**Xenoestrogen Effects During Premeiotic Stages of Spermatogenesis
Development in an *In Vitro* Test System and Molecular Markers of
Action**

Project Scope

The objective of this project was to test the hypothesis that
environmental estrogen-like chemicals (xenoestrogens) act directly on
cells of the testis to disrupt estrogen-dependent mechanisms of cellular
growth control during spermatogenesis. The study used the dogfish shark,
an animal model in which successive stages of spermatogenic development
and different cell populations were isolated. This allowed for both
direct observation and experimental manipulation to establish causal
relationships and mechanisms. The focus was on early (premeiotic)
spermatogenic stages when nuclear estrogen receptor concentrations are
highest.

The main objectives of this research were to:

-   Identify elements of the estrogen-activated gene response cascade
    for use as molecular markers of estrogen action;

-   Characterize estrogen\'s role in regulating cellular proliferation,
    survival, and development during spermatogensis.

The dogfish shark (*Squalus acanthias* ) was identified as a useful
model organism for a stage-by-stage analysis because of its cystic mode
of spermatogenesis and the simple linear arrangement of developing
spermatocysts across the diameter of the testis. In response to the
agonistic or antagonistic activity of particular xenoestrogens,
disruption of this natural developmental scheme was characterized based
on altered profiles of molecular markers and either a deficit in the
number of cells that complete spermatogenesis (infertility, sterility)
or uncontrolled expansion of the cell population (testicular cancer).

# 

**Project Results**

[Aim 1: Stage-by-Stage Documentation of Apoptosis and Mitosis during *In
Vivo* and *In Vitro* Spermatogenesis]{.underline} 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 light microscopy. Eight recognizable
sub-stages, from stem cell to mature spermatid stages of development
(based on distance from germinal ridge, cyst diameter, presence/absence
of a lumen, *etc*.) were identified. Month-by-month analysis of a
complete annual cycle provided to the following data: (1) the absolute
number and fraction of spermatocysts in given stages reflecting mitoses
and apoptosis in preceding generations; (2) the percentage of
proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl
transferase-mediated dUTP nick end-labeling (TUNEL) immunostained cysts
(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
(approaching meiosis) spermatogonial generations 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 showed that
overproduction of germ cell clones is the normal condition, and that the
final germ cell number is determined by programmed cell death in
spermatogonial generations 9 through 13. Although results indicate 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 pre-meiotic
cysts were determined for apoptotic and dead cells by staining with
acridine orange and propidium iodide, respectively, and incorporation of
3H-thymidine (DNA synthesis) as end points. The total RNA was prepared
from selected treatment groups for reverse transcription-polymer chain
reaction (RT-PCR) and PCR-differential display (DD). The results
indicated that serum increased 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 (ER) and
Cytochrome P450 Aromatase (Genbank accession #AF147746 and AF203106,
respectively)]{.underline} To characterize the intratesticular estrogen
signaling pathway (source and target of estrogen) during
spermatogenesis, a sequential PCR cloning strategy was designed to
isolate shark testis specific ER and cytochrome P450 aromatase cDNAs. An
1812 bp ER cDNA which 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 was of the
β-subtype with a single 7.1 kb transcript in testis. No evidence for a
second ER (α-subtype) was obtained, suggesting that the β-isoform is the
ancestral vertebrate molecule. Although low in abundance, ER mRNA was
highest in testis, kidney, and liver. Within testis, the expression was
stage-dependent: germinal zone (GZ) \> pre-meiotic (PrM) \>\> meiotic
(M) = post-meiotic (PoM). The data confirmed our earlier estrogen
binding studies and reinforce the view that hormonal estrogen and
xenoestrogens target stem cells and early spermatogonial generations.
The ER mRNA levels also were enriched in zone of degeneration (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.

Additionally, a 2118 bp P450 aromatase cDNA was isolated from the shark
ovary using a PCR cloning strategy. Phylogenetic analysis and sequence
comparisons showed that the shark aromatase was neither a β- nor an
α-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 the results of RT-PCR (PrM
\> M = 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.

[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)]{.underline} The regulation of
spermatogenesis occurs through an interaction of ER- and androgen
receptor (AR)-mediated signaling pathways and through the interaction of
certain environmental toxicants with AR. Thus a PCR cloning strategy
based on degenerate and sequence-specific primers was applied to obtain
a ![](media/image1.jpeg){width="0.16666666666666666in"
height="0.14583333333333334in"}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.
A RT-PCR analysis with gene-specific primers showed that AR expression
in testis was stage-dependent (PrM \> M \> PoM), agreeing with the
earlier androgen binding assays. Within the PrM region, AR mRNA
increased progressively from stem cell to late spermatogonial substages,
indicating the early stages of development are targeted by the effects
of hormonal androgen and toxicants that bind to AR.

Ligands that bind to the arylhydrocarbon receptor (AHR), such as dioxin
and other polycyclic aromatic hydrocarbons (PAHs), are known to be
spermatotoxicants, and induce testicular cancer or 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), RT-PCR was used to show the high
testicular AHR expression (relative to liver). It was observed that each
isoform had a unique stage-related distribution pattern. Pre-meiotic
stage spermatocysts cultured with AHR ligands (TCCD, BNF, 33\'44\'TCB)
showed ligand-specific, dose-response effects on apoptosis (AO staining)
and DNA synthesis. The data indicates that 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, a shark testis specific homolog of vasa (a known germ cell marker
and member of the DEAD box family of helicases) was cloned. Vasa was
expressed at the highest levels in the PrM and M stages. Additionally, a
shark β-tubulin cDNA was cloned, and sequence-specific primers and
probes were generated for normalizing RT-PCR and Northern analyses of
all genes of interest. The results indicated that the number and size of
multiple β-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, the
PCR-DD method of mRNA fingerprinting was applied. Five primer sets were
used to obtain a total of 49 stage-dependent and cadmium (Cd) regulated
bands. In the initial experiments, Cd was used because it is a known
mammalian spermatotoxicant which interferes with ER actions, and
accumulates 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, or Northern
analysis). In one cDNA, which was enriched in GZ/PrM stages and
upregulated five-fold *in vivo,* Cd was identified as a \~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. They also are reported to be stage-dependent and
androgen-responsive in rodent testis. A second band was ultimately
identified as the shark homolog of BRCA1-associated protein 2 (BRAP2,
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 of development.
A third PCR-DD identified band was found to be S-100 (Acc. \# AF421551),
one of a large family of Ca++ binding protein with diverse functions. In
shark testis, it was observed that the S-100 expression increased
progressively with maturation and was Cd-regulated.

[Aim 4: Applicability and general relevance of the Shark Testis
Model]{.underline} Findings of this project clearly demonstrated 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. 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 testis, results of
these studies suggest that 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 following factors
necessitated a focus on mainly *in* vivo treatment paradigms: the
seasonality of spermatocyst condition, functional variability from one
substage to another within a given preparation of staged cysts, and the
inability to optimize and stabilize the spermatogenic condition and
progression *in vitro* (as measured by apoptosis, mitosis, and mRNA
yield and quality) within the time frame of this project. The
experiments were especially successful with Cd; in contrast to
endogenous estrogen, Cd is virtually undetectable in controls, thereby
facilitating recognition of treatment effects in shark testis.

**Investigators**

Gloria V. Callard - Boston University, Boston, MA

**For More Information**

Laboratory web page:

<http://www.bu.edu/biology/Faculty_Staff/gvc.html>

NCER Project Abstract and Reports:
<http://cfpub2.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/136/report/0>

#