Mouse Model of Male Germ Cell Apoptosis in Response to a Lack of Hormonal Stimulation

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. The seminifero us (I : I 000) for 30 min at room temperature foll owed by tubule profile area (a) was determined by po int a 30 min incubation with ready-to-use avidin­ 2 counting using th e equation: a= p.u , where p is biotinylated horseradish peroxid ase (HRP) complex number of points per tubu lar profil e and u is th e (commercially available and prepared accord in g to di stance between two neighboring points in terms of the manufacturer's in structio ns). The pre-formed the magnification used to measure the area. The mean avidin-bi otin ylated HRP complex bi nds to the biotin d iameters of Sertoli cell nucleoli and germ cell nucle i on the secondary anti body and a lattice is formed, were obtained by direct measurements of their largest w hi ch locali zes the HRP to the areas where the cross-sectioned profiles in serial sections. The height primary antibody has specifical ly bound to its antigen of the smallest recognizable nuclear or nucleolar and visuali zed with d iaminobenzidin e tetrahydro­ profile was assumed to be one tenth of the diameter of chlori de (DAB) as per the manufactur~r's instructions the structure. The ab5-.olute number of these cells was (rabbit Un itect ABC Immunohistochemistry Detection then determined by multiplying their Nv by the fresh System; Calbiochem. San Diego. CA, USA). Slides vol ume of the testis. Cell counts were final ly were counterstained with hematoxylin . exr ,•·essed as the number of germ cells per Sertoli cell Activation of the executioner ca5-.pase 3 in germ (( m cell: Sertoli cell ratios). cel ls undergoing apoptosis was also detected by the \ssessment (~f opofJ/osis--ln sittt detection of cells confocal microscopy using double immunostaining ' ith DNA strand breaks was performed in glutar­ for caspasc 3 using rabbit polyclonal caspase 3 aldehyde-fixed. paraffi n-embedded testicular sections antibody (I: I 000, kindly provided by Dr. Annu by the terminal deoxynuclcotidyl transferase (TdT)­ Srinivasan. !dun Pharmaceuticals. San Diego. CA, mediated deoxy-UTP nick end labeling (TUNEL) USA) th at recognizes o nly the cleaved product of p18 3 7 technique · using an ApopTag-peroxidase kit and p 12 subunits of active casp:.~se 3, but not the (Chemicon International, San Francisco, CA, USA). .Inact .i ve zymogen anc I DNA f' ragmentat1o. n 7 · Ill . I n S.i tu Enumeration of the nonapoptotic Sertoli cell nuclei detecti o n of cel ls wi th DNA strand breaks was with distinct nucleoli and apoptotic germ cell popu­ performed in Bouin's fixed, paraffin -embedded lati on was caiTied out at stages VII -V III usin g an testi cul ar sections usi ng an ApopTag-flu orescein kit O lympus BH-2 mi croscop~ (New Hyde Park, NY, (Chemicon Internati onal). In brief, after USA) with a x iOO oil immersion objecti ve. For each cl eparaffini zation and rehydration, ti ssue secti ons mouse, at least I 0 tubules were used. Stages were were incubated w ith protein ase K for I 5 min at room identified according to the criteri a proposed by Russell te mperature and washed in PBS for 5 min at room 1 et a/. for paraffin sections. The rate o f germ cell apop­ temperature. Sections were incubated with a mi xture tosis or apoptotic index (AJ ) was expressed as the co ntaining di goxigenin-conjugated nucleotide and 3 6 17 number of apoptotic germ cell s per I 00 Sertol i ce ll s · . . TdT in a humidified chamber at 37°C for I hr and fm11w noh istoche11t ica I a 11d immltl w.fluo re sceJ! ce subsequently treated with antidigoxigenin-fluorescein ana/yses-Bouin's fi xed, paraffin-embedded for 30 min in the clark . After fluorescein staining, testicular sections were immunostained as described slides were washed in PBS and incubated with 8 10 17 previousJ/· · " . Briefly, testicular sections were blocking serum for 20 min to reduce non-specific deparaffinized, hydrated by successive series of antibody binding. For stain ing of caspase 3, slides ethanols, rinsed in phosphate buffered saline (PBS), were then incubated in a humidified chamber for I hr and then incubated in 2% H20 2 to quench endogenous with rabbit polyclonal active caspase 3 antibody peroxidase. Sections were then blocked with a follo~ed by goat-anti-rabbit Texas Red-labeled blocking serum containing I drop of normal goat secondary antibody for 45 min at roo m temperature. serum in I ml of PBS to suppress non specific binding Slides were washed and then mounted in ProLong of IgG and subsequently incubated with rabbit Antrfade (Molecular Probes Inc., Eugene, OR, USA). polyclonal caspase 9 (I :50; Cell Signaling For controls, sections were treated only with Technology, Beverly, MA, USA), which recognizes secondary antibody, and no signals were detected. both full length and the cleaved product of p 39 and Confocal imaging was performed usin g a Leica TCS- MENDIS-HANDAGAMA & ARIYARATNE: LEYDIG CELLS x THYROID HORMONES 951 whether the atrophic changes and malfunctional status in month old control rats. Reversibility (100%) in LH­ the aged Leydig cells are, at least in pa11, caused by the stimulated testosterone secretory capacity per testis and hypothyroid status in the aged rats. Also, it has been per Leydig cell in vitro and Leydig cell size is achieved by 163 167 163 167 demonstrated . that exogenous supplementation of the combined treatment ofT4 and LH ' . These studies thyroid hormone alone to aged Brown Norway rats (18 indicate that thyroid hormones are important in maintaining months of age) for 28 days could reverse the LH­ the steroidogenic function of Leydig cells. Representative stimulated testosterone secretory capacity per testis and Leydig cells of control rats of 3 and 19 month of age and per Leydig cell in vitro by 71 %, Leydig cell size by 82% T4-, and T4+LH- treated Brown Norway rats are shown and serum testosterone levels by 33% compared to three in Fig. 9.

