Sensitivity of Testicular Germ Cells to Toxicant-InducedApoptosis in gld Mice That Express a NonfunctionalForm of Fas Ligand*

JOHN H. RICHBURG, ADRIAN NANEZ, LISA R. WILLIAMS,MICHELLE E. EMBREE, AND KIM BOEKELHEIDE

Division of Pharmacology and Toxicology, University of Texas College of Pharmacy (J.H.R., A.N.),Austin, Texas 78712-1074; and Department of Pathology and Laboratory Medicine, Brown University(L.W., M.E., K.B.), Providence, Rhode Island 02912

ABSTRACTGerm cell apoptosis in testis is essential for functional spermato-

genesis. Recent evidence suggests that the Fas signaling system iscritical for the regulation of testicular germ cell apoptosis. To furtherevaluate the Fas signaling system in testis, we examined the inci-dence of germ cell apoptosis in gld mice that lack a functional Fas-signaling pathway. gld mice have a small, but significant, increase intestis weight and numbers of spermatid heads per testis comparedwith wild-type mice. In addition, gld mice have a small increase in thespontaneous incidence of germ cell apoptosis, as indicated by char-acteristic DNA fragmentation via the terminal deoxxynucleotidyl-transferase-mediated deoxy-UTP nick end labeling assay. To test therole of the Fas system in toxicant-induced germ cell apoptosis, mice

were exposed to either a Sertoli cell- or germ cell-specific toxicant[mono-(2-ethylhexyl)phthalate (MEHP; 1 g/kg) or 5 Gy radiation,respectively]. These two exposure paradigms induced extensive in-creases in germ cell apoptosis in wild-type mice. However, exposureof gld mice to MEHP caused only a minimal increase in germ cellapoptosis, whereas they were as sensitive as wild-type mice to radi-ation exposure. These data indicate that the Fas signaling pathwayis 1) involved in regulating the numbers of germ cells in the testis, 2)crucial for the initiation of germ cell apoptosis after MEHP-inducedSertoli cell injury, and 3) differentially active in the cell-specific reg-ulation of germ cell apoptosis that occurs as a consequence of Sertolicell vs. germ cell injury. (Endocrinology 141: 787–793, 2000)

APOPTOSIS OF testicular germ cells is critical for sper-matogenesis in mammals. Elimination of testicular

germ cells serves as a mechanism to limit their clonal ex-pansion to numbers of cells that can be supported by thesustentacular cells of the testis, the Sertoli cells (1–5). In-creases in germ cell apoptosis are often observed after ex-posure of laboratory animals to various testicular toxicants(3, 5–9). Increases in germ cell apoptosis have also beenreported in human testis after testicular injury or certaindisease conditions (10–12).

The mechanisms controlling spontaneous physiological ortoxicant-induced testicular germ cell apoptosis are currentlythe focus of many investigations (1, 4, 5, 7, 8, 13). Recentevidence indicates that the Fas/Apo-1/CD-95-mediated sig-naling system participates in the regulation germ cell apo-ptosis in rodent models (4, 14, 15) as well as in human testis(16). The Fas system is a receptor-ligand signaling system inwhich Fas ligand (FasL) binds to and activates the Fas re-ceptor (Fas) to initiate a cascade of intracellular events thatleads to the elimination of the Fas-bearing cells via apoptosis(for a recent review, see Ref. 17). In the rodent testis, FasL isfound constituitively expressed on Sertoli cells, and Fas is

localized on select germ cells (4). Experimentally inducedinhibition of the expression of Sertoli cell FasL in rat Sertolicell germ cell cocultures results in a significant reduction inthe incidence of germ cell apoptosis that normally occurswith time in culture (4). Furthermore, addition of the Fasactivating JO-2 antibody to mouse germ cells in vitro resultsin a significant increase in germ cell apoptosis (4). In additionto these rodent studies, it has recently been reported that bothFas and FasL are present in human testis, and disruption oftheir function leads to protection against germ cell apoptosis(16).

