Histopathology guidelines for the Fathead Minnow
(Pimephales promelas) 21-day reproduction assay
July 6, 2006
-------
ACKNOWLEDGMENTS
The following persons contributed time and effort toward the creation of this document:
Gerald Ankley, USEPA, USA
Christiana Grim, USEPA, USA
Stephen Duffell, Syngenta, UK
John Fournie, USEPA, USA
Anne Gourmelon, OECD
Rodney Johnson, USEPA, USA
Christine Riihl-Fehlert, Bayer AG, Germany
Christoph Schafers, Fraunhofer IME, Germany
Masanori Seki, CERI Environment, Inc., Japan
Leo van der Ven, RIVM, The Netherlands
Pieter Wester, RIVM, The Netherlands
Jeffrey Wolf, Experimental Pathology Laboratories, Inc., USA
Marilyn Wolfe, Experimental Pathology Laboratories, Inc., USA
INTRODUCTION
The purpose of this document is to provide guidelines for the preparation and
histopathological evaluation of gonads from fathead minnow (Pimephalespromelas).
The goals of these guidelines are to provide an updated source of direction for the
participating laboratories, to supply template text for laboratory protocols, and most
importantly, to facilitate non-biased comparisons of inter-laboratory results.
Throughout this document, the proposed procedures were derived from the consensus
opinions of various fish pathologists, recommendations from the Bilthoven 2002 and
Paris 2003 workshops, information distilled from previous guidelines, and the scientific
literature.
This guidance document is divided into three sections: I) Post-mortem and Histotechnical
Procedures; II) Gonadal Histopathology Glossary and Diagnostic Criteria; and III)
Gonadal Staging Criteria.
-------
I. POST-MORTEM AND HISTOTECHNICAL PROCEDURES
The purpose of this section is to outline all of the post-mortem steps and procedures that
occur prior to the evaluation of histologic sections on glass slides, to include euthanasia,
necropsy, tissue fixation, decalcification, tissue trimming, processing, embedding,
microtomy, staining, coverslipping, and slide labeling.
1. Substrate obtained for vitellogenin analysis.
Fathead Minnow (FHM): blood sample from the caudal vein/artery or heart
2. Tissue specimen for gonad histopathology. Techniques were selected that
would most optimally: 1) preserve the cellular structure of the gonads; 2)
maximize the amount of gonad tissue available for analysis; 3) sample the
gonads in a representative and consistent fashion; and 4) allow the pathologist
to examine at least three step sections of both gonads on a single glass slide.
In FUM, the gonads are excised from the fish.
Davidson's fixative is the recommended fixative. Compared to other common fixatives,
such as 10% neutral buffered formalin or Bouin's fixative, the advantages of Davidson's
fixative are: 1) the morphologic appearance of gonad sections is generally considered to
be comparable to sections fixed in Bouin's fixative and superior to sections fixed in
formalin; 2) compared to Bouin's fixative, which contains picric acid, Davidson's
fixative is generally considered to be less noxious, less hazardous, and more easily
disposed of; 3) there is anecdotal information which suggests that Bouin's fixative may
be difficult to obtain in the near future; 4) specimens fixed in Bouin's fixative require
multiple rinses prior to transfer to alcohol or formalin. Please see photographic
comparison of specimens fixed in Davidson's versus Bouin's fixatives (Appendix A, Fig.
I). Please be aware that different recipes and products that are designated as "Davidson's
fixative" may actually be modifications of the original formula (Appendix C); if a
modified Davidson's fixative is used, this should be noted by the laboratory. If
necessary, a recommended decalcification solution is listed (Appendix D). Factors that
may affect the need for decalcification include the size of the individual fish, the length
of time that the carcass was immersed in fixative, and the extent to which the abdominal
cavity came into contact with the fixative.
-------
I. Fathead Minnow
1. Euthanasia, Necropsy, and Tissue Fixation
Objectives:
1. Provide for the humane sacrifice of fish.
2. Obtain necessary body weights and measurements.
3. Obtain specimens for vitellogenin analysis.
4. Excise gonad specimens.
5. Evaluate secondary sex characteristics.
6. Provide for adequate fixation of the gonads and the carcass.
Materials:
1. Fish transport container (-500 ml, contains water from the experimental
tank or system reservoir).
2. Small dip net.
3. Euthanasia chamber (-500 ml vessel).
4. Euthanasia solution (Appendix C or FA-100 [Japan]).
5. Electronic slide caliper (minimum display: < O.lmm)
6. Electronic analytical balance (minimum display: < O.lmg) and tared
vessels.
7. Stereoscopic microscope.
8. Pins and corkboard.
9. Small scissors (e.g., iris scissors).
10. Small forceps.
11. Microdissection forceps.
12. Microdissection scissors.
13. Gauze sponges.
14. Davidson's fixative (Appendix C & Appendix D).
15. Plastic syringe (3ml).
16. Standard plastic tissue cassettes (one per fish).
17. Fixation containers (100 ml, one per fish).
Procedures:
1. Fish should be sacrificed within one to two minutes prior to necropsy.
Therefore, unless multiple prosectors are available, multiple fish should
not be sacrificed simultaneously.
2. Using the small dip net, a fish is removed from the experimental chamber
and transported to the necropsy area in the transport container. For each
test chamber, all male fish are sacrificed prior to the sacrifice of female
fish; the sex of each fish is determined by external body characteristics
(e.g., presence or absence of nuptial tubercles, dorsal pad, etc.).
3. The fish is placed into buffered MS-222 solution. The fish is removed
from the solution when there is cessation of respiration and the fish is
unresponsive to external stimuli.
-------
4. The fish is wet weighed, measured according to protocol, and a blood
sample is obtained from the caudal artery/vein or heart.
5. The fish is placed on a corkboard on the stage of a dissecting microscope.
Using iris scissors and small forceps, the abdomen is opened via a
carefully made incision that extends along the ventral midline from the
pectoral girdle to a point just cranial to the anus.
6. The fish is placed in dorsal recumbency and the opposing flaps of body
wall are pinned laterally to expose the abdominal viscera (Appendix A,
Fig. 2 & Fig. 3).
7. Using the small forceps and small scissors, the abdominal viscera (liver,
gastrointestinal tract, spleen, pancreatic tissue, and abdominal mesentery)
are carefully removed en masse in the following manner:
a. The intestine is severed proximal to the anus.
b. A forceps is applied to the terminal portion of the intestine. Using
gentle traction and taking care not to disturb the gonads, the viscera
are dissected out of the abdominal cavity in a caudal to cranial
direction.
c. The distal esophagus is severed just proximal to the liver.
8. Using a syringe, approximately 0.5 ml of Davidson's fixative is then
gently applied to the gonads in situ. Approximately 90 seconds following
the application of fixative, the liquid fixative within the abdomen is
removed with a gauze sponge, and the gonads are excised in a manner
similar to the abdominal viscera:
a. Using the microdissection scissors, the spermatic ducts or oviducts are
severed proximal to the genital pore.
b. Microdissection forceps are then applied to the spermatic
ducts/oviducts. Using gentle traction, the gonads are dissected out of
the abdominal cavity in a caudal to cranial direction, severing the
mesorchial/mesovarial attachments as needed using the
mi crodis section scissors. The left and right gonads may be excised
individually or they may be excised simultaneously and subsequently
divided at their caudal attachment.
9. The gonads (right and left) are placed into a pre-labeled plastic tissue
cassette which is then placed into an individual container of Davidson's
fixative accompanied by the abdominal viscera. The volume of fixative in
the container should be at least 10 times the approximated volume of the
tissues. The fixative container is gently agitated for five seconds to
dislodge air bubbles from the cassette.
10. Using the carcass, the secondary sex characteristics are assessed (e.g.,
dorsal nape pad, nuptial tubercles). The carcass is then added to the
fixative container.
11. All tissues remain in Davidson's fixative overnight followed by transfer
to individual containers of 10% neutral buffered formalin the next day.
Containers with cassettes are gently agitated for 5 seconds to ensure
-------
adequate penetration of formalin into cassettes (it is not necessary to rinse
with water or perform multiple changes in formalin).
2. Tissue Trimming
Tissue trimming is not required for FHM.
3. Tissue Processing
Objectives:
1. Dehydrate tissue to provide for adequate penetration of paraffin.
2. Impregnate the tissue with paraffin to maintain tissue integrity and create a
firm surface for microtomy.
Materials:
1. Tissue processor.
2. Paraffin heating pots.
3. Processing unit oven.
4. Activated charcoal.
5. Paraffin (Paraplast®, or equivalent, Appendix D).
6. 10% neutral buffered formalin.
