Final Report: Xenoestrogen Effects During Premeiotic Stages of Spermatogenesis Development of an InVitro Test System and Molecular Markers of Action

EPA Grant Number: R825434
Title: Xenoestrogen Effects During Premeiotic Stages of Spermatogenesis Development of an InVitro Test System and Molecular Markers of Action
Investigators: Callard, Gloria V.
Institution: Boston University
EPA Project Officer: Hahn, Intaek
Project Period: February 17, 1997 through February 16, 2000
Project Amount: $463,527
RFA: Endocrine Disruptors (1996) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals


The objectives of this research project were to: (1) test the hypothesis that environmental estrogen-like chemicals (xenoestrogens, XE) disrupt normal estrogen-dependent mechanisms of cellular growth control during spermatogenesis by direct actions on nuclear estrogen receptors (ER) in specific cell types and developmental stages; and (2) validate the utility of a novel animal model (the dogfish shark, Squalus acanthias) for recognizing and characterizing stage-specific toxicant genes, effects, and susceptibilities.

Summary/Accomplishments (Outputs/Outcomes):

The hypothesis and rationale of the project were tested by four specific aims:

Aim 1: Stage-by-Stage Documentation of Apoptosis and Mitosis In Vivo and In Vitro. Criteria for staging spermatocysts (follicle-like units comprising a single germ cell clone and second clonal population of Sertoli cells) in the developmental progression were established using the light microscope. Eight recognizable substages, from stem cell to mature spermatid stages of development (based on distance from germinal ridge, cyst diameter, presence/absence of a lumen, etc.) were described. Month-by-month analysis of a complete annual cycle showed: (1) the absolute number and fraction of spermatocysts in a given stage varied with time of year, which in turn reflected mitoses and apoptosis in preceding generations; (2) the percentage of proliferating cell nuclear antigen (PCNA)–and/or TUNEL–immunostained cysts (respectively, mitotic and apoptotic indices) varied independently by stage of development, time of year, cell type, and specific toxicant exposure, e.g., estradiol, cadmium, TCDD (see details below); (3) mature spermatogonial generations (i.e., approaching meiosis) were most susceptible to seasonal (hormone withdrawal) and toxicant effects as measured by an increased percentage of apoptotic clones; (4) germ cells, but not Sertoli cells, were affected by apoptosis; (5) cessation of mitoses in Sertoli cells preceded that in germ cells by several generations; and (6) within susceptible spermatogonial generations, apoptotic cell death was directly correlated with cell cycle activity (i.e., the death pathway is entered through cell cycle). The results clearly show that overproduction of germ cell clones is the normal condition, and that final germ cell number is determined mainly by programmed cell death in spermatogonial generations 9 through 13. Although results illustrate the utility of the shark testis model for obtaining new information of general relevance, they also underline the necessity of substage-by-substage analysis (rather than analysis of mixed stages) to identify and quantify toxicant effects on apoptosis/mitosis. A second component of this specific aim was the optimization/standardization of an in vitro spermatogenesis system. Effects of serum (FBS), estrogen, and other additives on premeiotic cysts were documented by staining of apoptotic (acridine orange labeling) and dead cells (propidium iodide) and incorporation of 3H-thymidine (DNA synthesis) as end points; and total RNA was prepared from selected treatment groups for reverse transcription-polymer chain reaction (RT-PCR) and PCR-differential display (DD). Results showed that serum increases both mitosis and apoptosis (see conclusion 6 above); however, steroid effects were both stimulatory and inhibitory, depending on dose and the presence or absence of serum.

Aim 2: Stage-Specific Expression of Nuclear Estrogen Receptors and Cytochrome P450 Aromatase (Genbank Accession #AF147746 and AF203106, respectively). To characterize the intratesticular estrogen signaling pathway (source and target of estrogen) during spermatogenesis, a sequential PCR cloning strategy was used to isolate shark testis specific ER and cytochrome P450 aromatase cDNAs. An 1812 bp ER cDNA, that included the full open reading frame was obtained, and gene specific probes and primers were designed for Northern analysis and RT-PCR. Phylogenetic analysis showed the shark ER is of the beta subtype, with a single 7.1 kb transcript in testis. No evidence for a second ER alpha subtype was obtained, suggesting that the beta isoform is the ancestral vertebrate molecule. Although generally low in abundance, ER mRNA was highest in testis, kidney, and liver. Within testis, the expression was stage-dependent: GZ > PrM >> M = PoM. The data confirmed our earlier estrogen binding studies and reinforce the view that hormonal estrogen and xenoestrogens preferentially target stem cell and early spermatogonial generations. Also, ER mRNA levels were enriched in ZD cysts containing Sertoli cells only (and degenerate germ cells), implying that Sertoli cells are the ER-expressing cell type in spermatogonial stages that contribute to ZD. Although digoxigenin-labeled shark specific ER probes were synthesized, precise localization of ER by cell type and stage is incomplete.

