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. 2011 Jan 5;84(5):957–965. doi: 10.1095/biolreprod.110.088575

Suppression of Stra8 Expression in the Mouse Gonad by WIN 18,4461

Cathryn A Hogarth 3, Ryan Evanoff 3, Elizabeth Snyder 3, Travis Kent 3, Debra Mitchell 3, Christopher Small 3, John K Amory 4, Michael D Griswold 3,2
PMCID: PMC3080421  PMID: 21209416

Abstract

Bis-(dichloroacetyl)-diamines (BDADs) are compounds that inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully investigated. It has been proposed that BDADs may function via inhibition of testicular retinoic acid (RA) biosynthesis. We employed an organ culture technique and the expression of a marker for RA activity, Stra8 (stimulated by retinoic acid gene 8), to investigate if the BDAD WIN 18,446 inhibited the biosynthesis of RA from retinol (ROL) in neonatal and adult murine testis and in the embryonic murine gonad. After culturing either whole testes or germ cells isolated from mice at 2 days postpartum (dpp) with WIN 18,446 or with WIN 18,446 plus ROL, Stra8 expression was suppressed, demonstrating that WIN 18,446 inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-responsive LacZ reporter gene to demonstrate limited RA induction of LacZ expression in 2-dpp testes cultured with WIN 18,446 plus ROL. The expression of Stra8 was downregulated in adult mouse testis tubules cultured with WIN 18,446 when compared to tubules cultured with the vehicle control. WIN 18,446 also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 h. These murine results provide critical insights regarding how the BDADs can inhibit spermatogenesis by blocking the ability of vitamin A to drive germ cell development. In addition, these techniques will be useful for screening novel inhibitors of RA biosynthesis as potential male contraceptives.

Keywords: Bis-(dichloroacetyl)-diamines, retinoic acid, spermatogenesis, Stra8, testis

The bis-(dichloroacetyl)-diamine, WIN 18,446, inhibits the enzymatic conversion of retinol to retinoic acid, and suppresses the retinoic acid-mediated expression of Stra8 in the neonatal and adult mouse testis and the embryonic gonad.

INTRODUCTION

Vitamin A plays an essential role in many different physiological functions in mammals. Dietary vitamin A is usually stored in the liver and transported throughout the serum as retinol (ROL), with metabolism to its active form, retinoic acid (RA), taking place in target tissues [1]. Vitamin A metabolism is under the control of two families of enzymes, the alcohol and aldehyde dehydrogenases (Fig. 1) [1, 2]. Within cells, RA binds to two families of intracellular receptors, termed either RA receptors or retinoid X receptors [3, 4]. These receptors, in the form of homo- and heterodimers, regulate gene expression by binding to specific elements in the promoter regions of genes under the control of RA.

FIG. 1.

FIG. 1.

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Vitamin A metabolism. The dietary form of vitamin A in animals, ROL, is metabolized to RA via a two-step enzymatic process. ROL is converted to retinaldehyde by the alcohol dehydrogenases and the short-chain dehydrogenases in a reversible manner. Retinaldehyde is then modified irreversibly to form RA by the aldehyde dehydrogenases. WIN 18,446, one of the BDAD compounds, can inhibit the enzymatic activity of at least one of the aldehyde dehydrogenases and prevent the conversion of ROL to RA [14].

Retinoic acid is essential for normal spermatogenesis. When adult male mice are made vitamin A deficient (VAD), all differentiated germ cells are lost from the seminiferous epithelium, and only type A spermatogonia and Sertoli cells remain [5]. However, this loss of differentiated germ cells is reversible. An injection of either ROL or RA into a VAD male mouse will reinitiate spermatogonial differentiation throughout the testis in these animals [6, 7]. Recent evidence generated using short-term cultures of neonatal testes and isolated undifferentiated spermatogonia show that RA treatment of these tissues and cells upregulates the expression of Stra8 and Kit [8, 9], which are markers of differentiated spermatogonia, and increases the number of cells containing nuclei reminiscent of leptotene and zygotene spermatocytes [9]. Likewise, the subcutaneous delivery of RA to mice at 2 days postpartum (dpp) or to adult, vitamin A-sufficient male mice induced Stra8 transcript and/or an RA-responsive LacZ transgene in the testis [10, 11]. Therefore, the importance of RA to germ cell development and the reversibility of the spermatogenic block that results from VAD suggest that the enzymes responsible for the conversion of ROL to RA could make interesting targets for the development of male contraceptives.