7. Ethyl alcohol (absolute and dilutions as required).
8. Proprietary clearing agent (Clear Rite-3™ or equivalent, Appendix D).
9. Xylene.
Procedures:
1. Labeled tissue cassettes are removed from formalin storage and are
washed in tap water.
2. The cassettes are placed in the processing basket(s) in a single layer. The
processing basket is loaded into the tissue processor.
3. The processing schedule is selected (see Appendix B, Schedule 1). The
"Gonad Program" or equivalent is selected for FHM.
4. After the tissue processor has completed the processing cycle, the
basket(s) may be transferred to the embedding station.
4. Embedding
Objective:
1. Properly orient the tissue in solidified paraffin for microtomy.
Materials:
1. Embedding station (thermal, dispensing and cryo consoles).
2. Paraffin heating pots.
3. Paraffin transfer pots.
4. Laboratory oven.
-------
5. Thermometer.
6. Embedding molds.
7. Block drawers.
8. Forceps.
9. Scraper.
10. Standard paraffin.
Procedures:
1. The cryo console of the embedding station is turned on. (Power to the
dispensing console and thermal console should remain on at all times.)
2. The basket(s) of cassettes is/are removed from the processor and
immersed in the paraffin-filled front chamber of the embedding station
thermal console.
3. The first cassette to be embedded is removed from the front chamber of
the thermal console. The cassette lid is removed and discarded, and the
cassette label is checked against the animal records to resolve potential
discrepancies prior to embedding.
4. An appropriately sized embedding mold is selected.
5. The mold is held under the spout of the dispensing console and filled with
molten paraffin.
6. The gonads are removed from the base of the cassette and are placed in the
molten paraffin in the mold. The two gonads (left and right) are oriented
horizontal to their long axis in the mold to allow for longitudinal
sectioning.
7. The base of the cassette is placed on top of the mold. Additional paraffin
is added to cover the bottom of the base.
8. The mold with the cassette base is placed on the cooling plate of the cryo
console.
9. After the paraffin has solidified, the block (i.e., the hardened paraffin
containing the tissues and the cassette base) is removed from the mold.
10. Steps 3 through 10 are repeated for each cassette to be embedded.
5. Microtomy
Objective:
1. Create and mount histologic sections for staining.
Materials:
1. Microtome.
2. Disposable microtome knives.
3. Lipshaw Pike® oil (or equivalent lightweight, machine oil).
4. Temperature-controlled water bath.
5. Ice.
-------
6. Microscope slides.
7. Staining racks.
8. Permanent slide marking pen.
9. Forceps.
10. Fine-tipped paint brush.
11. Temporary labels.
12. Slide warmer/oven.
Procedures:
1. The temperature in the water bath is allowed to stabilize so that ribbons
cut from the tissue blocks will spread out uniformly on the surface without
melting. This temperature assessment is a qualitative judgment made by
the microtomist before and during microtomy.
2. If necessary, a new blade is mounted onto the microtome and the
microtome is lubricated with oil.
3. The initial phase of microtomy is termed "facing" the block and is
conducted as follows:
a. The block is placed in the chuck of the microtome.
b. The chuck is advanced by rotating the microtome wheel and thick
sections are cut from the paraffin surface of the block until the knife
reaches the embedded tissues. This process is referred to as "rough
trimming" of the block.
c. The section thickness on the microtome is set between 4-10 microns.
The chuck is advanced and multiple sections are cut from the block to
remove any artifacts created on the cut surface of the tissue during
rough trimming. This process is termed "fast trimming" of the block.
d. The block is removed from the chuck and placed facedown on ice to
soak the tissue.
e. Steps a. through d. are repeated until all blocks to be microtomed have
been faced.
f. If it is determined during facing that any block is not of acceptable
quality for microtomy, it is returned for re-embedding before
proceeding with microtomy.
g. Any extraneous pieces of paraffin are removed from the microtome
and workstation periodically during facing and before proceeding with
the next phase of microtomy.
4. The next phase of microtomy is final sectioning and mounting of tissue
sections on slides. These procedures are conducted as follows:
a. Macroscopic lesions (if any) that are reported in the records are noted.
Care is taken to include any macroscopic lesions in the sections
collected during final sectioning.
b. The block is removed from the ice and placed in the chuck of the
microtome.
-------
c. With the section thickness on the microtome set to 4-5 microns, the
chuck is advanced by rotating the microtome wheel. Sections are cut
from the block until a "ribbon" containing at least one acceptable
section has been produced. As necessary during sectioning, the block
may be removed from the chuck, placed on ice to soak the tissue, and
replaced in the chuck.
d. Each ribbon is floated flat on the surface of the water in the water bath.
An attempt is made to obtain at least one section in the ribbon that
contains no wrinkles and has no air bubbles trapped beneath it.
e. A microscope slide is immersed beneath the best section in the floating
ribbon. The section is lifted out of the water using the slide. This
process is referred to as "mounting" the section on the slide.
f. A single slide is prepared for each fish. A total of three step sections
(each section consisting of both the right and left gonad) are mounted
on each slide. The first section is obtained at the point where
approximately half of the gonad has been removed and the size of the
section is maximized. For both the testis and the ovary, the second
and third sections are taken at 50 micron intervals following the first
section.
g. With a slide-marking pen, the block number from which the slide was
produced is recorded on the slide.
h. The slide is placed in a staining rack.
i. The block is removed from the chuck and placed facedown for storage.
j. Steps a. through h. are repeated for all blocks to be microtomed.
6. Staining, Coverslipping, and Slide Labeling
Objectives:
1. Differential staining of intra- and inter-cellular components of the gonads
to facilitate diagnostic examination by brightfield microscopy.
2. Permanently seal mounted and stained tissues.
3. Permanently identify stained sections in a manner that allows complete
traceability.
Materials:
1. Automated slide stainer (optional).
2. Robot coverslipping machine (optional).
3. Clarifier solution (Richard Allen or equivalent).
4. Bluing reagent (Richard Allen or equivalent).
5. Eosin-Y (Richard Allen or equivalent, Appendix C & Appendix D).
6. Hematoxylin-2 (Richard Allen or equivalent, Appendix C & Appendix D)
7. Xylene.
8. Absolute ethyl alcohol (100% ETOH).
9. 95% ETOH.
-------
10. 80%ETOH.
11. Coverslipping mountant (Permount or equivalent, Appendix D).
12. Glass coverslips, No. 1, 24 x 50 (or 60) mm (Appendix D).
13. Slide flats.
Procedures:
1. Staining
a. Slides are routinely air-dried overnight before staining.
b. An example H&E staining schedule for automated stainers is in
Appendix B, Schedule 2. A similar schedule can be adapted for
manual staining.
2. Coverslipping
a. Coverslips can be applied manually or automatically.
b. A slide is dipped in xylene, and the excess xylene is gently knocked
off the slide.
c. Approximately 0.1 ml of mounting medium is applied near the end of
the slide opposite to the frosted end.
d. A coverslip is tilted at a shallow angle as it is applied to the slide.
3. Labeling
a. Each slide label should contain the following information:
i. Laboratory name
ii. Species
iii. Specimen No. / Slide No.
iv. Chemical / Treatment group
v. Date (optional)
-------
II. GONADAL HISTOPATHOLOGY GLOSSARY AND DIAGNOSTIC CRITERIA
The purposes of this section are:
1) To provide general guidance for the light microscopic evaluation of tissue
sections;
2) To promote a common awareness of various pathological findings that may be
observed; and
3) To foster consistency in the use of diagnostic terminology.
General approach to reading studies
Studies are to be read by individuals experienced in reading toxicologic pathology
studies, and who are familiar with normal, small fish gonad histology, with gonadal
physiology, and with general responses of the gonads to toxicologic insult. Pathologists
may be board certified (e.g. American College of Veterinary Pathologists, The European
Centre of Toxicologic Pathology, or other certifying organizations), however certification
is not a requirement as long as the pathologist has obtained sufficient experience with,
and knowledge of, fish histology and toxicologic pathology. Technicians should not be
used to conduct readings due to the subtle nature of some changes and the need for
subjective judgments based on past experience.
It is recognized that there is a limited pool of pathologists with the necessary training and
experience that are available to read the gonadal histopathology for the 21-day
reproduction assay. If an individual has toxicological pathology experience and is
familiar with gonadal histology in small fish species, he/she may be trained to read the
fish assay. If pathologists with little experience are used to conduct the histopathological
analysis, informal peer review may be necessary.
Pathologists are to read the studies non-blinded (i.e. with knowledge of the treatment
group status of individual fish). However, it is expected that any potential compound-
related findings will be re-evaluated by the pathologist in a blinded manner prior to
reporting such findings, when appropriate. Certain diagnostic criteria, such as relative
increases or decreases in cell populations, cannot be read in a blinded manner due to the
diagnostic dependence on control gonads. As a rule, treatment groups should be
evaluated in the following order: Control, High-dose, Intermediate-dose, and Low-dose.
