Mouse Model of Male Germ Cell Apoptosis in Response to a Lack of Hormonal Stimulation
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Indian Journal of Experimental Biology Vol. 43, November 2005, pp. 1048-1057 Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation Ami ya P Sinha Hikim*, Yanira Vera, Rashid I Elhag, Yanhe Lue, Yu-Gui Cui , Vanisha Pope, Andrew Leun g, Vince Atienza, Christina Wan g & Ron ald S Swerdloff Di vision of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine at UCLA and Los Angeles Biomedical Research Institute, Torrance. Californi a. USA Received 5 August 2005 As a prerequisite for studies using mutant mi ce, we established a mouse model for induction of male germ ce ll apoptosis after depri vation of gonadotropins and intratesti c ul ar testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagoni st (GnRH-A), acyline, al one or in combinati on with an anti and rogen, flutamide for effective inducti on of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resul ted in almost th e same level of suppression of spermatogenesis, as judged by testi s weight and by germ cell apoptotic index, in 2 weeks as th at re ported for rats after treatment with 1.25 mg/kg BW Nai-Giu GnRH-A for the same time peri od. Within the study paradi gm, the maximum suppression of spermatogenesis occurred after a single sc injecti on of hi gh (20 mg/kg BW) dose of acyli ne with flutamide. The combined treatment resulted in complete absence of elongated spennatids. Germ cell counts at stages VII -VIII showed a signifi cant (P < 0.05) reduction in the number of preleptotene (27.1 %) and pachytene spennatocytes (8 1.9%), and ro und spennatids (96.6%) in acyline + tlutamide group in comparison with controls. In fact, treatment with a sin gle hi gh (20 mg/kg BW) dose of acyline combined with tlutamide in mice achi eved same or greater level of suppressio n, measured by germ cell counts at stages VII-Vlll, in two weeks when compared with those reported after dail y treatment with Nai-Giu GnRH-A fo r 4 weeks in rats. Both pl asma and testicular T levels were markedl y suppressed after administration of acyline alone ei th er by miniosmoti c pump or by a single sc injecti on. Addition of tlutamide to acyline had no di scernible effect on plasma or intratesticular T levels when compared with acyline al one. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is onl y possible in mi ce if total abolition of androgen action is achi eved and further emphasize the usefulness of acyline + tlutamide treated mice as a suitable model system to study hormonal regul ation of testi cular germ cell apoptosis. Keywords: Acyline, Flutamide, Germ Cell s, Apoptosis, Testi s Germ cell death has long been recognized a germ cells is an essential step towards th< 1 significant feature of mammalian spermatogenesis • In devel opment of novel therapeutic regimens to contra adult rat this loss is incurred mostly during accelerated apoptosis during abnorma spermatogonial development (up to 75%) and to a spermatogenesis, as well as more targeted approache: lesser extent during maturation divisions of to male contraception. 2 spermatocytes ar.d spermatid development . A growing body of evidence demonstrates that both Recently, using murine models of testiculru spontaneous (during normal spermatogenesis) and hyperthermia, we have demonstrated the involvemen increased germ cell death triggered by various of the mitochondria-dependent apoptotic pathway regulatory stimuli, including deprivation of characterized by Bax translocation, cytochrome < gonadotropins anrl intratesticular T by GnRH-A release, and activation of the initiator caspase 9 anc treatmene.4 or by exposure to local testicular the executioner caspases 3, 6, and 7, and poly (ADP 5 6 heating · in rats occur via apoptosis. Understanding ribose polymerase (PARP) cleavage, in heat-inducec 7 8 the molecular components of the apoptotic program in male germ cell apoptosis • . In additional studies using the gld and lprcg mice, which harbor loss-of 9 *Correspondent author: Division of Endocrinology, Harbor function mutations \n Pas L and Fas, respectively , W< UCLA Medical Center, Box 446, 1000 West Carson Street, showed that the Fas signaling system had little, if runy Torrance, California 90509. Phone: 310-222-8184: Fax: 310-533-0627 role in male germ cell death triggered by mile 10 E-mail: hikim @labiomed.org testicular hyperthermia . SINHA HIKIM e1 a/.: MALE GERM CELLS APOPTOSIS IN MICE 1049 An exciting advance in the understanding of the with the recommendation of the American Veter:.nary genetic modulation of programmed cell death is the Medical Association and were approved by the use of genetically altered mice either overexpressing Harbor-UCLA Medical Center and Los Angeles or harboring null or loss-of-function mutations of Biomedical Research In stitute animal care and use spec1"f" 1c genes II · p-. Th ese mutant animas. I wit. h review committee. additional manipulation (such as after exposure to a Blood collection and tissue preparation-Both mildly increased scrotal temperature or after hormone control and experimental animals were injected with deprivation) are invaluable tools not only for heparin (130 IU/100 g BW, ip) 15 min before a lethal confirming or refuting a proposed function of a injection of sodium pentobarbital (200 mg/kg BW, ip) particular gene in an in vivo setting, but also for to facilitate testicular perfusion using a whole body 16 uncovering novel functions for a gene that are not perfusion technique . Blood samples were collected anticipated in in vitro experiments. However, unlike from each animal by cardiac puncture immediately . l h h . d 17 8 10 d . d . test1cu ar ypert enn1a mo e · · , a equate 111 uct1on after euthanasia, and plasma was separated and stored of apoptosis was not achieved in mice after at -20°C for subsequent hormone assay. After exogenous administration of T or GnRH-A treat perfusion with saline, one testis was removed, ment 13. Thus, as a prerequisite for studies using decapsulated, weighed, snap frozen in liquid N", and mutant mice, we wish to establish a mouse model for stored at -70°C for subsequent measurement of induction of male germ cell apoptosis after intratesticular T. The contralateral testes were th en deprivati on of gonadotropins and intratesticular T. fixed by vascular perfusio n with either 5% Accordingly, in this study, we employed a new 14 glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) generation of a potent long acting GnRH-A, acyline , or Bouin's solution (Sigma Diagnostics, St. Louis, alone or in combination with an antiandrogen, 15 MO). The testes were removed and processed for tlutamide for effective induction of germ cell routine paraffin embedding for either in situ detection apoptosis. of apoptosis or immunohistochemistry. Portions of Materials and Methods glutaraldehyde-fixed testes were further diced into Allimals and experimental protocol-Seven-to small pieces, post-fixed into I % osmium tetroxide, eight-week-old male C57BL/6 mice were obtained and embedded in Epon 812 (Polysciences, from th e Jackson Laboratories (Bar Harbor, ME). Warrington, PA). Thin secti ons from selected tissue Animals were housed in a standard animal facility blocks were cut with an LKB ultramicrotome under controlled temperature (22°C) and photoperiod (Rockville, MD), stained with uranyl acetate and lead (12 hr of light, 12 hr of darkness) with food and water citrate, and examined with a Hitachi 600 electron ad libitum. Groups of 5 mice received one of the microscope (Tokyo, Japan). following treatments for two weeks: i) vehicle (sterile Viable germ cell counts- Numerical densities (Nv) distilled water); ii ) continuous delivery of GnRH-A, of Sertoli and germ cells (number per unit volume of acyline (k indly provided by Dr. Richard P. Blye, the seminiferous tubule) at stages VIJ-VIII of the Contraceptive and Reproductive Health Branch, cycle was determined by accepted stereological 6 17 NlCHHD, NJH ), at a dose of 3mg/kg BW per clay by techniques as described previousl/ · . For each testis miniosmotic pumps (Aiza Corp., Palo Alto, CA); iii ) sample, 10 round cross-sections of seminiferous continuous delivery of acyline (3 mg) + flutamide (in tubules were used. The Floclerus equation Nv = the form of sc pellets of 25 mg; lnnovati ve Research, N,)(T+D-2h) was used to calculate the Nv of germ Sarasota, FL); iv) a single sc injection of acyline (20 cell nuclei and Sertoli cell nucleoli, where NA is the mg/kg BW); and v) a single sc injection of acyline (20 number of nuclei or nucleoli counted per unit area of mg/kg BW) + tlutamicle (personal communication the seminiferous tubule profile, T is the secti on with Dr. Marvin L. Meistrich, University of Texas thickness, D is the average diameter of a given germ M.D. Anderson Cancer Center). All mice were killed cell nucleus or the Sertoli cell nucleolus, and h is th e 2 weeks after treatment. Five hypophysectomized height of the smallest recognizable nuclear or mice on the same background were purchased from nucleolar profile in the section. The nuclear profile of the Charles River Laboratories, Inc. (Wilmington, each germ cell (A 1 spermatogonia, preleptotene and MA) and were killed 2 weeks after surgery. Animal pachytene spermatocytes, and step 7 and 8 handling and experimentation were in accordance spermatids) and the number of Sertoli cell nucleoli 1050 INDIAN J EXP BIOL. NOVEMBER 2005 (thereby cells, since only one ty pical nucleolus is p 37 subunits. Testicular sectio ns were then washed present per nucleus or per cell ) in th e seminiferous three times in PBS and subsequently incubated with tubules were counted under a tOOOX magnification biotinylated goat anti-rabbit IgG secondary antibody using an oil-immersion objecti ve.