The bis-(dichloroacetyl)-diamines (BDADs) are a set of compounds that target the aldehyde dehydrogenases and prevent the oxidation of retinaldehyde to RA (Fig. 1). Nearly 50 yr ago, one particular BDAD, WIN 18,446, was shown to completely and reversibly inhibit spermatogenesis in men when dosed orally [12], and more recent studies with this compound in mice and rabbits [13, 14] have revealed that WIN 18,446-treated testes in these species resemble those of VAD mice [13]. The recent study in adult rabbits also demonstrated that both tissue RA and Stra8 expression were significantly reduced by treatment with WIN 18,446 [14]. However, the mechanism of action for WIN 18,446 has yet to be fully studied in the adult mouse testis, and whether WIN 18,446 is also active in the neonatal testis or the embryonic gonad is unknown. In addition to the marked impairment of spermatogenesis, men treated with WIN 18,446 experienced a “disulfiram reaction” when they drank alcohol, because this compound also inhibited the aldehyde dehydrogenase responsible for breakdown of the toxic acetaldehyde to acetic acid in the liver. Because of this adverse reaction, further development of this particular compound for use as a male contraceptive was abandoned without pursuing an understanding of how the BDADs effectively and reversibly inhibit spermatogenesis and whether other BDAD derivatives could act in a testis-specific manner.

The present study represents, to our knowledge, the first in-depth analysis of how WIN 18,446 inhibits spermatogenesis in the murine gonad. We utilized an agar mold testis culture technique to examine the effect of WIN 18,446 on spermatogenesis through the suppression of Stra8 expression and the use of transgenic mouse line that expresses LacZ (official symbol Tg(RARE-Hspa1b/lacZ)12Jrt, hereafter referred to as RARE-hsplacZ) in response to RA signaling as markers of RA activity. In the present study, we show, again to our knowledge for the first time, that WIN 18,446 can inhibit the conversion of ROL to RA in the whole neonatal testis, isolated neonatal germ cells, adult mouse testis tubules, and the embryonic murine gonad.

MATERIALS AND METHODS

Animals and Tissues

All animal experiments were approved by the Washington State University Animal Care and Use Committee and were conducted in accordance with the guiding principles for the care and use of research animals of the National Institutes of Health. BL/6–129 and CD1 mouse colonies were maintained in a temperature- and humidity-controlled environment with food and water provided ad libitum. The BL/6–129 mice, ranging in age from birth to adulthood (35–90 dpp), and the CD1 time-mated pregnant female mice used in the present study were collected from these colonies. The animals were euthanized by decapitation (fetuses and those aged 0–10 dpp) or using CO2 asphyxiation followed by cervical dissociation (10 dpp to adult), and their testes or ovaries were dissected. Fetal gonad tissues were collected from CD1 mice embryos staged by forelimb and hindlimb morphology [15]. Because it is visually impossible to determine the sex of an embryo at Embryonic Day 11.5, the tail of each embryo was removed during dissection and sexed using Sry PCR as previously described [16]. Cultured tissue samples for RNA preparation were snap-frozen immediately after collection and stored at −80°C until use.

Grooved Agar Mold Cultures

Isolated urogenital ridges, 2-dpp mouse testes cut into four pieces, or small pieces of stage-dissected adult mouse testis tubules were cultured on 1.5% agar blocks with a groove running down the center [17]. Each agar block was placed into a well of a 24-well plate containing 300 μl of Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and cultured at 37°C with 5% CO2. All tissues used for Stra8 expression analysis were cultured with 0.7 μM RA, 0.7 μM ROL, or 1 μM WIN 18,446. Tissues used for beta-galactosidase analysis were cultured with 0.7 μM RA, 1.4 μM ROL, or 2 μM WIN 18,446. The final concentrations of RA and ROL used were previously published [8, 18], and dose-curve analyses were performed to determine the optimal WIN 18,446 doses for real-time RT-PCR and colorimetric assay endpoints (data not shown). For each organ culture, the agar block was equilibrated in medium plus treatment or vehicle (dimethyl sulfoxide [DMSO]) for 24 h. Then, the medium and treatment were replaced and the tissue placed inside the groove. The neonatal testes and adult testis tubules were cultured for 24 h and the embryonic gonads for 48 h. During the 48-h culture, the medium was refreshed at the 24-h time point. For the experiments using whole neonatal testes, adult mouse testis tubules, or embryonic gonads, each treatment or vehicle-control incubation was performed at least in triplicate on testis tissue or embryonic gonad collected from at least three different animals or embryos. A Student t-test was performed to determine statistically significant differences between vehicle and treated samples.