It is suggested that the pathologists be provided with all available information related to
the study prior to conducting their readings. Information regarding gross morphologic
abnormalities, mortality rates, and general test population performance and health are
useful for pathologists to provide comprehensive reports and to aid in the interpretation
of findings. For a more comprehensive discussion of standard reading approaches for
toxicologic pathology studies, please refer to the Society of Toxicologic Pathology Best
Practices for reading toxicologic histopathology studies (Crissman JW et al. 126-31).
10
-------
Diagnostic Criteria
Histopathology is a descriptive and interpretive science, and therefore somewhat
subjective. However, histopathologic evaluations of the same study by any qualified
pathologist should identify the same treatment-related findings (Crissman JW et al. 126-
31). Therefore, we aim to define the diagnostic criteria that will likely be encountered
during the histopathologic analysis of the 21-day reproduction assay in fathead minnow.
A consolidated set of diagnostic criteria follow. These criteria are based on pathologists'
experience with certain consistent histopathologic changes that occur in fathead minnow
gonads in response to chemical exposure, however novel findings that are exposure-
related shall also be reported.
The criteria below have been divided into two sections: 1. Primary criteria, and 2.
Additional criteria. The criteria are graded for severity on a numerical scale. Any novel
findings are either graded on a numerical scale, or are qualitatively described.
Primary Criteria - Males:
1. Increased proportion of spermatogonia: Increases in the proportion of
spermatogonia are consequent of changes of the relative ratios of spermatogenic
cells. This could be due to an increase in the number of spermatogonia, or a
decrease in the number of other cell types, such as spermatocytes, spermatids, and
spermatozoa. Because the diagnosis of increased proportion of spermatogonia is
dependant on a comparison to controls, it is necessary to establish the normal
range of the ratios of spermatogenic cells in control male fish testes prior to
making determinations on relative proportions in dose groups.
Presence of testis-ova. The presence of one or more individualized or clustered
oogenic cells within the testis. Oocytes within the testis may be determined to be
perinucleolar, cortical alveolar, vitellogenic, or atretic. There is little or no
evidence of ovarian architecture. Whenever applicable, the term testis-ova should
be used in preference to less precise terms such as "intersex" or "hermaphrodite".
3. Increased testicular degeneration: Testicular degeneration is characterized by 1)
individual or clustered apoptotic germ cells; 2) vacuolated germ cells; and/or 3)
multinucleated (syncytial) cells in the germinal epithelium or testicular lumen.
Apoptotic germ cells are characterized by cell shrinkage, nuclear condensation,
and fragmentation into spherical, membrane-bound bodies, which are often
phagocytized by neighboring cells. There is no inflammation associated with
these cells. If possible, testicular degeneration should be differentiated from
necrosis, which is characterized morphologically by cytoplasmic coagulation or
swelling, nuclear karyorrhexis or pyknosis, associated inflammation, a locally
extensive pattern of tissue involvement, and/or the involvement of different local
11
-------
tissue elements (e.g., both germinal and stromal tissues). Extensive testicular
degeneration may lead to localized or generalized loss of the germinal epithelium.
4. Interstitial cell (Leydig cell) hyperplasia/hypertrophy: An increase in number
and/or size of the interstitial cells responsible for producing androgens.
Interstitial cells may have larger, more rounded nuclei, and interstitial cell
aggregates may occupy and expand some interstitial spaces.
Primary Criteria - Females:
1. Increased oocyte atresia: An increase in degradation and resorption of oocytes at
any point in development. Atresia is characterized by clumping and perforation
of the chorion, fragmentation of the nucleus, disorganization of the ooplasm,
and/or the uptake of yolk materials by perifollicular cells.
2. Perifollicular cell hyperplasia/hypertrophy: Increase in the size or number of
granulosa, theca, and/or surface epithelium cells involved in a developing follicle.
Abnormal perifollicular cell hypertrophy must be distinguished from the normally
enlarged granulosa and theca cells of a post-ovulatory follicle.
3. Decreased yolk formation: A decrease in the amount of vitellogenic/yolk material
that is deposited in the developing oocyte. Decreased vitellogenesis is
characterized by the presence of oocytes in which yolk material is not present
despite their relatively large size. Note that oocytes may be affected to varying
degrees. Some affected oocytes have extremely fine vitellogenic granules, and
this is interpreted as ineffective vitellogenesis.
4. Change in gonadal staging: Gonadal staging results are virtually meaningless in
terms of individual fish (versus treatment groups). This is because considerable
animal-to-animal variation in gonad cell proportions is to be expected, even
among fish of the control groups, as a consequence of spawning cycle
asynchrony. Consequently, following the gonadal staging of individual fish, each
treatment group is assessed as a whole and compared to the appropriate control
group to determine if a compound-related effect has occurred. Hence, gonadal
staging cannot be performed in a blinded manner. Because the cell distribution
pattern is likely to vary throughout a given tissue section, the gonad should be
staged according to the predominant pattern in that section. Similarly, both
gonads should be staged as a single organ according to the predominant pattern.
Gonads that cannot be reasonably staged for various reasons (e.g., insufficient
tissue, or extensive necrosis, inflammation, or artifact) should be recorded as UTS
(unable to stage).
12
-------
Criteria for Staging Ovaries
• Juvenile: gonad consists of oogonia exclusively; it may be difficult or impossible
to confirm the sex of these individuals.
• Stage 0 - Undeveloped: entirely immature phases (oogonia to perinucleolar
oocytes); no cortical alveoli.
• Stage 1 - Early development: vast majority (e.g., >90%) are pre-vitellogenic
follicles, predominantly perinucleolar through cortical alveolar.
• Stage 2 - Mid-development: at least half of observed follicles are early and mid-
vitellogenic.
• Stage 3 - Late development: majority of developing follicles are late
vitellogenic.
• Stage 4 - Late development/hydrated: majority of follicles are late vitellogenic
and mature/spawning follicles; follicles are larger as compared to Stage 3.
• Stage 5 - Post-ovulatory: predominately spent follicles, remnants of theca
externa and granulosa.
Secondary criteria - males:
1. Decreased proportion of spermatogonia: Decreased relative proportion of
spermatogonia to other spermatogenic cell types. This can be due to a decrease in
the number of spermatogonia, or an increase in the number of other cell types,
such as spermatocytes, spermatids, and spermatozoa. Because the diagnosis of
decreased proportion of spermatogonia is dependant on a comparison to controls,
it is necessary to establish the normal range of the ratios of spermatogenic cells in
control male fish testes prior to making determinations on relative proportions in
dose groups.
2. Increased vascular or interstitialproteinaceous fluid. Homogenous dark pink
translucent material, presumably vitellogenin, within the testicular interstitium or
blood vessels. The presence of this fluid may cause a thickening of interstitial
areas that might be misinterpreted as "stromal proliferation".
3. Asynchronous gonad development: The presence of more than one developmental
phase of spermatogenic cell within a single spermatocyst enclosed by a Sertoli
cell. For example, this term may be applied to a spermatocyst that contains a
mixture of spermatocytes and spermatids, or a spermatocyst that contains more
than one meiotic phase of primary spermatocyte (i.e., leptotene, pachytene, and/or
zygotene). It also refers to the presence of distinctly different populations (i.e.
developmental phases) of gametogenic cells in the right and left gonads.
4. Altered proportions of spermatocytes or spermatids: A change in the relative
proportions of spermatocytes or spermatids to other spermatogenic cell types.
Changes in relative ratios could be due to an increase in the number of
13
-------
spermatocytes or spermatids, or to a decrease in the number of other cell types.
Relative changes may also occur between spermatocytes and spermatids.
5. Gonadal staging: Gonadal staging results are virtually meaningless in terms of
individual fish (versus treatment groups). This is because considerable animal-to-
animal variation in gonad cell proportions is to be expected, even among fish of
the control groups, as a consequence of spawning cycle asynchrony.
Consequently, following the gonadal staging of individual fish, each treatment
group is assessed as a whole and compared to the appropriate control group to
determine if a compound-related effect has occurred. Hence, gonadal staging
cannot be performed in a blinded manner. Because the cell distribution pattern is
likely to vary throughout a given tissue section, the gonad should be staged
according to the predominant pattern in that section. Similarly, both gonads
should be staged as a single organ according to the predominant pattern. Gonads
that cannot be reasonably staged for various reasons (e.g., insufficient tissue, or
extensive necrosis, inflammation, or artifact) should be recorded as UTS (unable
to stage).
Criteria for Staging Testes
• Juvenile: gonad consists of spermatogonia exclusively; it may be difficult or
impossible to confirm the sex of these individuals.