A .'

•

.. ST ST

"'. 7* ~- ST

Fig. 9 - Representative light micrographs to demonstrate Leydig cells in 3, 6, 12 and 19 month old (A, B, C, and D, respectively) ami LH . (E), T4. (F) and, LH+ T4.treated (G) Brown Norway rats. Aging from 3 to 19 months causes atrophy of Leydig cells. The reduced steroidogenic potential of these aged Leydig cells, in vitro was partially recovered, (partial rejuvenation) by exogenous treatment of either LH (E) or T4 (F) and fully recovered (100% rejuvenation) by LH+ T4 [Pennission taken from the publisher, Bioi Reprod, 66 (2002) 1359] 1052 TNDJAN J EXP BJOL, NOVEMBER 2005 comparatively large cytoplasmic-to-nuclear volume in various treatment groups is summarized in Table 2. ratio. Mitochondria, lipid droplets, and smooth Spontaneous apoptosis of germ cells, differentiatin g endoplasmic reticula, characteristic of active Leydig spermatogonia and dividing spermatocytes in cells, were abundant. The Leydig cells from acyline particular were readily observed in the control mice. treated mice were markedly reduced in size and Administration of acyline alone either by miniosmotic exhibited irregular nuclei with more heterochromatin pump at a dose of 3 mg/kg BW per day or by a single and scanty amount of cytoplasm (Fig. lB) as sc injection at a dose of 20 mg/kg BW for 2 weeks compared with that of control animals (Fig. lA). resulted in a significant ( P < 0.05) increase in the Mitochondria, lipid droplets, and smooth endoplasmic number of apoptotic germ cells involving pachytene reticula were markedly less in acyline-treated Leydig spermatocytes and step 7 or 8 spermatids at stages cells (Fig. I B) as compared with that of control mice VII-VIII; such apoptotic cells are occasionally (Fig. lA). observed in control mice (Table 2). A further increase Tubule morplwlogy and germ cell apoptosis­ (2.1-fold) in the number apoptotic germ cells, Histoiogical examination of the seminiferous tubules compared to acyline alone, was noted by a combined in various groups of acyline with or without flutamide treatment of continuous release of acyline and treated mice for 2 weeks showed maximum involution flutamide (Fig. 3 and Table 2) and was not of ~.; permatogenesis after a combined treatment of a significantly different from values measured atter si ngle sc injection of high dose (20 mg/kg) of acyline hypophysectomy. However, a combination of a single ar; , flutamide. The seminiferous tubules were smaller high (20 mg/kg BW) dose of acyline with flutamide In diameter and exhibited fewer pachytene resulted in no further increase in the incidence of spermatocytes and round · spermatids and complete germ cell apoptosis compared to acyline alone as absence of elongated spermatids (Fig. 2). most of the apoptotic cells had been lost through In situ detection of germ cells undergoing apoptosis phagocytosis by the Sertoli cells (Table 2). was performed by a modified TUNEL assay (Fig 3). Since the principal intracellular effectors of Quantitative analysis in the incidence of germ cell apoptosis are caspases, we further examined the apoptosis (expressed as numbers per 100 Sertoli cells) activation of the initiator caspase 9 and the