Gonocyte Cultures

Testes were collected from 2-dpp mice into Hanks balanced salt solution and the tunica removed. MicroBeads with mouse CD90 (THY1) antibodies and MACS Separation columns (Miltenyi Biotec) were used in the isolation according to manufacturer's instructions. The culture was performed as previously described [8]. Isolated cells were cultured under feeder cell-free, serum-free, and growth factor-free conditions for 24 h with only vehicle (DMSO), 0.7 μM RA, 0.7 μM ROL, or 1 μM WIN 18,446. Cell viability was monitored by staining a small aliquot of cells after culture with 0.4% trypan blue solution, with less than 2% of cells found to be dead in each aliquot (data not shown). For each cell isolation, 12–15 mice at 2 dpp were used to obtain the necessary number of gonocytes. Each treatment or vehicle-control incubation was performed on at least three different cell isolations. A Student t-test was performed to determine statistically significant differences between vehicle and treated samples.

Real-Time RT-PCR

A two-step, real-time RT-PCR was used to measure the expression of candidate genes as previously described [19]. The RNA samples were analyzed in triplicate with primers specific for the target genes. Stra8 primers amplified a 151-bp product (primers, 5′-GTTTCCTGCGTGTTCCACAAG-3′ and 5′-CACCCGAGGCTCAAGCTTC-3′); Cidea primers amplified a 150-bp product (primers, 5′-GCAGCCTGCAGGAACTTATC-3′ and 5′-CCAAGATCATGAAATGCGTG-3′); Fstl3 primers amplified a 118-bp product (primers, 5′-CTTCCGGCAACATCAACAC-3′ and 5′-ACTCCGTCGCAGGAATCTTT-3′); and control Rps2 primers amplified a 112-bp product (primers, 5′-CTGACTCCCGACCTCTGGAAA-3′ and 5′-GAGCCTGGGTCCTCTGAACA-3′). Expression of Stra8 was normalized to expression of Rps2. Student t-test was used to analyze all results. Data are presented as the mean ± SEM.

Microarray Analysis

Array output was normalized via the GeneChip robust multiarray method, and data analysis was conducted using GeneSpring (Version 11.0.2; Agilent Technologies). Genes were considered to be regulated by BDAD treatment if they 1) had a raw score of greater than 50 in at least one sample, 2) were determined to be significantly different versus controls by ANOVA (P = 0.05), with a 5% false-discovery rate multiple-test correction, and 3) showed a 2-fold or greater increase or decrease versus controls. Data were deposited with the National Center for Biotechnology Information gene expression and hybridization array data repository (GEO accession no. GSE25610; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25610).

Functional annotation cluster analysis was performed using tools in the Database for Annotation, Visualization, and Integrated Discover (DAVID) Bioinformatics Resources (http://david.abcc.ncifcrf.gov) [20, 21]. Affymetrix Gene Chip Probe IDs identified in the microarray analysis as being regulated by WIN 18,446 were uploaded to DAVID Bioinformatics Resources, and functional annotation clustering was used to examine biological process gene ontology (GO) terms defined as being significantly overrepresented. Enrichment scores for each cluster are reported, as are the most biologically relevant GO term in the cluster and the number of transcripts represented.

Germ Cell Nuclear Antigen Immunofluorescence

Immunofluorescence using germ cell nuclear antigen (GCNA) antibody (George Enders, Department of Anatomy and Cell Biology, The University of Kansas Medical Center, Kansas City, KS) was performed on the THY1-positive gonocytes isolated from the 2-dpp testes to determine cell purity. The isolated cells were left to settle onto a glass coverslip for 1 h, fixed in Bouin solution for 30 sec, and then rinsed three times in PBS. The cells were then blocked with 10% normal rabbit serum diluted in PBS (blocking solution) for 30 min before an overnight incubation in GCNA antibody (1:50 dilution in blocking solution) at 4°C. The cells underwent four 5-min washes with PBS before being incubated in rabbit anti-rat immunoglobulin M biotin-conjugated secondary antibody (1:500 dilution in blocking solution; Cortex Biochem). After four more 5-min PBS washes, bound antibody was detected using a 1-h incubation in an Alexa Fluor 488-conjugated streptavidin compound (1:1000 diluted in blocking solution; Invitrogen). Visualization followed mounting under coverslips using Vectashield with 4′,6′-diamidino-2-phenylindole (Vector Laboratories) and a Leica confocal microscope at the Washington State University Imaging facility.

Beta-Galactosidase Staining and Counting

Cultured RARE-hsplacZ testes were washed twice in PBS, then fixed in 4% paraformaldehyde with a 0.25% gluteraldehyde additive in PBS for 1 h at 4°C. Fixed tissue was washed and stained in bromo-chloro-indolyl-galactopyranoside (X-gal) as previously described [22]. Samples were washed in LacZ buffer and then in PBS three times before soaking in an equal-parts solution of 70% ethanol and PBS. Testis pieces and embryonic gonads were photographed in PBS before being dehydrated and embedded in paraffin for sectioning.