• Stage 0 - Undeveloped: entirely immature phases (spermatogonia to spermatids)
with no spermatozoa.
• Stage 1 - Early spermatogenic: immature phases predominate, but spermatozoa
may also be observed; the germinal epithelium is thinner than it is during Stage 2.
• Stage 2 - Mid-spermatogenic: spermatocytes, spermatids, and spermatozoa are
present in roughly equal proportions; the germinal epithelium is thinner than
Stage 1 but thicker than Stage 3.
• Stage 3 - Late spermatogenic: all stages may be observed, however, mature
sperm predominate; the germinal epithelium is thinner than it is during Stage 2.
• Stage 4 - Spent: loose connective tissue with some remnant sperm.
. Granulomatous inflammation: This process is characterized by the presence of
epithelioid macrophages that typically form sheets or nodules (granulomas) due to
desmosome-like cytoplasmic attachments (Noga et al., 1989). When compared to
histiocytic-type macrophages, epithelioid macrophages have larger, more open-
faced, centralized nuclei and less abundant cytoplasm. During resolution of
inflammation, the epithelioid macrophages may become flattened into fibrocyte-like
cells. Lymphocytes, granulocytes, and multinucleated giant cells may also be
components of granulomatous inflammation. Granulomatous inflammation is
intrinsically a pathologic process that is often associated with reactions to infectious
agents, foreign materials or the aftermath of necrosis; therefore, it is important to
distinguish this, if possible, from the presence of histiocytic cells in the lumen of
the testis.
14
-------
Secondary criteria -females:
1. Interstitial fibrosis: The presence of increased fibrous connective tissue (collagenous
fibers and fibrocytes or fibroblasts) within the ovarian interstitium (stroma). Collagen
may be difficult to appreciate in early phases of fibrosis.
2. Egg debris in the oviduct: The presence of inspissated-appearing, homogenous,
irregular, dense pink material, presumed to be yolk, within the oviduct.
3. Granulomatous inflammation: This process is characterized by the presence of
epithelioid macrophages that typically form sheets or nodules (granulomas) due to
desmosome-like cytoplasmic attachments (Noga et al., 1989). When compared to
histiocytic-type macrophages, epithelioid macrophages have larger, more open-faced,
centralized nuclei and less abundant cytoplasm. During resolution of inflammation,
the epithelioid macrophages may become flattened into fibrocyte-like cells.
Lymphocytes, granulocytes, and multinucleated giant cells may also be components
of granulomatous inflammation. Granulomatous inflammation is intrinsically a
pathologic process that is often associated with reactions to infectious agents, foreign
materials or the aftermath of necrosis; therefore, it is important to distinguish this, if
possible, from the presence of macrophage aggregates in the ovary.
4. Decreasedpost-ovulatory follicles: A decrease in the number of collapsed
perifollicular sheaths, or membranous structures lined by granulosa cells, theca cells
and surface epithelium, following release of oocytes, in comparison to control fish.
The granulosa cells are often hypertrophic, although this appears to be species
dependent (Saidapur, 1982).
Severity Grading
In toxicologic pathology, it is recognized that compounds may exert subtle effects on
tissues that are not adequately represented by simple binary (positive or negative)
responses. Severity grading involves a semi-quantitative estimation of the degree to
which a particular histomorphologic change is present in a tissue section (Shackelford et
al., 2002). The purpose of severity grading is to provide an efficient, semi-objective
mechanism for comparing changes (including potential compound-related effects) among
animals, treatment groups, and studies.
Severity grading will employ the following system:
Not remarkable
Grade 1 (minimal)
Grade 2 (mild)
15
-------
Grade 3 (moderate)
Grade 4 (severe)
A grading system needs to be flexible enough to encompass a variety of different tissue
changes. In theory, there are three broad categories of changes based on the intuitive
manner in which people tend to quantify observations in tissue sections:
Discrete: these are changes that can be readily counted. Examples include atretic
follicles, oocytes in the testis, and clusters of apoptotic cells.
Spatial: these are changes that can be quantified by area measurements. This
includes lesions that are typically classified as focal, multifocal, coalescing, or
diffuse. Specific examples include granulomatous inflammation and tissue necrosis.
Global: these are generalized changes that would usually require more sophisticated
measurement techniques for quantification. Examples include increased hepatocyte
basophilia, Sertoli cell/interstitial cell hypertrophy, or quantitative alterations in cell
populations.
General severity grading scale:
• Not Remarkable: This grade is used if there are no findings associated with a
particular diagnostic criterion.
• Grade 1: Minimal. Ranging from inconspicuous to barely noticeable but so
minor, small, or infrequent as to warrant no more than the least assignable grade.
For discrete changes, grade 1 is used when there are fewer than 2 occurances per
microscopic field, or 1-2 occurances per section. For multifocal or diffusely-
distributed alterations, this grade is used for processes where less than 20% of the
tissue in the section is involved.
Grade 2: Mild. A noticeable feature of the tissue. For discrete changes, grade 2
is used when there are 3-5 occurrences per microscopic field or per tissue section.
For multifocal or diffusely-distributed alterations, this grade is used for processes
where 30-50% of the tissue in the section is involved.
• Grade 3: Moderate. A dominant feature of the tissue. For discrete changes,
grade 3 is used when there are 6-8 occurrences per microscopic field or per tissue
section. For multifocal or diffusely-distributed alterations, this grade is used for
processes where 60-80% of the tissue in the section is involved.
• Grade 4: Severe. An overwhelming feature of the tissue. For discrete changes,
grade 4 is used when there are more than 9 occurrences per microscopic field or
per tissue section. For multifocal or diffusely-distributed alterations, this grade is
used for processes where greater than 80% of the tissue in the section is involved.
At the discretion of the pathologist, the severity of a given change should be scored
16
-------
according to one of the following two methods: 1) score compound-exposed animals
relative to the severity of the same change in control animals, or 2) score all animals
relative to "normal" as determined by the pathologist's experience. For each important
(i.e., treatment-associated) finding, the method that was used should be stated in the
Materials and Methods section of the pathology narrative report (see Histopathology
Report Format). By convention, severity grading should not be influenced by the
estimated physiologic importance of the change. For example, the presence of two
oocytes in the testis should not be graded as "severe", even if the pathologist considers
this finding to be highly significant in terms of endocrine modulation.
Data recording
An Excel worksheet form has been created that includes worksheets for primary,
secondary, and additional diagnoses to facilitate histopathology data collection. In this
worksheet, each data entry cell represents an individual fish. Additional sheets are
available for comments and additional findings. For each fish, the pathologist records a
severity score associated with the diagnosis (see Severity Grading). Diagnostic criteria
with non-remarkable findings shall be denoted using (-). If there is no reasonably
appropriate diagnostic term for a particular finding, the pathologist can create a term that
can be recorded in the "Additional diagnoses" worksheet. If insufficient tissue is
available for diagnosis, this should be recorded as IT (insufficient tissue). If a target
tissue is missing, this should be recorded as MT (missing tissue).
Adding a Modifier term to a diagnosis may help to further describe or categorize a
finding in terms of chronicity, spatial distribution, color, etc. In many instances,
modifiers are superfluous or redundant (e.g., fibrosis is always chronic); therefore, the
use of modifiers should be kept to a minimum. An occasionally important modifier for
evaluating paired gonads is unilateral (UNI); unless specified in this manner, all gonad
diagnoses are assumed to be bilateral. Other modifier codes can be created as needed by
the pathologist.
17
-------
Histopathology Report Format
Each histopathology narrative report should contain the following five sections:
Introduction, Materials and Methods, Results, Discussion, Summary/Conclusions.
A References section can also be included if applicable. The Introduction section
briefly outlines the experimental design. The Materials and Methods section describes
any items or procedures that are essentially different from Section 1: Post-mortem and
Histotechnical Procedures. As applicable, specific severity grading criteria (see Severity
Grading) should also be listed in this section. The Results section should report findings
that are: 1) compound-related; 2) potentially compound-related; 3) novel or unusual.
Detailed histomorphologic descriptions need only be included for findings that differ
substantially from diagnoses presented in Section IIB, Glossary and Diagnostic Criteria.
It is intended that the Results section should be as objective as possible (i.e., opinions and
theories should be reserved for the Discussion section). The Discussion section, which
contains subjective information, should address relevant findings that were reported in
the Results section. Opinions and theories can be included in this section, preferably
backed by references from peer-reviewed sources, but unsupported speculation should be
avoided. The Summary/Conclusions section should encapsulate the most important
information from the Results and Discussion sections.