In the present study we use the gld (for generalized lympho-proliferative disease) mutant mice to evaluate the functionalimportance of the Fas system in the regulation of germ cellapoptosis in the testis. The gld mice have a point mutation in theintracellular C-terminus of FasL. This mutation abolishes theability of FasL to bind Fas and initiate the apoptotic pathwaywithin the cell (21, 22). These mice display diseases resultingfrom systemic autoimmunity and lymphadenopathy, indicat-ing the importance of the Fas system in the function of theimmune system (18, 20). In addition, these mice are fertile anddisplay apparently normal spermatogenesis. One aim of thepresent study was to evaluate the spontaneous rate of germ cellapoptosis in gld mice to gain insights into the role of the Fassystem in the regulation of spermatogenesis.

To further characterize the involvement of the Fas systemin germ cell apoptosis, we examined the response of gld miceto two cell-specific toxicants. Exposure to mono-(2-ethyl-hexyl)phthalate (MEHP) specifically inhibits Sertoli cellfunction, leading to germ cell apoptosis (for reviews, see

Received July 29, 1999.Address all correspondence and requests for reprints to: John H.

Richburg, Ph.D., Division of Pharmacology and Toxicology, Universityof Texas College of Pharmacy, Austin, Texas 78712-1074. E-mail:john_richburg@mail.utexas.edu.

* This work was supported in part by grants from the NIEHS, NIH(ES-09145, to J.H.R.; ES-05033, to K.B.), the Burroughs Wellcome Fund,and NIH Center Grant ES-07784.

0013-7227/00/$03.00/0 Vol. 141, No. 2Endocrinology Printed in U.S.A.Copyright © 2000 by The Endocrine Society

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Refs. 23–25), and irradiation of testis specifically targets thedifferentiating germ cells (9). These two model systems havebeen shown to cause increases in both germ cell apoptosis aswell as in the expression of either Fas or FasL (4, 7, 14, 15).The results of the present study implicate the Fas signalingsystem in the regulation of spontaneous germ cell apoptosisas well as increases in germ cell apoptosis after toxicant-induced Sertoli cell injury. However, the failure to see dif-ferences between gld and wild-type mice in the incidence ofapoptosis after irradiation suggests that the Fas system par-ticipates in differentially triggering germ cell apoptosis afterSertoli cell vs. germ cell injury.

Materials and MethodsAnimals

Adult 8-week-old male and 21-day-old wild-type C57BL/6 (B6) andB6.SMNC3H-Fasgld,gld (B6-gld, gld) mice (The Jackson Laboratory, BarHarbor, ME) were given water and standard lab chow ad libitum. An-imals were allowed to acclimatize for at least 1 week before experiments.The animal room climate was kept at a constant temperature (74 6 2 F)at 30–70% humidity with a 12-h alternating light-dark cycle. All pro-cedures involving animals were performed in accordance with theguidelines of either the University of Texas-Austin’s institutional animalcare and use committee or Brown University’s institutional animal careand use committee in compliance with the guidelines established by theNIH.

MEHP exposure protocol

MEHP was purchased from TCI America (Portland, OR) and certifiedto be more than 94% pure by gas chromatography. In these experiments,prepubertal 28-day-old mice were used. Young rodents are more sen-sitive to the effects of the MEHP (26, 27) and show a robust increase ingerm cell apoptosis after MEHP exposure (7). Mice received a single dose(1 g/kg) of MEHP in corn oil by gavage at a volume equal to 4 ml/kg.Control mice received a similar volume of corn oil vehicle. The primarycellular site of MEHP toxicity is the Sertoli cell, and this exposure modelhas been extensively employed in the investigation of testicular injurythat results from the disruption of Sertoli cell function (for review, seeRef. 23). Control and MEHP-treated mice were killed by CO2 asphyx-iation after 0, 3, 6, 12, and 24 h, and the testes were quickly removed. Onetestis was immersed in Tissue-Tek OCT embedding medium (MilesLaboratories, Inc., Elkhart, IN) and quickly frozen via submersion inliquid nitrogen and then stored at 280 C until analyzed. The other testiswas submerged and stored in neutral buffered formalin after gentlyperforating the tunica with a 27.5-gauge needle.