The paraffin-embedded, cultured RARE-hsplacZ neonatal testes were sectioned (thickness, 4 μm), melted onto glass slides, and then stained with Harris hematoxylin. For beta-galactosidase activity analysis, germ cells were considered to be beta-galactosidase positive if they contained two or more foci of beta-galactosidase activity (light blue staining) or definitive, diffuse cytoplasmic staining. Quantification was performed on at least 50 tubule cross sections from a minimum of three different pieces of cultured testis for each sample. The frequency of beta-galactosidase-positive germ cells per tubule cross section was determined for each treatment. The ratio of this value for each treatment compared to that of the vehicle controls was then used for all statistical analysis. A Student t-test was performed to determine statistically significant differences between vehicle and treated samples. In all cases, a minimum of three biological replicates and technical duplicates were utilized for analysis.

RESULTS

WIN 18,446 Inhibited Conversion of ROL to RA in Whole Neonatal Testes

Previous studies have demonstrated that daily treatment of adult male mice with WIN 18,446 results in a VAD-like phenotype in the testis [13], and very recent experiments in adult male rabbits have shown that the levels of tissue RA and Stra8 transcript levels are reduced in the testis after daily oral WIN 18,446 treatment [14]. However, the mechanism of WIN 18,446 action that generates the VAD-like phenotype in mice has yet to be described, and whether WIN 18,446 is also active in younger animals is unknown. To examine the effect of WIN 18,446 on RA activity in the neonatal testis, testes were isolated from 2-dpp, wild-type male mice, with each then cut into four pieces and incubated in grooved agar molds for 24 h in the presence of RA, ROL, WIN 18,446, or a combination of RA or ROL plus WIN 18,446. Total RNA was extracted from these samples, and Stra8 expression was measured using real-time RT-PCR. RA was found to significantly induce Stra8 expression in neonatal testes cultured in grooved agar molds (Fig. 2). This result matches what was observed previously when neonatal testis pieces were cultured with RA on floating filters in our laboratory [8]. ROL significantly induced Stra8 expression in cultured whole testes; however, the incubation of testes with WIN 18,446 alone or in combination with ROL significantly reduced Stra8 expression. As a control sample, testes were also cultured with a combination of RA and WIN 18,446, and an induction in Stra8 expression comparable to that seen in samples cultured with RA alone was observed.

FIG. 2.

FIG. 2.

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Inhibition of Stra8 expression with WIN 18,446 treatment of neonatal testes. Graph depicts Stra8 expression within 2-dpp neonatal testis pieces after 24 h in culture with RA, ROL, WIN 18,446, or combinations of these treatments. Relative real-time RT-PCR expression level is given on the y-axis with the treatment group outlined on the x-axis. Asterisks represent statistical difference of a treatment group compared to control except where black lines indicate statistical significance between treatment groups. Four independent samples were incubated with each treatment. Error bars represent SEM. *P < 0.05, **P < 0.001.

The effect of WIN 18,446 treatment on gene expression in the testis is currently unknown. To determine whether global changes occur in gene expression in the testis in response to WIN 18,446 treatment, total RNA isolated from the 2-dpp, wild-type testis cultures was processed and hybridized to Affymetrix Mouse Genome 430 2.0 microarrays. The arrays were analyzed to determine which genes were up- or downregulated in these testis cultures in response to WIN 18,446 treatment. Arrays identified 250 transcripts representing 217 unique genes that were downregulated, and 111 transcripts representing 108 unique genes that were upregulated, in response to a 24-h WIN 18,446 treatment. The analysis tool DAVID (http://david.abcc.ncifcrf.gov/) [20, 21] was used to determine which ontology clusters were enriched in the lists. Of the 250 downregulated transcripts, 148 were associated with significantly overrepresented GO terms in a biological process. Functional annotation clustering revealed clusters associated with cytoskeletal protein binding (16 transcripts, enrichment score of 4.62), cell junction (18 transcripts, enrichment score of 2.4), and reproductive developmental process (12 transcripts, enrichment score of 2.06). Of the 111 upregulated transcripts, 64 were associated with overrepresented GO terms in a biological process. Annotation clustering revealed clusters associated with protein glycosylation (33 transcripts, enrichment score of 2.31) and proteolysis (11 transcripts, enrichment score of 1.55).