Glossary / Diagnostic Criteria
The purposes of this section are: 1) to provide photomicrographs of normal gonadal
structure in fathead minnow, 2) to provide a common technical "language" and 3) to
create a reference atlas of both microanatomical structures and potential pathological
findings. The information in this section is derived from a number of sources including
scientific articles, conference proceedings, related guidelines, toxicologic pathology
textbooks, medical dictionaries, and the personal experience of various fish pathologists.
Regarding the last, opinions were solicited via a questionnaire that was circulated among
conference participants following the October 2003 meeting of the histopathology
subcommittee of the Fish Discussion Group in Paris. Consensus replies to this
questionnaire form the basis for naming many of these terms. Other considerations
include traditional usage and scientific precedence, and attributes such as clarity and
brevity.
The section is arranged as follows:
1. Normal testicular architecture in fathead minnow
2. Normal ovarian architecture in fathead minnow
3. Primary diagnoses - males
4. Primary diagnoses - females
5. Additional diagnostic criteria and an illustrated glossary of microanatomical
and diagnostic terms
18
-------
Normal Testicular Architecture in Fathead Minnow
Spermatogenic Cell Types:
Spermatogonia. Spermatogonia A in a male
FHM (GMA, H&E).
Syermatosonia: The largest of the spermatogenic
cells (~ 5-10 um), Spermatogonia generally have pale
vesicular nuclei, prominent nucleoli, variably distinct
nuclear membranes, perinuclear cytoplasmic granules,
and moderate amounts of granular cytoplasm.
Spermatogonia B are smaller than Spermatogonia A,
and Spermatogonia B are usually present in larger
clusters (e.g., > 4 cells). If at all possible, an attempt
should be made to classify these cells as
Spermatogonia rather than to label them with a non-
specific term such as "pale cells" or "light cells".
Spermatocytes. FHM testis (GMA, H&E).
Syermatocytes: Derived from Spermatogonia,
spermatocytes are of intermediate cell size (~ 4-6 um), and
have comparatively dense nuclei and minimal to moderate
amounts of indistinct cytoplasm. Spermatocyte nuclei are
usually evident in one of three meiosis phases: pachytene,
leptotene, or zygotene. Primary spermatocytes are larger
than secondary spermatocytes, and the latter are derived
from primary spermatocytes following the first meiotic
division. Spermatocytes are usually one of the most
abundant spermatogenic cells, and they tend to contribute
to the largest spermatocysts.
Spermatids. Spermatids in a male FHM. Intercellular
attachments are lost just prior to rupture of the spermatocyst
and release of these cells as spermatozoa (GMA, H&E).
Spermatids: Derived from spermatocytes
following the second meiotic division,
these cells have dense nuclei and narrow
rims of eosinophilic cytoplasm. They are
the smallest cells within the germinal
epithelium (~ 2-3 um), and the cells lose
their cytoplasmic attachments to one
another during spermiogenesis.
19
-------
Spermatozoa. Spermatozoa in a
male FHM (GMA, H&E).
Spermatozoa: These cells have dark, round nuclei and
minimal or no apparent cytoplasm. Tails are generally not
apparent in histologic sections. Spermatozoa are the
smallest spermatogenic cells (~ 2 um), and they exist as
scattered individual cells within tubular lumen.
Sertoli cells: These cells tend to have sharply-defined
elongated or triangular nuclei, variably evident
nucleoli, and cytoplasm that is often indistinct. The
cytoplasmic arms of a Sertoli cell encircle a clonal
group of spermatogenic cells, forming a spermatocyst.
Compared to germinal cells, Sertoli cells are usually
present in low numbers, usually as single cells located
adjacent to lobular septa. In some instances,
hypertrophic (enlarged, swollen) Sertoli cells may
resemble spermatogonia.
Sertoli cells (FHM, GMA, H&E).
20
-------
Interstitial (Leydis) cells: These
cells have dense, dark round or
oval nuclei with little detail and
moderate amounts of variably-
evident, faintly vacuolated
cytoplasm. Compared to
germinal cells, interstitial cells are
usually present in low numbers,
usually as single cells or small
aggregates, within the interlobular
interstitium. Although they may
resemble spermatocytes,
interstitial cells are only present
in interlobular areas.
Interstitial cells (FHM, H&E). Interstitial cells (small arrows)
are only found in interlobular areas. Note the resemblance
between these cells and spermatocytes (large arrow).
Spermatocyst: The functional unit of the testis, this
structure consists of a clonal group of spermatogenic
cells (spermatogonia, spermatocytes, or spermatids)
that are surrounded by the cytoplasmic arms of
(usually) one Sertoli cell. Cells within spermatocysts
exist as syncytia, maintained by intercellular
attachments (cytoplasmic bridges), until final
maturation and release of spermatozoa occurs
(spermiogenesis) (Grier, 1976).
Spermatocyst (FHM, adult male, plastic, H&E).
A group of dissociated spermatids are surrounded
by the cytoplasmic "arms" of a single Sertoli
cell (arrow). This arrangement is usually not
as obvious as it is in this photograph. The
nucleus of this particular Sertoli cell appears
enlarged (hypertrophic).
21
-------
am
wSa t».
Spermatocysts (FHM, adult male, GMA, H&E). Spermatocysts outlined in red and green contain
spermatocytes and spermatids, respectively. Spermatogonia (black arrows) and spermatozoa within tubular
lumina (blue arrows) are also indicated.
%'4sgp ¥m^m^ ;*ss®m •
$P* *--id
k, ,4lS
Germinal epithelium (male): The
germinative intratubular (intralobular)
parenchyma of the testis, this
membrane-bound structure consists of
multiple Spermatocysts in various
phases of development. For FHM,
boundaries of the germinal epithelium
at various locations throughout the
testis include the interlobular
interstitium, the lobular lumina,
collecting ducts, and the tunica
albuginea.
Germinal epithelium, male. Normal testis from an adult FHM.
Double arrow indicates width of germinal epithelium, which
extends from the interlobular interstitium to the lobular lumen
(GMA = glycol methacrylate, H&E, bar = 25 urn).
22
-------
Normal Ovarian Architecture in Fathead Minnow
Oogenic Cell Types:
Oosonia: These cells represent the replicative
pool of the ovary. Unlike mammalian oogonia
(although this dogma may soon change based
on recent data from rodent research), piscine
oogonia continue to divide in juveniles and
adults. The smallest of the oocytic cells,
oogonia reside within the ovarian germinal
epithelium, usually in comparatively low
numbers. Oogonia are characterized by a
relatively large nucleus with small or
inapparent nucleolus, and minimal amounts of
cytoplasm.
Oogonia (FHM, paraffin, H&E, bar = 10 um). A small cluster
of oogonia reside within a portion of germinal epithelium; the
nucleus of only one oogonium is visible (small arrow). The
oogonia are dwarfed by a perinucleolar oocyte (large arrow).
Chromatin nucleolar oocytes: Slightly larger
than an oogonium, this oocyte is formed when
an oogonium becomes surrounded by
prefollicle cells (presumptive granulosa cells)
and the resulting complex buds from the
germinal nest as a primordial follicle. The
chromatin nucleolar oocyte has a relatively
large nucleus that contains a single, large
nucleolus. Compared to an oogonium, there is
more cytoplasm which is slightly more dense
and finely granular.
Chromatin nucleolar oocyte (FHM, paraffin, H&E, bar = 10 um).
A single chromatin nucleolar oocyte protrudes from the germinal
epithelium (arrow).
23
-------
Perinucleolar oocytes: Concomitant with
oocyte growth, the nucleus (germinal vesicle)
increases in size and multiple nucleoli appear,
generally at the periphery of the nucleus. The
cytoplasm stains uniformly dark, although late
perinucleolar oocytes may have small clear or
amphophilic vacuoles in the cytoplasm. These
cells tend to be abundant in normal adult
ovaries.
Perinucleolar oocytes. Several perinucleolar oocytes
in the ovary of a FHM. Arrows indicate nucleoli at the
periphery of the germinal vesicle (paraffin, H&E, bar
= 25 um).
Cortical alveolar oocytes: Generally larger
than perinucleolar oocytes, this phase is
characterized by the appearance of cortical
alveoli (yolk vesicles) within the ooplasm. The
cortical alveoli are technically not yolk, as they
do not provide nourishment for the embryo
(Selman and Wallace, 1989). The chorion
becomes distinctly evident in this phase, and
the perifollicular cells are more easily
visualized.
Cortical alveolar oocytes. FHM ovary demonstrating
multiple cortical alveolar oocytes. The cytoplasm is
predominately filled by numerous cortical alveoli, which
are amphophilic with this preparation. Evident are oocytes
in transition from the perinucleolar to cortical alveolar
phase (small arrow), and from the cortical alveolar to early
vitellogenic phase (large arrow) (paraffin, H&E, bar
= 100 um).