Fig. 2- Representative light micrographs of testis sections from control (A) and mice after 2 weeks of treatment with a single sc injection of high dose (20 mg/kg BW) acyline and flutamide (B). Note a marked suppression of spermatogenesis after acyline + flutamide treatment. Magnification x 200. SINHA HlKTM eta/.: MALE GERM CELLS APOPTOSTS IN MICE 1053 executioner caspase 3 in hormone deprivation­ was activated in those germ cells undergoing induced germ cell apoptosis 111 mtce. apoptosis after acyline with or without flutamide Immunocytochemical analysis revealed a cell type­ treatment as evidenced by immunofluorescence specific increase in caspase 9 immunoreactivity in the staining of active caspase 3 (Fig. 5). susceptible germ cells, including those undergoing Electron microscopic observations of testicular apoptosis after acyline treatment (Fig. 4). Activation materials further confirmed the identity of various of the executioner caspase 3 in germ cells undergoing apoptotic germ cells characterized by TUNEL as say apoptosis was also detected by confocal microscopy (Figs 6 A-D). Clusters of step 16 spermatids, some of using double immunostaining of active caspase 3 which also underwent apoptosis, were frequently (red) and DNA fragmentation (green). The caspase 3 observed deep within the seminiferous epithelium next to the limiting membrane of the tubule (Fig. 6 Table 2-Apoptoti c index (ex pressed as number per 100 Serto li cells) in mice after 2 weeks of treatment with vehicle, C). Some of these apoptotic germ cells were in th e acy line, alone or in combination with Outamide, or process of being phagocytosed by the Sertoli cells hypophysectomy (Fig. 6 D). .[Values are mean± SEM] Viable germ cell counts-The effectiveness of a Group Apoptotic Index combined treatment of a single high (20 mg/kg BW) dose of acyline with flutamide on spermatogenesis Vehicle 3.1 ± 2.29" was further assessed by enumeration of various germ Acyline (3mg/kg-pump) 68.8 ± 13.8c cells supported by individual Sertoli cells at stages Acyline (3 mg/kg-pump + Outamide) 146.0 ± 24.9d Acyline (20 mg/kg-sc) 32.1 ± 5.1 b VII-VTII of the seminiferous epithelial cycle. Data Acyline (20 mg/kg-sc + Outamide) 25.9 ± 6.6" reported in Table 3 revealed a significant (P < 0.05) Hypophysectomy I 33.3 ± 23.3 t1 reduction in the number of preleptotene (27 .1 %) and Means with unlike superscripts are significantly (P <0.05) pachytene spermatocytes (81.9% ), and round different. spermatids (96.6%) in acyline + flutamide group in

Fig. 3--In situ 3' end- labeling of DNA strand breaks in apoplotic genn cells at stage Vll from a mouse treated with continuous release of acyline at a dose of 3 mg/kg BW/day and flutamide for 2 weeks by TUNEL assay. Methyi green was used as a counterstain. Compared with the control (A), where no germ cell apoptosis is detected, combined treatment with acyline and Outamide causes a marked increase in the number apoptotic germ cells (B). Magnification: x 600. 1054 LNDIAN J EXP BIOL, NOVEMBER 2005 companson with controls. This is consistent with T able 3-Serto li cell number and germ ceii-Sertoli cell rati os previous data showing no further increase in the at stages VII-Vlll in mice after 2 weeks of treatment with incidence · of germ cell apoptosis in this group acyline combined with tlutamide [Values are mean ± SEM.] compared to acyline alone as most of the apoptotic cells had been lost through phagocytosis by the Cell types Control Acyline (20 Sertoli cells (Table 2). mg/kg-sc + tlutamide)

Discussion Sertoli cell s (xl06/testi s) 1.39 ± 0.12 1.1 8 ± 0. 27 Gonadotropin-releasing hormone antagonists Germ ceii-Sertoli cell ratios (GnRH-As) are synthetic analogs of GnRH th at Spermatogonia 0. 10 ± 0.02 0.12±0.01 through competitive blockade of pituitary GnRH Preleptote ne spermatocytes 2.29 ± 0.09 1.67±0.12' 18 0.51±0.18* receptors inhibit the secretion of LH and FSH . Pachytene spermatocytes 2.82 ± 0.27 0.28 ± 0.1 2* Numerous studies have demonstrated the ability Round spermatids 8. 16± 0. 3 1 of these antagonistic analogs of GnRH to suppress *Significantly lower than controls

Fig. 4----Immunocytochemical analysis of in vivo changes in caspase 9 expression at stage VII in mice treated with contin uous release of acyline at a dose of 3 mg/kg BW for 2 weeks. An affinity purified caspase 9 antibody, which detects both th e inactive zymogen and the cleaved products p 38 and p 17 of active caspase (Cell Signalin g Technology, Beverly) was used. In control mice, a strong cytosolic caspase 9 immunostaining is found in the Sertoli cells, while weak to moderate is detected in germ cells (A). Note an appreciable increase in caspase 9 im munoreactivity in the susceptible germ cell s at stage Vll after acyline treatment (B). Magnification: x 800.