Tables 1 and 2 present the 10 annotated transcripts that displayed the highest fold-changes, up- or downregulated, when vehicle-control and WIN 18,446-treated samples were compared. Of the transcripts most highly downregulated by WIN 18,446 (Table 1), cell death-inducing DNA fragmentation factor, alpha subunit-like effector a (Cidea), and follistatin-like 3 (Fstl3) have been found to be upregulated by RA treatment of testis tissue in a previously published microarray dataset [8], and our laboratory has identified the induced expression of these two genes in RA-treated testis samples collected from mice of various ages (Hogarth, Evanoff, Small, and Griswold, unpublished data). In addition, the expression of essential meiotic endonuclease 1 homolog 2 (Eme2), a protein thought to be involved in DNA recombination, was also downregulated by WIN 18,446 (Table 1). Of the transcripts most highly upregulated by WIN 18,446 (Table 2), the expression of potassium voltage-gated channel, Isk-related subfamily, member 1 (Kcne1) has been found to be enriched in undifferentiated spermatogonia, in normal rat testis tissue, and in human seminoma samples characterized by the overproliferation of undifferentiated germ cells [23]. In addition, the expression of genes with known roles in spermatogenesis, such as reproductive homeobox 4 cluster (Rhox4), nuclear receptor subfamily 0, group B, member 1 (Nr0b1), also known as Dax1, and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2), were also regulated by WIN 18,446.

TABLE 1.

Top ten annotated transcripts downregulated in response to WIN 18,446 treatment.a

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TABLE 2.

Top ten annotated transcripts upregulated in response to WIN 18,446 treatment.a

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To confirm the data generated by the arrays, real time RT-PCR for two of the WIN 18,446-regulated genes that have been shown previously to be regulated by RA [8], Cidea and Fstl3, was performed. The expression of both Cidea and Fstl3 was upregulated in the RA-treated sample and downregulated in the samples treated with WIN 18,446 alone and with ROL plus WIN 18,446 (Fig. 3). We also compared our lists of WIN 18,446-regulated transcripts with transcripts reported to be RA-regulated in the testis [8, 9] (Table 3). Of the published RA-upregulated transcripts, five (one which being Stra8) were significantly downregulated by WIN 18,446, and of the RA-downregulated genes, two were significantly upregulated by WIN 18,446 treatment (Table 3).

FIG. 3.

FIG. 3.

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Inhibition of Cidea and Fstl3 expression with WIN 18,446 treatment of neonatal testes. Graphs depict the expression of Cidea (A) and Fstl3 (B) within 2-dpp neonatal testis pieces after 24 h in culture with RA, ROL, WIN 18,446, or combinations of these treatments. Relative real-time RT-PCR expression level is given on the y-axis with the treatment group outlined on the x-axis. Asterisks represent statistical difference of a treatment group compared to control except where black lines indicate statistical significance between treatment groups. Four independent samples were incubated with each treatment. Error bars represent SEM. *P < 0.05, **P < 0.001.

TABLE 3.

RA regulated gene expression in response to WIN 18,446 treatment.a

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To visualize the effect of WIN 18,446 on RA activity, the RARE-hsplacZ transgenic mouse line was utilized [24]. This mouse expresses the LacZ gene under the control of the hsp core promoter and a RA-response element (RARE). In these animals, beta-galactosidase will be produced in cells containing RA and the necessary RA-response machinery and can be visualized using a colorimetric assay to detect enzyme activity. Testes were isolated from 2-dpp RARE-hsplacZ transgenic males and cultured for 24 h in the same manner described for the wild-type mice. The tissue was then stained for beta-galactosidase activity. At the level of the whole tissue, beta-galactosidase staining appeared to increase in RA- and ROL-treated testes as compared to control (Fig. 4, A–F), whereas beta-galactosidase staining appeared to be reduced by a combination of ROL and WIN 18,446 (Fig. 4, G and H). After whole-tissue examination, cultured RARE-hsplacZ testes were processed for histology, and the number of beta-galactosidase-positive germ cells per tubule cross section was determined. To correct for inherent culture and litter variation, the frequency of beta-galactosidase-positive germ cells within a given treatment was compared to that of the vehicle controls. In the case of both RA and ROL treatment, the frequency of beta-galactosidase-positive cells was much greater than that observed in vehicle controls. Conversely, treatment with both ROL and WIN 18,446 resulted in a lower frequency of beta-galactosidase-positive cells as compared to ROL treatment alone. Although not significant, all samples exposed to WIN 18,446, either alone or in conjunction with ROL, had reduced frequencies of beta-galactosidase-positive cells as compared to vehicle controls (Fig. 5).

FIG. 4.

FIG. 4.

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WIN 18,446 inhibits the expression of a RA-driven LacZ expression construct. Panels depict vehicle (DMSO) control-treated testis pieces (A) and a representative cross section (B) as well as 2-dpp RARE-hsplacZ mouse testis pieces and a representative cross section cut from each of these tissues treated with RA (C and D), ROL (E and F), or ROL plus WIN 18,446 (G and H). The black arrows indicate beta-galactosidase-positive germ cells. Bars = 500 μm (A, C, E, and G) or 50 μm (B, D, F, and H).