Radiation exposure

Adult 8-week-old wild-type C57 or gld mice were used for experi-ments. Mice were given half-body irradiation to a single dose of 5 Gywithout anesthesia at a dose rate of 98.5 rads/min using a Philips250kVp x-ray machine. Mice were restrained in polystyrene chambers,and the upper body was shielded using lead. Mice were killed by carbondioxide asphyxiation 12 h after irradiation, and the testes were excised.This treatment protocol has been previously been shown to stimulatemassive apoptosis of spermatogonia and spermatocyte germ cells (9).

In situ terminal deoxxynucleotidyltransferase-mediateddeoxy-UTP nick end labeling assay (TUNEL) staining andquantitation

Germ cell apoptosis was detected in 8-mm cryosections of fresh-frozen testis by the TUNEL labeling method using the ApopTag kit(Intergen, Purchase, NY). Tissue was counterstained with methyl green.Tissues sections were viewed using a Nikon E800 microscope (Melville,NY) using differential interference contrast microscopy. The imageswere captured with a Kodak DC120 digital camera equipped with aMDS120 adapter (Eastman Kodak Co., Rochester, NY) and processedusing Adobe Photoshop 5.0 software (Adobe, San Jose CA). TUNEL-positive germ cells were quantitated in each tissue section by countingthe number of TUNEL-positive cells in each essentially round seminif-erous tubule. For each testis section, approximately 100–200 tubuleswere counted from each of three different mice. The incidence of apo-ptosis was then categorized into either of two groups, defined as zeroto three or more than three TUNEL-positive germ cells per seminiferoustubule cross-section. In the control mouse testis, the percentage of sem-iniferous tubules with more than three TUNEL-positive cells is less than5%, so that an increase in apoptosis is easily determined using this datapresentation. The data, calculated as a percentage of the total, are ex-pressed as the mean 6 sem.

Testicular sperm head counts

Testicular sperm head numbers were assessed by the procedure ofBlazak et al. to evaluate the numbers of mature elongate spermatids inthe testis (28). Briefly, testes were homogenized in an 8-ml solution of0.9% NaCl and 0.05% Triton X-100, and sperm heads were counted usinga hemocytometer. Each sample was counted four times and averaged.

Histopathology

To examine the morphological appearance of the seminiferous tu-bules after MEHP treatment, testes were fixed in 10% neutral bufferedformalin and embedded in glycol methacrylate using a Historesin em-bedding kit (Reichert-Jung, Heidelberg, Germany). Sections (2 mm) werestained with periodic acid-Schiff reagent and hematoxylin.

Statistics

Significance between groups (P , 0.05) was determined by singlefactor ANOVA with Fisher’s least significant differences test comparisonusing StatView software (SAS Institute, Inc., Cary, NC).

ResultsAssessment of gld vs. wild-type testicular parameters

To initially characterize the testis of the gld mice, bodyweights, testis weights, testicular spermatid head counts,and incidence of germ cell apoptosis were compared withthose observed in wild-type mice (Table 1). A significantdifference in both the body and testis weights of adult gldmice was observed compared with that in wild-type controls.The gld mice had an increase in both body and testis weightsof approximately 10%. Measurement of testicular spermhead counts in the testis, a useful indicator of differences in

TABLE 1. Comparison of wild-type and gld mice baseline testicular parameters

Mouse strain Body weight (g) Testis wt (g) Sperm head/testis (3106)

% of seminiferous tubules with the no. ofTUNEL-positive germ cells indicated

0–3 .3

C57 22.59 6 0.38 0.090 6 0.001 19.41 6 0.46 96.71 6 0.93 3.29 6 0.93gld 25.93 6 0.63a 0.095 6 0.002a 22.71 6 0.78a 92.76 6 1.27a 7.24 6 1.27a

Values represent the mean 6 SEM determined from 17 C57 mice and 15 gld mice.a P , 0.05, by ANOVA with Fisher’s protected least significant differences test.

