Transcript Endocrine Disruptors

OVARIAN STRUCTURE PROTEIN 1: A
SENSITIVE MOLECULAR BIOMARKER
OF GONADAL INTERSEX IN FISH
Dr. Cecon T. Mahapatra
What is Gonadal Intersex?
An organism of fixed sex possessing both
testicular and ovarian tissue simultaneously or
sequentially
in ovarian tissue
Histological appearance of intersex fish
Cause of such alterations?
Extensive lab studies indicates that chemical
exposures contribute to endocrine disorders
in humans and wildlife. Particularly the socalled endocrine-disrupting compounds
(EDCs).
Background
• An endocrine disruptor is an exogenous substance or mixture
that alters function(s) of the endocrine system and
consequently causes adverse health effects in an intact
organism or its progeny or populations"
• Exposure to Endocrine Disrupting Chemicals (EDCs) can lead
to alterations in gonadal development and function
• Substances inducing feminizing effects are estrogenic and
those inducing masculinizing effects are androgenic
• Release of EDCs into the environment have increased in the
past few years
Background
• EDC can be extremely effective at low doses
• Timing of exposure to EDC is critical in terms of
developmental and reproductive stage
Background
• Exposure to EDCs during early development can
lead to later-in-life, potentially irreversible
reproductive effects.
• There is an absolute need for the development of
high throughput assays that are both biologically
relevant (in vivo) and measure endpoints that can
be applied for estimating health risks of EDCs (that
induce gonadal alterations in humans and animals)
noninvasively.
Objective 1
Identify a strong molecular biomarker that is
responsive to a xenoestrogen / xenandrogen on
a vertebrate animal model that can be sexed
non-invasively very early during development
Japanese medaka as the model
species for our research
Advantages:
•Medaka is the only small fish model for which
sex is chromosomally determined (just as in
humans) and thus genetic sex can be known
very early during embryo development (2-3 dpf)
without having to sacrifice the animal for
examining the gonads histologically.
•In the SK2MC strain, gender can be noninvasively determined almost immediately after
fertilization by the presence of leucophores only
in males
•Genders can also be determined nondestructively by fin clipping in older
larvae/juveniles by determining the presence of a
male-specific gene (dmy1)
Hypothesis
•
Genes involved in sex differentiation show strong
gender specific expression
•
Down-regulation of female specific gene expression
will occur after a short-term exposure to a potent
androgen (17ß-trenbolone, TRB) and up-regulation of
male gene expression will occur after exposure to a
potent estrogen (ethinyl estradiol, EE2)
•
Short-term exposures of synthetic hormones during
sex differentiation will result in phenotypic changes in
gonadal development
Gene expression during sexual differentiation
• Gene expression during development (5 dpf to 30 dpf) was quantified
using real time quantitative PCR (qPCR) for four genes (osp1, vtg, cyp19a
and esr2) in both male and female embryo/larvae
Sexual dimorphic expression of osp1 was observed during early stages
of development
Abdel-Moneim et al. 2015. Environ. Toxicol. Chem. In Press.
Female specific expression of osp1 in
medaka ovaries
Immunohistochemistry
Western Blotting
B
A
P
V
C
100
µm
56.2 kDa
ACTIN
C
29 kDa
21 kDa
OSP1
(23.5 kDa)
100
µm
• OSP1 was detected only in ovary
• OSP1 was abundant in the cytoplasm of oogonia and perifollicular oocytes. In
mature follicles, OSP1 was localized primarily in the granulosa and theca cells
of the follicular layers.
Abdel-Moneim et al. 2015. Environ. Toxicol. Chem. In Press.
Osp1 is sensitive to short-term EDC
exposure
• Female larvae were exposed to 4
ng/LTRB and male larvae were
exposed to 6 ng/L EE2
• Exposure period: 10 days (15 to 25
dpf).
Exposure of female medaka larvae to TRB significantly
decreased the transcription levels of osp1 suggesting the high
sensitivity of this gene to androgen exposure
Abdel-Moneim et al. 2015. Environ. Toxicol. Chem. In Press.
Early exposure to EDC vs gonadal phenotype
• Histological analysis of gonads was conducted in 60 dpf juveniles
• EE2 exposed testis exhibited a delay in gametogenesis compared to control
• In TRB-exposed females, spermatogonia and spermatocyte nests interspersed
between ovarian follicles were observed indicating the development of intersex
as a result of this short-term exposure to a potent androgen
60 dpf control male
Abdel-Moneim et al. 2015. Environ. Toxicol. Chem. In Press.
60 dpf EE2-exposed male
60 dpf control female
60 dpf TRB-exposed female
Summary
• Ovarian structural protein 1, OSP1 is only expressed in ovaries of females
that are at least 12 dpf.
•
In 20 dpf larvae, there is > 200 fold increase in osp1 expression
compared to 5 dpf embryos.
• Decreased expression of osp1 is observed in females upon exposure to a
synthetic androgen (trenbolone)), which supports the use of this platform
for screening EDCs that are antiestrogenic and/or androgenic.
• We could not detect this protein in testes by western blot . We also were
not able to induce osp1 gene expression significantly in males when
exposed to a potent synthetic estrogen (ethinyl estradiol).
•
This is important because all of the current estrogenic/androgenic
“biomarkers” target genes or proteins that are predominately expressed in
one gender, but can be expressed in either testes or ovaries.
Objective 2
Our next logical step is to build an eGFP transgenic
line targeting the osp1 gene that will allow for a
cost-effective and non-lethal way for screening
EDCs
Hypothesis
Osp1-eGFP female larvae will respond
with a down-regulation in the expression of
osp1 upon exposure to antiestrogenic
and/or androgenic EDCs which will be
non-invasively visualized through decrease
in GFP expression (i.e. fluorescence)
levels
Approach
A. Establishment of the osp1-GFP/SK2MC strain:
Medaka osp1
Chr 6
21,751,000
21,755,000
Tg(osp1:GFP)
Approach
B. Validate the transgenic females from Tg(osp1:GFP) strain
using a known androgen (TRB) and a putative androgen
(tributyltin)
Female embryos
from Tg(osp1:GFP)
Larvae
Larvae
15 dpf
30 dpf
Quantification of GFP
Juveniles
45 dpf
Gonad histology
Exposed to
different
concentrations
of the androgens
Experimental design of EDC exposure experiment using see through
Tg(osp1:GFP)
Expected Outcomes
• Successful generation of this transgenic line will be
a novel diagnostic tool and help in the in vivo noninvasive visualization of antiestrogenic/androgenic
exposure associated with a very specific molecular
target (osp1)
• This fluorescent see-through transgenic fish can be
used for high-throughput screening of EDCs in a
cost-effective manner as well as decrease the
number of animals used for
experimentation.
Acknowledgements
Dr. Marisol S. Sepulveda
Ahmed Abdel-Moneim
THANK YOU