FIG. 5.

FIG. 5.

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WIN 18,446 reduced the number of beta-galactosidase-positive germ cells in cultured neonatal testes. Graph depicts the numbers of beta-galactosidase-positive cells per tubule, relative to the vehicle control, after 24 h in culture with RA, ROL, WIN 18,446, or ROL plus WIN 18,446. The relative numbers of beta-galactosidase-positive cells per tubule are in given on the y-axis with the treatment group outlined on the x-axis. Values represent the ratio of beta-galactosidase-positive germ cells per tubule in the vehicles compared to beta-galactosidase-positive germ cells per tubule for each treatment. Asterisks represent statistical difference of a treatment group compared to control except where black lines indicate statistical significance between treatment groups. Six independent samples were incubated with each treatment, and all were used to count beta-galactosidase-positive germ cells. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

WIN 18,446 Can Act Directly on Undifferentiated Germ Cells to Suppress Stra8 Expression

WIN 18,446 was able to suppress Stra8 expression in whole-testes cultures that contained both germ and somatic cells. To determine whether germ cells alone were susceptible to WIN 18,446 treatment, THY1-positive gonocytes were isolated using magnetic cell sorting and then cultured for 24 h with or without RA, ROL, and ROL plus WIN 18,446. After each cell isolation, GCNA immunofluorescence was performed to determine the purity of the isolated cells. In each case, greater than 95% of the isolated cells were GCNA positive, indicating that a nearly pure preparation of gonocytes had been obtained (data not shown). After treatment, RNA was isolated and Stra8 expression analyzed by real-time RT-PCR. Both RA and ROL treatment alone significantly increased Stra8 levels; however, the presence of WIN 18,446 decreased Stra8 expression in the isolated germ cells (Fig. 6).

FIG. 6.

FIG. 6.

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Inhibition of Stra8 expression with WIN 18,446 treatment of isolated gonocytes. Graph depicts Stra8 expression within isolated gonocytes after 24 h in culture with RA, ROL, or ROL plus WIN 18,446. Relative real-time RT-PCR expression level is given on the y-axis with the treatment group outlined on the x-axis. Asterisks represent statistical difference of a treatment group compared to control except where black lines indicate statistical significance between treatment groups. Three independent samples were incubated with each treatment. Error bars represent SEM. *P < 0.05, **P < 0.001.

WIN 18,446 Only Affects Stra8 Expression in Adult Mouse Testis Tubules in Cells Associated with Clusters 2 and 3

The cells within an adult mouse testis tubule are arranged such that specific subpopulations of differentiating germ cells are in association. Under a dissection microscope, distinct groups (clusters) of these associations can be distinguished, because the germ cells will refract light differently depending on their differentiation state [25]. Using this dissection technique, adult mouse testis tubules can be physically separated into four distinct clusters, which represent groups of germ cell associations (stages) that are present within that piece of tubule: Cluster 1 comprises stages XII and I; cluster 2, stages II–VI; cluster 3, stages VII–VIII; and cluster 4, stages IX–XI. This technique is known as stage dissection [25], and it was utilized to test whether particular stages in the adult mouse testis respond to RA, ROL, or WIN 18,446 treatment. The testes were removed from adult male mice and the tubules dissected into clusters. Pieces of tubules representing clusters 1–4 were placed in grooved agar mold cultures for 24 h with RA, ROL, or WIN 18,446 alone or with combinations of either RA or ROL and WIN 18,446. RNA was extracted from these samples, and quantitative RT-PCR was performed to determine the expression of Stra8 in each of the treated samples compared to the vehicle control. No significant differences in Stra8 expression were seen in clusters 1 and 4 in any of the five treatments (data not shown). Treatment of cluster 2 with RA induced Stra8 expression 6-fold, whereas treatment with WIN 18,446 alone downregulated Stra8 (Fig. 7A). Interestingly, neither ROL nor RA treatment had a significant effect on the expression of Stra8 in cluster 3; however, incubation of this cluster with WIN 18,446, alone or in combination with ROL, did significantly reduced the amount of detectable Stra8 transcript (Fig. 7B).

FIG. 7.

FIG. 7.

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Inhibition of Stra8 expression with WIN 18,446 treatment of stage-dissected clusters. Graphs depict Stra8 expression within adult mouse testis tubules staged at cluster 2 (A) and at cluster 3 (B) after 24 h in culture with RA, ROL, WIN 18,446, or combinations of these treatments. Relative real-time RT-PCR expression level is given on the y-axis with the treatment group outlined on the x-axis. Asterisks represent statistical difference of a treatment group compared to control except where black lines indicate statistical significance between treatment groups. Three independent samples were incubated with each treatment. Error bars represent SEM. *P < 0.05, **P < 0.001.