A 2118 bp P450 aromatase cDNA, which includes the full open reading frame, was isolated from shark ovary using a PCR cloning strategy. Phylogenetic analysis and sequence comparisons showed that the shark aromatase is neither a B- nor an A-isoform as shown in teleost fish, but instead is more closely related to the ancestor that gave rise to the single copy avian and mammalian aromatase genes. Northern analysis confirmed results of RT-PCR (M > PrM = PoM), a stage-related distribution that corresponded to our previously measured aromatase enzyme activity. As with ER, aromatase mRNA was enriched in ZD, indicative of preferential localization of aromatase in Sertoli cells. Although digoxigenin-labeled probes were synthesized, conditions for in situ hybridization to obtain direct evidence for this idea are still in progress.

Aim 3: Identification of Known and Unknown mRNAs that Correlate with Xenoestrogen Effects, Stage of Development and/or Cell-Type (Genbank Accession #AF421550 and 421555). Because the regulation of spermatogenesis is an interplay of ER and androgen receptor (AR) mediated signaling pathways, and certain environmental toxicants interact with AR specifically, a PCR cloning strategy based on degenerate and sequence-specific primers was applied to obtain a 3 kb AR cDNA. The isolated cDNA fragment encodes a protein that begins at the DNA binding domain at the 5'-end and extends through the poly-A tail of the 3' UTR, which was very long. RT-PCR analysis with gene-specific primers showed that AR expression in testis is stage-dependent (PrM > M > PoM), which agrees with our earlier androgen binding assays. Within the PrM region, AR mRNA increased progressively from stem cell to late spermatogonial substages, indicating that effects of hormonal androgen and toxicants that bind to AR target these early stages preferentially.

Ligands that bind to the arylhydrocarbon receptor (AHR), such as dioxin and other polycyclic aromatic hydrocarbons (PAHs), are known to be spermatotoxicants, and/or to induce testicular cancer, and/or to interfere with estrogen signaling pathways. In collaboration with Mark Hahn (Woods Hole Oceanographic Institution, WHOI), who has cloned fragments of two AHR cDNA variants (1 and 2) from qualus, we used RT-PCR to show high testicular AHR expression (relative to liver), but each isoform had a unique stage-related distribution pattern. Premeiotic stage spermatocysts cultured with AHR ligands (TCCD, BNF, 33'44'TCB) showed ligand-specific dose-response effects on apoptosis (AO staining) and DNA synthesis. These data indicate the testis is a direct target of certain AHR-binding xenobiotics, and that growth control processes in early developmental stages are preferentially targeted.

To evaluate direct and indirect effects of diverse toxicants on germ cells, we cloned a shark testis specific homolog of vasa, a known germ cell marker and member of the DEAD box family of helicases. DEAD represents the one letter code for the tetrapettide, Asp-G1u-Ala-Asp. Vasa was expressed at the highest levels in the PrM and M stages. Additionally, a shark beta-tubulin cDNA was cloned, and sequence-specific primers and probes were generated for normalzing RT-PCR and Northern analyses of all genes of interest. Interestingly, the number and size of multiple beta-tubulin mRNAs varied in a stage-specific manner.

To establish the utility of the shark testis model for identifying novel unknown stage-specific/dependent and toxicant sensitive genes, we applied the PCR-DD method of mRNA fingerprinting. Five primer sets were used to obtain a total of 49 stage-dependent and cadmium (Cd)-regulated bands. We used Cd in these initial experiments because it is a known mammalian spermatotoxicant, is reported to interfere with ER actions directly or indirectly, and accumulates preferentially in ER expressing stages in shark testis, where it increases the number of apoptotic cysts. Of the identified bands, three were subjected to further analyses (e.g., sequence analysis, cDNA library enrichment and screening, 5' and 3' RACE, RT-PCR, Northern analysis). One cDNA, which was enriched in GZ/PrM stages and upregulated five-fold by in vivo Cd was identified as an approximately 400 bp fragment of the control region of mitochondrial (mt) DNA, implying that it is a marker of ongoing transcriptional activity on the H strand. It may be relevant that, of the 12 proteins encoded by mtDNA, the cytochrome oxidase subunits appear to be involved in caspase activation leading to apoptosis, and also are reported to be stage-dependent and androgen-responsive in rodent testis. A second band was ultimately identified as the shark homolog of BRAP-2 (Acc. #AF421550). The high degree of sequence identity between the shark, human, and bacterial BRAP2 genes implies an important conserved function. There is good evidence that BRAP2 is a novel cytosolic protein that specifically binds to nuclear localization signals, thereby regulating cytosolic-nuclear shuttling of transcription factors and other nuclear proteins. In shark testis, BRAP2 expression increased with spermatocyst maturation but decreased after Cd exposure in vivo, suggesting a possible toxic mechanism for observed effects on apoptosis in PrM stages and additional, still to be identified, effects on transcriptional regulation in maturing and mature stages. A third PCR-DD identified band was found to be S100 (Acc. #AF421551), one of a large family of Ca++ binding proteins with diverse functions. In shark testis, the S-100 expression increased progressively with maturation and was Cd-regulated.