24
-------
Early vitellogenic oocytes. In this FHM ovary,
numerous fine pale pink granules (large arrow), and a
few larger dark red granules (small arrow), are evident
in the central region of an early vitellogenic oocyte.
Although nuclei are present they are not apparent in
every oocyte due to the comparatively vast amount of
cytoplasm (paraffin, H&E, bar = 100 um).
Late vitellogenic oocytes. Late vitellogenic oocyte
in a FHM ovary. The yolk granules almost fill the
ooplasm. The nucleus has not yet begun to migrate
peripherally (paraffin, H&E, bar =100 um).
Early vitellosenic oocytes: Larger than
cortical alveolar oocytes, these cells are
characterized by the centralized appearance of
spherical, eosinophilic, vitellogenic yolk
granules / globules. In H&E sections,
accumulations of fine yolk granules in the
central region of the oocyte may somewhat
resemble (and thus be confused with) the
reddish nucleus.
Late vitellosenic oocytes: These cells are
characterized by an increased accumulation of
vitellogenic granules that displace the cortical
alveolar material to the periphery of the
cytoplasm. It is during this stage that the
nucleus begins to migrate toward the periphery
of the cell.
25
-------
Mature/spawning oocytes. Two mature/spawning
oocytes in a FHM ovary. The oocytes and the yolk
granules have attained their maximum size just prior
to spawning, and the nucleus is not evident (paraffin,
H&E, bar = 100 urn).
Zona radiata
Mature/spawning oocytes: In this phase of
development, vitellogenesis has reached its peak,
the cell has become larger and more hydrated,
and the nucleus has migrated toward the
periphery of the cell and is in the process of
dissolution. The loss of nucleus is not a very
helpful diagnostic feature, however, as the
nucleus is often not visible in larger oocytes due
to the plane sectioning. Because of the transient
nature of these cells in fractional spawning fish,
mature/spawning oocytes are uncommonly
observed.
Ooplasm
Grant losa
Oocyie
Follicle
Diagram of an ovarian follicle. From Tyler and
Sumpter, 1996.
Ovarian follicle: The functional unit of the ovary,
this term generally refers to an oocyte plus its
surrounding sheath of perifollicular cells (granulosa
cells, theca cells, and surface epithelium cells)
(Tyler and Sumpter, 1996). However, there are
subtypes of follicles in which the oocyte is not
present or may be difficult to appreciate; these
include post-ovulatory (spent), empty, and atretic
follicles. A post-ovulatory follicle (the follicle has
ruptured to release an oocyte during spawning) is
collapsed and often has enlarged (hypertrophic)
granulosa and theca cells. Conversely, an empty
follicle (in which the oocyte has been dislodged
from the histologic section as a post-mortem
artifact) generally retains the shape of the oocyte
and may or may not have enlarged granulosa and
theca cells. An atretic follicle must be distinguished
from both spent follicles and empty follicles; the
presence of at least some ooplasmic material (often
heterochromatic) within a follicle indicates that it
contains an atretic oocyte.
26
-------
Perifollicular cells. In this photomicrograph, the perifollicular cells
are compared to the cells of the ovarian wall epithelium, which
contains dark brown (melanin) pigment (arrow) and is comprised of
ciliated columnar cells in FHM. FHM, adult female, paraffin, H&E,
bar = 25 urn)
Perifollicular cells: These cells form a three-
layered sheath around each oocyte, and combined
with the oocyte itself comprise the ovarian
follicle. These layers are more easily visualized
as the oocyte matures. The innermost layer
consists of the granulosa cells, the middle layer
consists of the theca cells, and the outermost layer
consists of the surface epithelial cells. The
granulosa cells especially may become enlarged
and vacuolated following ovulation or during
oocyte atresia. The perifollicular sheath should
not be confused with folds of the ovarian wall
epithelium.
Chorion: Usually pale to dark eosinophilic and
refractile, the chorion is the thick external layer of
an oocyte that surrounds the ooplasm. The terms
zona radiata and vitelline envelope have been
used synonymously. In mature, unspent follicles,
the chorion is noticeably surrounded by
perifollicular cells (granulosa cells, theca cells,
and surface epithelial cells). As viewed by light
microscope, the chorion is often minimally
apparent or inapparent prior to the cortical
alveolar phase of oocyte development.
Chorion. The chorions of two oocytes are indicated by large arrows.
A smaller arrow denotes a post-ovulatory follicle. (FHM, paraffin,
H&E, bar = 25 urn).
27
-------
Post-ovulatory follicle: A collapsed
perifollicular sheath following release of the
oocyte; this is a membranous structure lined by
granulosa cells, theca cells, and surface
epithelium. The granulosa cells are often
hypertrophic, although this appears to be species
dependent (Saidapur, 1982). Mammalian terms
such as "corpus lutea" and "Graafian follicles",
are probably less desirable, due to structural and
functional differences between these entities and
piscine post-ovulatory follicles. Whenever
possible, post-ovulatory follicles should be
differentiated from atretic follicles, the latter of
which contains oocyte debris.
Post-ovulatory follicle. Situated between three oocyte-containing
follicles is a collapsed follicle that does not contain oocyte remnants
(arrows) (FHM, adult female, paraffin, H&E, bar = 25 um).
28
-------
Primary Diagnoses - males:
Increased proportion of spermatogonia:
Increased cells, spermatogonia. A:. Testis from adult male
FHM negative control (GMA, H&E, bar = 25 urn).
BI Spermatogonia dominate the germinal epithelium in this
testis from adult male FHM exposed to 10 nM E2 for 10 days.
Other diagnoses for this section include "Decreased cells,
spermatocytes", "Decreased cells, spermatids" (GMA, H&E,
bar = 25 urn).
Increased proportion of spermatosonia: It is
recognized that endocrine active compounds may
alter the proportional distribution of gametogenic
cell types in the testis or ovary. Certain types of
alterations (for example, the proliferation or
absence of single cell population) may not be
adequately documented by gonadal staging. This
diagnostic term provides a mechanism for
documenting such changes. Quantitative
alterations are: 1) relative to other cell types in
the gonad; 2) relative to cell numbers in control
animals; and 3) estimates only, versus actual cell
counts.
29
-------
Grade 1
Grade 2
Grade 3
Grade 4
Testis from an adult male FHM. There is a minimal
(Grade 1) increase in the proportion of spermatogonia
(arrows). H&E.
Testis from an adult male FHM. There is a slight/mild
(Grade 2) increase in the proportion of spermatogonia
throughout the germinal epithelium. H&E.
Testis from an adult male FHM. There is a moderate
(Grade 3) increase in the proportion of spermatogonia.
H&E.
Testis from an adult male FHM. There is a severe (Grade
4) increase in the proportion of spermatogonia. H&E.
30
-------
Presence of testis-ova: An example of testicular oocytes is not available for FHM.
Increased testicular degeneration:
Testicular degeneration. Multiple clusters of apoptotic germ
cells (black arrows) and vacuolated germ cells (red arrow) within
the germinal epithelium. (FHM, adult male, GMA,H&E, bar =
25 urn).
Testicular degeneration: Examples of
degenerative findings in the testis include: 1)
individual or clustered apoptotic germ cells; 2)
vacuolated germ cells; 3) multinucleated
(syncytial) cells in the germinal epithelium or
testicular lumen. These diagnoses may be
"lumped" together under the term testicular
degeneration. Apoptotic germ cells are
characterized by cell shrinkage, nuclear
condensation, and fragmentation into spherical,
membrane-bound bodies, which are often
phagocytized by neighboring cells. There is no
inflammation associated with these cells. If
possible, testicular degeneration should be
differentiated from necrosis, which is
characterized morphologically by cytoplasmic
coagulation or swelling, nuclear karyorrhexis or
pyknosis, associated inflammation, a locally
extensive pattern of tissue involvement, and/or
the involvement of different local tissue elements
(e.g., both germinal and stromal tissues).
Extensive testicular degeneration may lead to
localized or generalized loss of the germinal
epithelium.
Interstitial cell (Leydig cell) hyperplasia/hypertrophy:
Non-Remarkable
Non - remarkable testis from males FHM. Interstitial
areas contain small aggregates of interstitial (Leydig) cells
(arrows). Most interstitial cells have wispy, pale
cytoplasm. H&E.
31
-------
Grade 1
_
f*-;n£^
Grade 2
Testis from an adult male FHM. Interstitial cell
aggregates (arrows) in the testis of this fish are larger than
in control fish, and the cytoplasm of these cells is slightly
more dense. This was diagnosed as Increased Cells,
Interstitial Cells, Grade 1 (minimal) severity. H&E.