Stra8 Expression in Embryonic Ovary Is Susceptible to Treatment with WIN 18,446

Retinoic acid is also essential for meiosis in the embryonic ovary, but to our knowledge, no published reports have examined whether the BDAD compounds could also affect female fertility. In addition, evidence suggests that RA is important for the mitotic arrest of gonocytes in the embryonic testis; therefore, the effect of WIN 18,446 treatment on embryonic gonads from both sexes was investigated. Murine embryonic gonads were collected at Embryonic Day 11.5, and additional tissue was harvested from each embryo for determination of sex by PCR. The gonads were cultured for 48 h with or without RA, ROL, and ROL with WIN 18,446. Stra8 real-time RT-PCR analysis was conducted on RNA isolated from these samples. In the presence of RA, Stra8 significantly increased in the cultured testes but not in the ovaries. Stra8 levels decreased significantly when the embryonic ovary or testis was treated with WIN 18,446 (Fig. 8).

FIG. 8.

FIG. 8.

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Inhibition of Stra8 expression with WIN 18,446 treatment of embryonic gonads. Graph depicts Stra8 expression within embryonic mouse ovaries (open bars) and embryonic mouse testes (solid bars) after 24 h in culture with RA, ROL, or ROL plus WIN 18,446. Relative real-time RT-PCR expression level is given on the y-axis with the treatment group outlined on the x-axis. The relative expression levels for the testis and ovary treatment groups have been compared only to the control group of the same sex. Asterisks represent statistical difference of a treatment group compared to control except where black lines indicate statistical significance between treatment groups. Three independent samples were incubated with each treatment. Error bars represent SEM. *P < 0.05, **P < 0.001.

DISCUSSION

The present study is, to our knowledge, the first in-depth analysis of how one particular BDAD compound, WIN 18,446, could inhibit murine spermatogenesis. In the 1960s, WIN 18,446 was shown to safely, effectively, and reversibly inhibit spermatogenesis in men; however, further research into how WIN 18,446 exerted this effect on spermatogenesis was suspended because of the serious side effects observed when this compound was taken with alcohol [12]. A more recent study with this compound in mice revealed that murine testes treated chronically with WIN 18,446 closely resembled a VAD testis, in which tubules were populated by only Sertoli cells and undifferentiated spermatogonia [13]. Our report strongly suggests that the likely cause of this phenotype is the blockage of the conversion of ROL to RA by WIN 18,446 in germ cells and that this block is effective not only in the adult testis but also in the neonatal testis, isolated undifferentiated spermatogonia, and the embryonic gonad.

In each of the age groups and tissues tested, WIN 18,446 was able to block the conversion of ROL to RA. Expression of both Stra8 and an RA-driven reporter gene in a transgenic mouseline were downregulated after treating the neonatal testis with both ROL and WIN 18,446; however, WIN 18,446 was not able to negate the stimulatory effect of RA on these markers in culture. Further analysis of the RARE-hsplacZ reporter gene demonstrated that significantly more gonocytes were positive for beta-galactosidase staining in the RA- and ROL-treated, 2-dpp testes, whereas the addition of WIN 18,446 significantly reduced the ROL-induced beta-galactosidase staining in germ cells. A microarray analysis of the wild-type, 2-dpp cultures determined that transcripts coding for gene products participating in a multitude of different cellular functions were regulated by WIN 18,446. For example, WIN 18,446 altered the expression of known regulators of spermatogenesis, including Dax1 and Sohlh2, yet this analysis has also identified transcripts that either have no known function in the testis or are yet to be described as being regulated by RA, such as potassium voltage-gated channel, subfamily H (eag-related), member 1 (Kcnh1). A second gene encoding a potassium-channel protein that is known to be enriched in undifferentiated spermatogonia but yet to be reported as RA regulated, Kcne1 [23], was also identified as being regulated by WIN 18,446 in the present study, suggesting that further investigation of how retinoids regulate the expression of genes involved in forming ion channels in the testis is required.

The analysis of transcripts published as being either up- or downregulated by RA demonstrated that WIN 18,446 was also able to affect the expression of these transcripts, but in the opposite direction. In other words, transcripts that have been published as being upregulated by RA were found to be downregulated by WIN 18,446 in our array analysis, and vice versa. Further investigation of the transcripts, which are usually upregulated by RA treatment but were downregulated by WIN 18,446, may provide further insight regarding why WIN 18,446 results in a VAD-like phenotype. Two examples of such transcripts are Cidea and Fstl3, and this regulation was confirmed by real-time RT-PCR analysis of the RNA samples derived from the 2-dpp, whole-testis cultures. Cidea encodes a cell-death protein that has been linked to regulating insulin sensitivity and the maintenance of adipose tissue in mice [26], but a role for this protein in testis development has yet to be described. FSTL3 appears to also play a role in fat and glucose homeostasis [27]; however, this protein has been shown to be present in Leydig cells, spermatogonia, and mature spermatids in the testis [28]. In addition, the fact that WIN 18,446 was able to alter the expression of such a range of different transcripts in the absence of added retinoids is very interesting. This suggests that while the testes are in culture, the conversion of endogenous ROL to RA can take place and that the introduction of WIN 18,446 is able to block this process. Why and how blocking the synthesis of RA regulates gene expression in the testis, and whether this regulation has a direct effect on germ cell development, will be the focus of future studies in our laboratory.