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the size of the germ cell population in the testis (28), alsorevealed an approximately 10% increase in the number ofspermatid heads per testis in the gld mice compared withwild-type animals.

The incidence of germ cell apoptosis was determined bythe detection of DNA fragmentation via the in situ TUNELtechnique. To compare the incidence of germ cell apoptosisbetween gld and wild-type mice, the percentage of seminif-erous tubules displaying either zero to three or more thanthree TUNEL-positive cells was determined (Table 1). Sig-nificant differences between the gld and wild-type mice in theincidence of TUNEL-positive germ cells were evident. Thegld mice showed a significant increase in the percentage ofseminiferous tubules with greater than three TUNEL-posi-tive cells (7.24%) relative to wild-type mice (3.29%).

MEHP-induced histopathology in wild-type and gld mice

Histopathology was evaluated in 2-mm cross sections ofplastic-embedded testis stained with periodic acid-Schiff re-agent and hematoxylin. The seminiferous epithelium ap-peared similar in untreated wild-type and gld mice (Fig. 1, Aand D, respectively). At 12 h (Fig. 1, B and E) and continuingto 24 h (Fig. 1, C and F) after MEHP exposure, a similarincrease in the incidence of characteristic features of MEHP-induced Sertoli cell injury (germ cell sloughing into the lu-men, enlarged lumen size, and increases in the incidence ofSertoli cell vacuoles) was evident in both wild-type and gldmice, respectively. However, at higher magnification, germcells from wild-type mice, but not gld mice, display the char-acteristic apoptotic morphology of cell shrinkage and chro-matin condensation after MEHP exposure (data not shown,see Fig. 2).

MEHP-induced germ cell apoptosis

To evaluate the extent of MEHP-induced germ cell apo-ptosis, apoptosis in the testes of 28-day-old treated gld micewas compared that in testes of similarly treated wild-typemice. In wild-type mice a time-dependent increase inTUNEL-positive germ cells was observed after MEHP ex-posure (Fig 2, A–D). This increase in apoptosis resulted in amarked reduction in the number of cells evident in the sem-iniferous epithelium by the 24 h point (Fig. 2D). In gld mice,exposure to MEHP did not result in an increase in the inci-dence of TUNEL-positive germ cells (Fig. 2, F–H). The testiscross-sections from the gld mice 24 h after MEHP exposureshowed no apparent decline in the numbers of cells in theseminiferous epithelium (Fig. 2H).

To quantitatively evaluate the amount of germ cell apo-ptosis above baseline induced by MEHP treatment, the dif-ference in the percentages of seminiferous tubules displayinggreater than three apoptotic cells per seminiferous tubulewere compared (Fig. 3). After MEHP treatment of wild-typemice, a time-dependent increase in TUNEL-positive germcells was evident. A sharp increase in apoptosis occurredbetween 3–12 h after MEHP exposure and leveled off by 24 hafter exposure. In MEHP-treated gld mice, only a small in-crease in TUNEL-positive cells was observed (Fig. 3). A smallincrease in germ cell apoptosis occurred between 3–12 h,

although the peak incidence of apoptosis at 12 h was ap-proximately 50% less than that in the wild-type mice.

Radiation-induced germ cell apoptosis

Radiation exposure results in a direct injury of the differ-entiating germ cells. Exposure of mice to 5 Gy of radiationinduces a massive increase in the incidence of apoptosis inboth spermatogonia and spermatocytes 12 h after irradiation(9). Therefore, radiation was used to test the apoptotic re-sponse of gld testicular germ cells to direct injury. A similarmassive increase in the incidence of germ cell apoptosis wasobserved in both wild-type and gld mice after exposure to 5Gy of ionizing radiation (Table 2). Approximately 50% of theseminiferous tubules in wild-type and gld mice had morethan three TUNEL-positive cells compared with approxi-mately 5% in untreated mice (Table 1).