Fig. 5--Activation of caspase 3 in mouse testicular germ cells undergoing apoptosis after administration of continuous release of acyl inc (3 mg/kg BW/clay) + tlutamide for 2 weeks. Confocal images of germ cells at stage VII from mouse treated w ith acyline for 2 weeks show TUNEL (green) and active caspase 3 (red). In the merged panels, green corresponds to TUNEL, and reel to active caspase 3; co­ localization between TUNEL and active caspase 3 in the nucle us is detectable as yellow. Magnifications:x 1000. SINHA HI KIM eta/.: MALE GERM CELLS APOPTOSIS IN MICE lOSS

Fig. 6--Electron mi crographs of portions o r stage VII tubules from a mouse treated with continuous release of acyline (3 mg/kg BW) for 2 weeks showi ng an apoptotic pachytene spermatocyte (A), a step 7 spennatid, which can still be recognized by a relatively less dense acrosome region (B), and several apoptotic step 16 spennatids (C) deep within the seminiferous epithelium next to the limiting membran.: of th e tubule. Some of these apoptoti c cells arc in the process of being phagocytosed by th e Sertoli cell (D). Magnificati on: x 8.000. circulating levels of FSH, LH, and T and, as a add flutamide to the treatment of continuous rel ease consequence, testicular function in a variety of animal of acyline to achieve the same level of suppression of 16 19 20 22 23 24 models including, rat . . , monke/ 1. , and man · . spermatogenesis, as judged by testis weight and by GnRH-A-treated rats have long been used as a activation of germ cell apoptosis, in 2 weeks as th at suitab!t: model for studying the underlying reported for rats after daily treatment with I .2S mg/kg 4 1 mechanism of germ cell death after hormone BW Nal-Giu GnRH-A for the same time period · ('. 3 depri vation .4. In contrast to rats, mice are resistant to Importantly, the observed suppression of spermato­ . . d . . . . 1125 26 G n RH -A w h en a d mmtstere vta sc 111J ectton · · · . genesis was identical to that of hypophysectomy. This Accordingly, GnRH-A was administered to mice by finding is in agreement with that of our previous Alzet miniosmotic pumps. In th e rat treatment with report on the rat, which showed that the effects of Nal-Glu GnRH-A alone at a dose of 1.2S mg/kg BW Nal-Glu GnRH-A treatment, at least at given doses daily for 2 weeks decreased testicular weight to about and time point, were identical to those of 16 6 S6.8% of the control value . In the mouse even with hypophysectom/ . Of further importance, we found continuous administration of a more potent GnRH-A even a greater suppression of spermatogenesis after a (acyline) for the same time period reduced testicular combined treatment of a single higl: dose (20 mg/kg weight to only 79.6% of the control value. We had to · BW) of acyline with tlutamide for 2 weeks. In fact, 1056 INDIAN J EXP IJI OL, NOVEMBER 2005 treatment with a single high dose of acyline combined Acknowledgement with flutamide in mice achieved the same or g reater This work is supported by a grant from the Nati.:nal level of suppression, measured by germ cell counts at Institute of Health (ROI HD 39293) to the author stages VII-Vlll, in 2 weeks when compared with (APSH) Miss Vera is supported by Research those reported after treatment with Nai-Giu GnRH-A Supplements for the Underrepresented Minority 16 ( 1.25 mg/kg BW) daily for 4 weeks in rats . The Program of the National In stitute of Health (HD results of the present study thus confirm and extend 39293-02 S I ). Mr. Elhag and Mi ss Pope are earli er reports on rats by demonstrating a rapid supported through the IMSD (GM 56902) and RISE suppression of spermatogenesis through increased (GM 62252) programs, respectively, by the National germ cells apoptosis after gonadotropin derivation by In stitute of Health. a potent GnRH-A treatment. References The optimum suppression of spermatogenesis is I Ru ssell L D, Ettlin R A, Sinha Hik im A P & Clegg E D. Histolor; ical and Histopat/wlogical Evahtcllion of the Testis most likely attributed to a decrease in intratesticular T (Cache River Press, Clearwater, Florida) 1990. levels induced by the