Testis from an adult male FHM. Interstitial cell aggregates
(arrows) in the testis of this fish are larger than in control
fish, and the cells tend to fill and expand the interstitial
spaces. This was diagnosed as Increased Cells, Interstitial
Cells, Grade 2 (mild) severity. H&E.
32
-------
Primary diagnoses - females:
Increased oocyte atresia:
LV = late vitellogenic
EA = early atretic
LA = late atretic
ve = vitelline envelope
LA
ve
Oocyte atresia, increased, immature /mature:
Degradation and resorption of an oocyte at any
point in development. Histopathologically,
atresia is often characterized by clumping and
perforation of the chorion, fragmentation of the
nucleus, disorganization of the ooplasm, and/or
the uptake of yolk materials by perifollicular
cells (FHM, e.g.). Separate diagnoses and
severity grades can be given to atretic oocytes
that are mature ("Oocyte atresia, increased,
mature") versus immature ("Oocyte atresia,
increased, immature"). In this context, oocytes
will be considered "mature" if they are appear
to have been interrupted in either the late
vitellogenic oocyte phase or mature / spawning
phase of development.
Oocyte atresia, mature oocytes. Note clumping and pore-formation
in the vitelline envelope (chorion) of the early atretic oocyte (large
arrow), and the vacuolar hypertrophy of its surrounding granulosa
cells (small arrows). Compare these with granulosa cells that
surround a non-atretic late vitellogenic oocyte (arrowheads). In FHM,
granulosa cells of atretic oocytes often appear to contain phagocytized
material, whereas the granulosa cells of non-atretic oocytes do not.
(FHM, adult female, paraffin, H&E).
Non Remarkable
Ovary from a control group female. H&E.
33
-------
Grade 3
Ovary from an adult female FHM. Numerous atretic
oocytes are evident (arrows). H&E.
Grade 4
.
Stage 4 ovary from an adult female FHM. This
ovary is characterized by severe oocyte atresia.
Asterisks indicate the relatively few non-atretic
oocytes. H&E.
Perifollicular cell hyperplasia/hypertrophy: An example of perifollicular cell
hyperplasia/hypertrophy is not currently available for FHM.
Decreased yolk formation:
Not Remarkable
-
Ovary (Stage 3) from a control group female. A single
atretic ovary is evident (arrow). H&E, bar = 250 microns.
34
-------
Grade 3
„ f
>:.-
O'
Ovary from an adult female FHM. Decreased yolk
formation is characterized by the presence of oocytes in
which yolk material is not present despite their relatively
large size (large arrows). Note that oocytes are affected to
varying degrees. Some affected oocytes have extremely
fine vitellogenic granules (small arrow), and this is
interpreted as ineffective yolk formation and deposition.
H&E. bar = 250 microns.
Change in ovarian staging: Photographic examples are not currently available that
demonstrate changes in ovarian staging.
35
-------
Additional diagnostic criteria and an illustrated glossary of microanatomical and
diagnostic terms
Asterisks denote "secondary" diagnoses for male and female FHM
** Asynchronous development, gonad (male or female): The presence of distinctly
different populations (i.e., range of developmental phases) of gametogenic cells in
different regions of a gonad.
Asynchronous development, spermatocyst (male): The presence of more than one
developmental phase of spermatogenic cell within a single spermatocyst. For example,
this term may be applied to a spermatocyst that contains a mixture of spermatocytes and
spermatids, or a spermatocyst that contains more than one meiotic phase of primary
spermatocyte (i.e., leptotene, pachytene, and/or zygotene).
Asynchronous development, right and left gonads (male or female): The presence of
distinctly different populations (i.e., developmental phases) of gametogenic cells in the
right and left gonads.
**Egg debris, oviduct: The presence of inspissated-appearing, homogenous, irregular,
dense pink material, presumed to be yolk, within the oviduct.
D = debris
Ov = oviduct
Egg debris, oviduct. (FHM, adult female, paraffin, H&E).
Gender: Because the genetic sex of a fish cannot be determined within the context of a
screening assay, and because the external phenotypic sex may be an unreliable indicator
and/or is not easily determined in some species, by convention the gender of a fish will
be assigned according to the most abundant mature cell type that is present in the gonad.
36
-------
Germinal epithelium (female): The germinative parenchyma of the ovary is a
membrane bound structure constitutively contains oogonia, prefollicular and prethecal
cells, epithelial cells, and occasionally small chromatin nucleolar (primary growth)
oocytes (Norberg et al., 2000; Parent and Grier, 2003). The germinal epithelium
separates the ovarian lumen from the stroma, the latter of which often contains
perinucleolar, cortical alveolar, and vitellogenic follicles within a variably-apparent
extravascular space.
Germinal epithelium, atrophy / hypoplasia (male): Indicating loss or
underdevelopment of germinal epithelium, respectively, this condition may be associated
with interstitial fibrosis and increased prominence of interstitial cells in affected areas of
the testis. It may be difficult to distinguish atrophy from hypoplasia. Care should be
taken to avoid mistaking areas of collecting ducts for atrophy. Severity of this finding can
vary from Grade 1 (minimum, focal) to Grade 4 (severe, diffuse). If thinning of the
epithelium appears to be caused by degenerative changes that are obvious in the section,
the diagnostic term testicular degeneration should be used instead.
Germinal epithelium, atrophy. A: Atrophy
of germinal epithelium in an adult male
FHM. Also note the prominence of
interstitial (Leydig) cells (red circles) and
interstitial fibrosis. (GMA, H&E, bar = 25
Dm). BI Normal FHM testis (GMA, H&E,
bar = 25 Dm). Ci Normal collecting duct
region in an adult male FHM (GMA, H&E,
bar = 25 Dm). The presence of pigment in
the duct walls and the lack of interstitial cells
are distinguishing features.
37
-------
**Granulomatous inflammation: In the early stages of inflammation, this process is
characterized by the presence of epithelioid macrophages that typically form sheets or
nodules (granulomas) due to desmosome-like cytoplasmic attachments (Noga et al.,
1989). When compared to histiocytic-type macrophages, epithelioid macrophages have
larger, more open-faced, centralized nuclei and less abundant cytoplasm. During
resolution of inflammation, the epithelioid macrophages may become flattened into
fibrocyte-like cells. Lymphocytes, granulocytes, and multinucleated giant cells may also
be components of granulomatous inflammation. Granulomatous inflammation is
intrinsically a pathologic process that is often associated with reactions to infectious
agents, foreign materials, or the aftermath of necrosis; therefore, it is important to
distinguish this, if possible, from the presence of macrophage aggregates in the ovary or
histiocytic cells in the lumen of the testis.
Granulomatous inflammation. A^ Sheets of macrophages and other inflammatory cells eclipse much of
the germinative tissue in this testis (FHM, adult male, paraffin, H&E). B: Relatively few viable-appearing
oocytes remain in this ovary. As in the testis photo, the inciting cause of the inflammation is not evident at
this magnification (FHM, adult female, paraffin, H&E).
Hepatocyte basophilia, increased / decreased: A generally diffuse increase in
hepatocyte cytoplasmic basophilia has been observed in male fish that have been exposed
to compounds that are able to interact with hepatic estrogen receptors, including E2 and
17a-methyldihydrotestosterone (Wester et al., 2003). This increase in basophilia, which
is correlated with increased vitellogenin production, presumably mimics the heightened
metabolic state (e.g., increased endoplasmic reticulum) that is required for the production
of vitellogenin in the reproductively-active female fish.
38
-------
Hepatocyte basophilia A:. The liver from an adult male FHM
control. In addition to the overall coloration, note the
hepatocyte cytoplasmic vacuolization as indicated by the arrows.
Ifc Liver from an adult male FHM that was exposed to a compound
with estrogenic activity. There is a diffuse increase in hepatocyte
basophilia, a loss of cytoplasmic vacuolization, and hepatic blood
vessels contain proteinaceous fluid (arrows). (FHM, paraffin, H&E).
39
-------
Histiocytic cells (male): The presence of individual or clustered cells with small
eccentric nuclei and moderate to abundant, pale or vacuolated cytoplasm within the
testicular lumen, germinal epithelium, efferent ducts and/or ductus deferens. Such cells
may contain intracytoplasmic cellular debris (presumably phagocytized). The origin of
the histiocytic cells in each particular case may not be clear; for example, they may be
hematogenous macrophages or Sertoli cells. Histiocytic cells should be differentiated
from macrophage aggregates (these variably pigmented cells are primarily interstitial)
and granulomatous inflammation (which is predominately comprised of "epithelioid"
macrophages and/or flattened, fibrocytic cells).
ym •*& m
]^p.c ^t • .4m
^•tf*" "^^i''^'''-*. »^1.
't:r-' ;.
