administrative offices to request a wide variety of information. T he St>ciety functictJ,s to pr<;1t1ipte $dentific itittrchaitge atid knowledge of the male reproductive syste»� and to 11dv1:mc� the

-_. - · m_ i de__ts__ __ta1 __ _ fo-,:_:c ;i go ,,� a.ndr __ _ o l ogy t'hr___ 01g t h _ a ni iua- l ASA Newsletter - ntceti1ig,s1 pt:Jstgtadtiate 2cUt$C$- - tmd the p-,:�bUcatiQn of meritori om� stt1dies in the he ASA Newsletter is circulated 2-4 times per Jot�mtr;l Pf A.1tefrplpgy. _, T year and updates members on the many -- " activities of the Society as well as news on NIH and private funding.

Journal of Andrology Student Benefits

ublished bimonthly, ASA members receive the tudents are the future of any research field - and P Journal of Andrology, a major source of scientific S Andrology is no different. With this in mind, the reports and topical reviews covering all aspects of male ASA offers special student discounts on membership and reproduction and sexual function. The Journal continues registration fees for the high quality postgraduate to be the strongest journal in the field of andrology training programs that are offered by the society. A based upon impact factor and its contents are indexed in student newsletter is mailed to all student members in Cunenl Conlenls and Index Meuicus. A $175 value, the conjunction the ASA newsletter. Travel funds are journalis provided as a part of membership. available for qualified students who present research at the Annual Meeting. Annual Meeting and Postgraduate Course Other Membership Benefi ts

SA's Annual Meeting offers an unequaled Membership Directory .. .Includes all ASA members, A opportunity to learn about the latest advances listed and referenced by name, location, research in basic and clinical andrological research in the form interests and specialities. of symposia, lectures and research reports (oral and poster). The meeting is designed to serve the interests of Handbook of Andrology ...An 81-page soft-bound ASA's diverse membership and state-of-the-art handbook that presents the basic science and clinical lectures supported by industry attract prominent aspects of the male reproductive system. Free for speakers to accomplish this task. The annual ASA members. Andrology Laboratory Workshop, Women In Andrology Luncheon, Student Colloquium and Awards Presentation are also featured events. Continuing medical education

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Membership Application • • • • • • • • • • • • • • • • • • • • • •

MIDDLE INITIAL

'You will receive a certificate with Name and De ree

EDUCATION 1under11raduate, graduate, postdoctoral - in chronological order NSTITUTION DEGREE YEAR

SPECIALTY (circleone) : . !"!i''.ll.�:�!���::���::11:::1!1!�11. ���·1:1111.:. ::�'::·:,�if.;. :,,,,.Q:::.�t��:�� , :;qi\!! '.!��ri�;. BR Basic Research u Urology EACH APPLICATION MUSTINCLUDE E Endocrinology OG Obstetrics I Gy i:iecology •Completed form IM Internal Med1cine •Check, drawn on a US bank and payable in US dollars only to: p Pediatrics American SociehJ of A11drology for th e annual dues of:

CLINICAL PRACTICE (c ircle one) Active Student Life Member Member Member fSing!epaym ent) C Oinical Practice 0 o Clinical Practice Residents of North America $100/yr $30/yr $2,000 Residents outsideof North America $110/yr $40/yr $2,200 DISCJPLINESup fsjrde to four)

a Advanced Laboratory Diagnostic Tech. •Surcharge to receive journal of Andrology by Air Mail: {U.S. $) b Biochemistry Mexico or Canada $12 c InununolC>gy South America, Europe or North Africa $28 d IVF/ GIFT South Africa, Middle East, Australia, New Zealand or Asia $35 e Impotence f Electroejaculation •Letter from advisor for any student member, stating that he/she is a student Cellular Biology /Sperm Physiology seeking any degree or is a postdoctoral fellow, intern or resident. fi Sperm Banking Infectious Diseases •Signature of one current ASA member OR one letter of recommendation. j Infertility k Oncology I Nutrition 111 Sp erm Anatomy/Biology Signature of Sponsor II Te stis Anatomy/Biology 0 Accessory Glands p Toxicology Signature of Applicant Date q Endocrinology r Contraception MAIL TO: American Society of Andrology • 74 New Montgomery, Ste. 230 • 5 Microsurgery San Francisco. CA 94105 or FAX (415) 764-4915 t Animal SCience

· ·· ber · fii · aa t· 6 . , . Mem...... s .. , P· ". eg . .. ne.s, ' ' •ACTJV� MEMBE� ¥>Pertt� : �� q�alifi p:\lysidan o:r·$¢1entist With ;;m interes� in �h� fie19 ofandr ology, ·�LI� MeMflER �pen y.: ? : :. tu, ari a� v� metrtbe� who m�� ii\ $lngl�paytn¢n t equivale.nt to twenty (20) yeaxs of "siubeNtMEMBER . ��ijfo !�ys;���t fulP�2gt:amt�admg to a degte¢,a post-