Aim 4: Applicability and General Relevance of the Shark Testis Model. Findings of this project clearly demonstrate the utility of modern methods of cellular and molecular biology as applied to the favorable anatomy of the shark testis model for identifying developmentally programmed and toxicant-sensitive genes. Although the effort involved in cloning and characterizing shark-specific cDNAs required a greater investment of effort than anticipated, eight new shark cDNAs were isolated and characterized, contributing significantly to available resources and the body of knowledge in the field. All of the identified genes have homologs in other vertebrates, including man. Indeed, sequences are remarkably conserved, signifying important functions and adaptive value. Although the mRNA and protein products of BRAP2 and S-100 have not previously been studied in testes, results of our studies suggest they could serve as molecular markers of spermatotoxicant effect and point to new hypotheses for testing in laboratory mammals. While results at the start of this project indicated that spermatocysts in defined stages would have utility for high throughput in vitro toxicant testing, the seasonality of spermatocyst condition, functional variability from one substage to another within a given preparation of staged cysts, and our inability within the time frame of this project to optimize and stabilize the spermatogenic condition and progression in vitro (as measured by apoptosis, mitosis, and mRNA yield and quality), necessitated a focus mainly on in vivo treatment paradigms. Experiments were especially successful with Cd. In contrast to endogenous estrogen, Cd is virtually undetectable in controls, thereby facilitating recognition of treatment effects. Due to the challenge of working with an unconventional animal model, our main focus has been to complete experiments rather than to progress to full publication, a deficiency that has highest priority during the next 12 months.

Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 13 publications 9 publications in selected types All 5 journal articles
Type Citation Project Document Sources
Journal Article Betka M, Callard GV. Stage-dependent accumulation of cadmium and induction of metallothionein-like binding activity in the testis of the dogfish shark, Squalus acanthias. Biology of Reproduction 1999;60(1):14-22. R825434 (Final)
  • Abstract from PubMed
  • Full-text: Biology of Reproduction - Full text HTML
  • Abstract: Biology of Reproduction - Abstract
  • Other: Biology of Reproduction - PDF
  • Journal Article Betka M, Welenc A, Franks DG, Hahn ME, Callard GV. Characterization of two aryl hydrocarbon receptor (AHR) mRNA forms in Squalus acanthias and stage-dependent expression during spermatogenesis. The Bulletin, Mount Desert Island Biological Laboratory 2000;39:110-112. R825434 (Final)
    not available
    Journal Article McClusky LM. Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias). Reproduction 2005;129(1):89-102. R825434 (Final)
  • Abstract from PubMed
  • Full-text: Reproduction-Full-Text
  • Abstract: Reproduction-Abstract
  • Other: Reproduction-PDF
  • Journal Article McClusky LM. Stage-dependency of apoptosis and the blood-testis barrier in the dogfish shark (Squalus acanthias): cadmium-induced changes as assessed by vital fluorescence techniques. Cell and Tissue Research 2006;325(3):541-553. R825434 (Final)
  • Abstract from PubMed
  • Abstract: Springer-Abstract
  • Journal Article Wang C, Callard GV. Polymerase chain reaction (PCR)-differential display identifies a subset of stage-dependent and cadmium-regulated genes during spermatogenesis in Squalus acanthias. The Bulletin, Mount Desert Island Biological Laboratory 2001;40:96-98. R825434 (Final)
  • Abstract: The Bulletin
  • Supplemental Keywords:

    water, exposure, effects, dose response, animal, cellular, enzymes, sex, susceptibility, chemicals, toxics, PAHs, dioxin, heavy metals, organics, biology, northeast., RFA, Health, Scientific Discipline, Environmental Chemistry, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Children's Health, Biology, Endocrine Disruptors - Human Health, dogfish shark, adverse outcomes, fish, infertility, endocrine disrupting chemicals, exposure studies, testicular cancer, molecular markers, animal models, developmental processes, xenoestrogen, screening methods, premeiotic stages, cellular growth, spermatogenesis, in vitro test methods

    Progress and Final Reports:

    Original Abstract
  • 1997
  • 1998