Discussion

The Fas signaling system in the testis has been reported byseveral investigators to play an important role in regulatinggerm cell apoptosis in the testis (4, 14–16, 29–31). We havepreviously demonstrated that Sertoli cells constituitively ex-press FasL, whereas select germ cells express Fas (4). Thislocalization has led us to hypothesize that Sertoli cells, viatheir expression of FasL, regulate the numbers of germ cellsby eliminating Fas-positive cells. It is further hypothesizedthat Sertoli cells use the Fas signaling system as a paracrinesignaling mechanism to reduce the population of germ cellsto a level that they can support (4, 14). Therefore, based onour previous findings, we would predict that gld mice woulddisplay a testicular germ cell hyperplasia and abnormal sper-matogenesis. However, gld mice are fertile and display ap-parently normal spermatogenesis. In fact, evaluation of thehistopathology of the seminiferous epithelium does not re-veal any significant differences between the wild-type andgld mice (Fig 1, A and D, respectively). These findings sug-gest that either the mutant gld mice have adapted to theFasL-deficient system, perhaps by using a parallel apoptosisregulatory pathway, or that the Fas signaling system is notrequired for spermatogenesis.

The observations of this study that gld mice have signif-icantly increased testicular weights and testicular spermatidheads per testis indicate that the numbers of germ cells in thegld testis are maintained at a higher baseline level than inwild-type animals. However, the low absolute expansion ofthe germ cell population (;10%) suggests that the number ofthe germ cells in the testis is tightly limited. The observationof an increased incidence of baseline apoptosis in gld miceimplies that apoptosis is serving as a mechanism to modulatean expanded population of the germ cells in these mice.These findings suggest that germ cell apoptosis in the gldtestis is triggered by an alternate mechanism or that the Fassystem in testis is only involved in triggering apoptosis of asmall subpopulation of germ cells in the testis.

The decreased sensitivity of gld mice to MEHP-inducedgerm cell apoptosis provides strong evidence that the Fassignaling system plays a direct role in initiating germ cellapoptosis after Sertoli cell injury. The Sertoli cells are widelyrecognized to maintain the viability of the germ cells by

gld MICE AND GERM CELL APOPTOSIS 789

providing appropriate nutritional, hormonal, and physicalsupport. Therefore, injury to Sertoli cells could compromisetheir supportive capacity. A potential explanation for the

decreased sensitivity of germ cells to apoptosis after MEHPexposure could be that Sertoli cells of gld mice are insensitiveto MEHP-induced injury. The similar MEHP-induced

FIG. 1. Progressive stages of MEHP-induced testicular histopathology. Wild-type (A–C) or gld mice (D–F) were exposed to MEHP (1 g/kg), andtestis were collected after 0 (A, D), 12 (B and E), or 24 (C, F) h and processed for histopathological analyses as described in Materials and Methods.Note the similar MEHP-induced increases in seminiferous tubule lumen size, appearance of Sertoli cell vacuoles (arrows), and presence ofsloughed germ cells in the lumen of both wild-type and gld mice after MEHP exposure. Bar, 300 mm.

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changes in testicular histopathology in wild-type and gldmice shown in this report argue that the Sertoli cells of thegld mice are not protected from MEHP-induced injury. Wehave previously reported increases in the expression of bothFas and FasL in young rats and mice after MEHP exposure(4, 14, 15). The protection of young gld mice against MEHP-induced apoptosis further argues for the participation of theFas system in triggering germ cell apoptosis after Sertoli cellinjury. These data also indicate that the injured Sertoli cellsthemselves, via the expression of FasL, are actively involved

in eliminating germ cells. We hypothesize that the reductionin the number of germ cells via apoptosis occurs as a mech-anism to match their numbers to the reduced supportivecapacity of the injured Sertoli cells and preserve functionalspermatogenesis by the remaining germ cells.