GnRH-A, acylin e, and 2 Huckins C, The morphol ogy and kineti cs of spermatogoni al . h"b". f d . b fl "d 15ry027 111 1 1t1on o an rogen act1on y utamt e ·- · . degeneration in normal ad ult rats: An an alysis usin g a Flutamide alone has been reported to increase serum simplified classifi cati on of the ge rminal epithelium, Anal gonadotropin and T levels by blocking the androgen­ Rec, 190 ( 1978) 905. dependent feed back inhibition of GnRH pulse as well 3 Sinha Hikim A P, Wang C. Leung A & SwerdlofT R S. In vo lvemelll of apoptosis in the in duc ti on of germ cell as pitUitary. . LH secreti. o nryo- ·-?S . H owevcr, a t. ter degenerati on in adult rat s after go nadotropin-releasi ng inhibition of the gonadotropin and T secretion with hormone antagoni st treatment, Endocrinology, 136 ( 1995) acyline, the addition of flutamide produced no such 2770. increases in gonadotropin and T levels. The present 4 Sinha Hikim A P. Rajavas hi sth T B, Sinha Hikim I. Luc Y, findings of marked suppression of spermatogenesis Bonavera J J, Leun g A, Wang C & Swerdloff R S. Si gnifi can ce of apoptosis in the temporal and stage-spec ifi c o nly after combined treatment with acyline and loss of germ ce ll s in th e ad ult rat after gonadotropin flutamide suggest that unlike rats a total abolition of dcpri vation, Bioi Rep rod, 57 ( 1997) I 193. testicular androgen action is required for effective S Lue Y H, Sinha Hi kim A P, Swerdloff R S, lm P, Taing K S, suppression of spermatogenesis in mjce. Consiste nt Bui T, Leung A & Wang C, Sin gle exposure to heat induces with this is the demonstration that luteinizing stage-specifi c germ cell apoptosis in rat s: Role of intratesti cular testosterone (T) on stage specifi city, hormone receptor knockout (LuRKO) mice at th e age Endocrinology. 140 ( 1999) 1709. of 12 months show completion of qualitatively normal 6 Lu e Y H, Sinha Hi kim A P, Wang C, lm M, Leung A & spermatogenesis even in th e presence of dramatically Swcrdloff R S, Testi cu lar heat exposure enhances the reduced (2% of control level) intratesticular T levels suppression of spermatogenesis by testosterone in rat s: The due to lack of LH s timula t i on"'~. However, the "two-hit" approach to mal e co ntr :~cep ti vc development, completion of spermatogenesis up to the elo ngated Endocrinology. 14 1 (2000) 14 14. 7 Sinha Hikim A P, Luc Y. Yamamoto C M, Vera Y. spermatids of late steps 13- 16 was blocked by 29 Rodriguez S. Yen P H. Soeng K. Wang C. Swerdl off R S. flutamide , indicating that very low endoge nous T Key apoptotic pathways for heat-i nduced programmed germ levels that is produced without LH stimulation is cell death in th e testi s, Endocrinology. 144 (2003) 3167. sufficient to maintain spermatogenesis in mice. 8 Sinha Hikim A P, Luc Y. Diaz- Romero M. Yen P H, Wang Therefore, optimum suppression of spermatogenesis C & Swerdloff R S, Deciphering the pathways of ge rm cell apoptosis in the testi s, J Steroid Mol Bioi, 85 (2003) 175. through increased germ cell death is o nly possible in 9 Nagata S & Gol stei n P. The Fas death factor, Science, 267 mice if total abolition of androgen action is achieved. (1995) 1449. In summary, th e present study further emphasizes 10 Vera Y, Diaz- Romero M. Rodri guez S, Lue Y, Wang C, the usefulness of GnRH-A + tlutamide treated mice as Swerdloff R S & Sinha Hi kim A P, Mitochondria- dependent a suitable model system to study hormonal regulation pathway is involved in heat-induced male germ cell dea th: of testicular germ cell death m m1ce. This lessons from mutant mice, Bioi Rep rod, 70 (2004) I 534. pharmacological model with additional manipulati on II Sinha Hikim A P & Swerdloff R S, Hormonal and genetic control of germ cell apoptosis in th e tes tis, Rev Reprod, 4 (such as using transgenic or gene-ablated mice) can ( 1999) 38. be used to further study the genetic regulation of 12 Ranger AM, Malynn B A & Korsmey::r S J, Mouse mod els programmed germ cell death. of cell death, Nature Genet, 28 (200 I) 113. SINHA HIKIM eta/.: MALE GERM CELLS APOPTOSIS IN MICE 1057

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