Gonocytes appear to contain the machinery necessary to convert ROL to RA. Serum free-cultures of gonocytes isolated from 2-dpp testes increased Stra8 expression in response to ROL treatment; however, WIN 18,446 was able to significantly negate this effect. The ability of isolated gonocytes to upregulate Stra8 in the presence of ROL has previously been demonstrated [8], and recent data suggest that WIN 18,446 can target the testis-specific aldehyde dehydrogenase 1A2 (ALDH1A2) [14]. Aldh1a2 has been shown to be expressed by spermatogonia in the adult mouse testis [29], but further investigation of whether the protein is present in germ cells of the neonatal testis is required to determine whether this is the aldehyde dehydrogenase that is being acted upon by WIN 18,446 in gonocytes.

To our knowledge, the present study is also the first to investigate how the individual clusters of stages of the seminiferous epithelium can respond to RA. A previous study from our laboratory demonstrated that STRA8 could be induced by RA in stages other than stages VII and VIII [11], and the current study narrows this to stages II–VI. Of the four adult mouse testis tubule clusters, only the cells present in cluster 2 significantly upregulated Stra8 expression in response to RA in culture. The current model for spermatogonial differentiation in the adult testis proposes that spermatogonial stem cells are located at random throughout the testis tubules and that at a single point along any given tubule, one of these spermatogonial stem cells receives the necessary signals to differentiate and then commits to undergo meiosis. This decision occurs at stage VIII within cluster 3 and is most likely driven by RA, because the testes of VAD mice contain only Sertoli cells and undifferentiated spermatogonia. Treating VAD mice with RA stimulates the differentiation of the undifferentiated spermatogonia throughout the testes of these animals simultaneously [6]. The data in the present study suggest that the undifferentiated spermatogonia in cluster 2 are primed to respond to RA and that the lack of available RA to these cells is one of the factors required to keep them in an undifferentiated state. Whether this limitation is imposed by regulating the delivery of RA to the germ cells, by the precise expression of the retinoid metabolism enzymes and receptors, or by a combination of both needs to be the focus of future research to clearly define how the cycle of the seminiferous epithelium is regulated.

The stage-dependent response to WIN 18,446 is also informative regarding where the synthesis of RA may be taking place across the stages of the cycle. Given that Stra8 expression was not induced after RA treatment of cluster 3 but WIN 18,446 was able to downregulate Stra8 transcript production in this cluster suggests that sufficient endogenous RA production occurs to induce Stra8 expression in cluster 3 but also that this production can be inhibited by WIN 18,446. The observation that WIN 18,446 alone can inhibit Stra8 expression in cluster 2 implies that the enzymes responsible for the conversion of ROL to RA are present and that the availability of ROL limits RA production. Clearly, further analysis of the localization of the retinoid-metabolizing enzymes throughout the cycle of the seminiferous epithelium is required to enhance our understanding of how WIN 18,446 exerts a stage-specific effect in the adult mouse testis.

The present study demonstrates that WIN 18,446 can effectively inhibit the conversion of ROL to RA in the postnatal testis and in the embryonic gonad, making it an excellent tool for continuing research into how RA regulates germ cell development in both sexes. Until now, the elimination of RA signaling has been controlled by inhibiting the retinoid receptors, either through gene knockout studies or receptor antagonists. However, these methods rely on a particular receptor being expressed in a specific cell type and do not eliminate possible functional redundancy that may occur between receptors. WIN 18,446 will provide researchers with another means of removing RA signaling from the gonad and allow experiments in the juvenile and adult testis and in the ovary to be streamlined through using the same compound.

Acknowledgment

The authors thank Professor George Enders, University of Kansas Medical Center, for supplying the GCNA antibody.

Footnotes

1

Supported by Contraceptive Center grant U54 42454 and by HD 10808 from the National Institutes of Health (NIH) to M.D.G. and by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, a division of the NIH through cooperative agreement U01 HD060408 to J.K.A. Data were deposited with the National Center for Biotechnology Information gene expression and hybridization array data repository (accession no. GSE25610; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25610).

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