To further examine the role of the Fas system in germ cellapoptosis after toxicant-induced injury, we used a radiationexposure model that directly injures mitotically active sper-matogonia and spermatocytes (9). Increases in Fas, but notFasL, messenger RNA (mRNA) in rats and mice have been

FIG. 2. Protection against MEHP-in-duced increases in germ cell apoptosisin gld mice. DNA fragmentation, a char-acteristic feature of apoptosis, was de-tected in testis cryosections via theTUNEL technique. Wild-type (A–D) orgld mice (E–H) were exposed to MEHP(1 g/kg), and testis were collected after0 (A and E), 6 (B and F), 12 (C and G),or 24 (D and H) h and processed forTUNEL analysis as described in Mate-rials and Methods. Arrows indicateTUNEL-positive cells. The TUNEL-stained sections were counterstainedwith methyl green and viewed using dif-ferential interference contrast micros-copy. Bar, 100 mm.

gld MICE AND GERM CELL APOPTOSIS 791

observed 6 h after irradiation with 5 Gy (14), a time point justbefore the observation of large increases in apoptosis (14, 32).Although these data suggest the participation of Fas in ra-diation-induced germ cell apoptosis, no differences in themassive radiation-induced increases in germ cell apoptosiswere observed between wild-type and gld mice. The exper-iments presented in this report exposed adult mice to radi-ation. However, preliminary experiments in our laboratoryirradiating young gld mice show the same massive increasein germ cell apoptosis as that in wild-type mice (unpublishedobservations). Direct damage to germ cells elicited by radi-ation may activate an autocrine pathway within the germ cellthat is Fas independent. The reason for the up-regulation ofFas mRNA after irradiation and the role that it may play inelimination of germ cells is uncertain. However, an increasein expression of Fas mRNA in germ cells occurs after expo-sure to a variety of toxicants whose primary targets areSertoli cells or germ cells (14). The expression of Fas by thegerm cells may ensure their elimination after various testic-ular injuries by the constitutive expression of FasL by theSertoli cells. In the case of radiation exposure, the damageinduced by 5 Gy may initiate a signaling pathway that over-rides the effects of the Fas signaling system.

Although the participation of the Fas signaling system inthe regulation of testicular germ cell apoptosis has beenproposed by several investigators (4, 14, 30, 33), an under-standing of the exact populations of cells that are regulatedby this pathway is lacking. The subtypes of germ cells that

undergo apoptosis after MEHP or radiation exposure are notthe same. The germ cells that undergo apoptosis after MEHPexposure are primarily spermatocytes and early spermatids,whereas after irradiation the majority of cells undergoingapoptosis are differentiating spermatogonia. Therefore, it ispossible that the Fas system plays a more predominant rolein triggering apoptosis in certain subtypes of germ cells. Adifferential role of the Fas signaling system in various germcell types may explain the protection of gld mice against germcell apoptosis induced by MEHP, but not against that causedby radiation.

In summary, the present study demonstrates the followingthree novel observations: 1) testis of gld mice are larger, withmore spermatid heads; 2) gld mice are insensitive to MEHP-induced germ cell apoptosis; and 3) germ cells of wild-typeand gld mice are similarly sensitive to the radiation-inducedincreases apoptosis. The increase in testis weights and sper-matid head counts in gld mice suggests the involvement ofthe Fas signaling system in triggering the spontaneous germcell apoptosis associated with normal testicular homeostasis.The insensitivity of gld mice to MEHP-induced germ cellapoptosis further underscores the participation of the Fassystem in the regulation of germ cell apoptosis after MEHP-induced Sertoli cell injury. Finally, our findings also revealthat the Fas system participates differentially in the cell-specific regulation of germ cell apoptosis that occurs as aconsequence of Sertoli cell vs. germ cell injury.

Acknowledgment

We gratefully acknowledge Dr. J. Leith for his assistance with theradiation exposure experiments.

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