••»-/ '
Histiocytic cells. (FHM, adult male, GMA, H&E). A^ Cells with small
peripheral nuclei and abundant vacuolated cytoplasm are present within
the germinal epithelium and are scattered throughout the tubule lumen
(arrows). Some of these cells contain phagocytized cellular debris.
Ifc Similar cells are evident within the lumen of the collecting duct.
40
-------
Interstitial fibrosis (male or female): The presence of increased fibrous connective
tissue (collagenous fibers and fibrocytes or fibroblasts) within the testicular or ovarian
interstitium (stroma). Collagen may be difficult to appreciate in early phases of fibrosis.
In most cases, this term should be used in preference to terms such as "stromal
hyperplasia."
Macrophage aggregates: These cell clusters are constitutively present in the interstitium
of the ovary primarily, although they may also be found in the fish testis (unusual for
tank-raised FHM). These phagocytes usually have small condensed eccentric or
peripheralized nuclei and various brown, yellow, red, or gold pigmented granules
(lipofuscin, ceroid, hemosiderin, and/or melanin) that often impart a slightly crystalline
appearance to their comparatively abundant pale cytoplasm. In the normal ovary,
macrophage aggregates are thought to be involved in the processing of breakdown
products associated with atresia of unspawned oocytes. It has been demonstrated that
macrophage aggregates may become larger and/or more numerous following exposure to
certain toxicants or infectious agents (Blazer et al., 1987). Whenever possible,
macrophage aggregates should be distinguished from granulomatous inflammation,
which is characterized by the presence of epithelioid macrophages. This is not always
easy, as macrophage aggregates often proliferate with, and become incorporated into,
granulomatous inflammation.
I*,- ^ **"
Macrophage aggregates. The arrows indicate multiple aggregates within
the ovarian interstitium. (FHM, adult female, paraffin, H&E).
41
-------
Nephropathy: Degenerative renal disease has been observed in a variety of fishes that
have been exposed to compounds with estrogenic activity (Herman & Kincaid, 1988;
Zillioux et al., 2001; Palace et al., 2002). Renal impairment presumably occurs due to
increased production of vitellogenin (especially in males) that stresses the kidney via
protein overload. Microscopic lesions may include swelling of tubular epithelial cells,
tubular necrosis, dilation of Bowman's capsule, interstitial fibrosis, casts, and hyaline
droplets in tubules or glomeruli.
.
**^»
••• /%;*-.'
.. -*.- .»••. - .'
•Sfe
Nephropathy. A:. Kidney from an untreated adult male FHM. Ifc Kidney from an adult male FHM
exposed to a compound with estrogenic activity. Changes include glomerular epithelial cell hypertrophy,
vacuolar swelling and necrosis of the tubular epithelium, and hyaline droplets within glomerular and
tubular epithelia (paraffin, H&E, bar = 25 um).
Ovarian spermatogenesis: The presence of non-neoplastic spermatogenic cells, usually
immature, within the ovary. There is little or no evidence of lobular or tubular testicular
architecture. Care should be taken to distinguish ovarian spermatogenesis from
mitotically dividing oogonia; a key feature of ovarian spermatogenesis is the presence of
multiple spermatogenic phases.
42
-------
Mitotically dividing oogonia. Packets of cells that resemble spermatocytes (arrow)
are situated between perinucleolar and cortical alveolar oocytes. This should not be
mistaken for spermatogenesis. (FHM, adult female, paraffin, H&E).
**Proteinaceous fluid, interstitial (male or female): Homogenous dark pink
translucent material, presumably vitellogenin, within the testicular or ovarian interstitium.
In male fish especially, this finding has been associated with exposure to estrogenic
substances. The presence of this fluid may cause a thickening of interstitial areas that
might be misinterpreted as "stromal proliferation".
Proteinaceous fluid, interstitial. There is homogenous
dark pink material in interstitial spaces (arrows). (FHM, adult
female, paraffin, H&E, bar = 50 um).
43
-------
**Proteinaceous fluid, intravascular (male or female): Homogenous dark pink
translucent material, presumably vitellogenin, within testicular or ovarian blood vessels.
In male fish especially, this finding has been associated with exposure to estrogenic
substances.
Proteinaceous fluid, intravascular. There is
homogenous dark pink material within large and small
bloodvessels (arrows). Also note the increased
proportion of spermatogonia in the testis. (FHM,
adult male, GMA, H&E, bar = 25 urn).
Sertoli cell hypertrophy: Exposure of male fish to estrogen-active compounds has been
reported to cause enlargement of Sertoli cells, with or without Sertoli cell proliferation
(Miles-Richardson et al., 1999a; Miles-Richardson et al., 1999b; Kinnberg et al., 2000;
van der Ven et al., 2003). In the scientific literature, the light microscopic appearance of
hypertrophic Sertoli cells tends to be ambiguous, as Sertoli cells resemble spermatogonia
in some descriptions and images.
Vitellogenic oocyte: An oocyte that contains microscopically visible yolk material.
Generally, such material is strongly eosinophilic and slightly refractile in hematoxylin-
and eosin-stained sections. This material may be present in the form of spherical,
globular, yolk granules. In some scholarly sources (e.g., Iwamatsu, et al., 1998), the term
"vitellogenic" has been applied to cortical alveolar oocytes, which lack eosinophilic yolk
granules/globules (although their amphophilic or clear cortical alveoli are also known as
yolk vesicles).
GONDAL STAGING CRITERIA
The goal of gonadal staging is to determine if the administration of a particular
endocrine-active substance affects the reproductive cycle status of adult male and female
fathead minnows. The purpose of this section is to describe a rapid, semi-quantitative
method for assessing the proportions of various gametogenic cell types (gonadal staging)
based on the light microscopic examination of hematoxylin and eosin-stained histologic
sections.
44
-------
Semi-quantitative gonadal staging has been proposed for, or employed in, studies
involving fathead minnows (Ankley et al., 2002; Jensen et al., 2001; Miles-Richardson et
al., 1999a; Nichols et al., 2001; US EPA, 2002), among other fishes. Although such
studies generally included excellent descriptions of the different gametogenic maturation
stages (e.g., spermatogonium through spermatozoa for the testis), they did not incorporate
pre-defined categorical guidelines for evaluating and reporting the reproductive cycle
status of an individual fish. To maintain scientific integrity across the board in a program
that involves multiple studies, multiple laboratories, and large numbers of animals, it is
essential that observations are recorded on a fish-by-fish basis. The use of a
categorization system can improve the consistency and objectivity of reported
observations within and among experiments; consequently, comparisons of the results are
more meaningful. Categorization systems also have some drawbacks and limitations, the
most significant of which are: 1) the potential loss of discriminatory data when similar,
but not identical, types of observations are combined (binned) into a single class; 2) the
questionable biological relevance of the classification criteria in some cases; and 3) the
inability of any single classification system to address every type of observation (either
predicted or unforeseen). To address this last limitation, gonadal staging is accompanied
by a complete histopathological evaluation of the gonads; in this manner, the loss or
overabundance of a specific gametogenic cell type, for example, can be documented.
The semi-quantitative gonadal staging scheme selected for analysis of FHM gonads is a
modification of a system adopted by the United States Department of the Interior, U.S.
Geological Survey, Biological Resources Division as part of the "U.S. Biomonitoring of
Environmental Status and Trends (BEST) Program" (McDonald et al., 2000). The
authors of the BEST system credit previous work by Treasurer and Holiday (1981),
Nagahama (1983), Rodriquez et al. (1995), and Goodbred et al. (1997). The foremost
benefits of this system are speed and ease of use, especially when compared to fully-
quantitative staging. The basis of the BEST system is a visual assessment of the density
of gametogenic precursors as compared to mature gametocytes in one or more gonad
sections. Accordingly, the stage numbers (testis: Stages 0 to 4; ovary: Stages 0 to 5)
increase in direct relationship to the relative proportion of mature cells. Although the
BEST system was initially developed to assess reproductive function in seasonal
spawners such as carp (Cyprinidae) and black basses (Centrarchidae), the same stage
categories can be applied to fractional spawners such as fathead minnow. This was
demonstrated at the October 2003 meeting of the histopathology subcommittee of the
FDG in Paris, at which the participants were asked to briefly evaluate the applicability of
a modified BEST system using actual histologic specimens of these three species. In
general, the participants agreed that they could readily recognize the various gonadal
stages as defined by modified BEST criteria. The participants acknowledged that the
terminal stages of the system (testis: Stage 4, "Spent"; ovary: Stage 5, "Post-ovulatory")
would be minimally present (or possibly not present at all) among reproductively-active
adult FHM because they are not seasonal spawners. Similarly, it was thought that Stage
4, "Late development/hydrated", would be rarely observed in the test fishes, due to the
very transient nature of this stage in fractional spawners.
A few modifications have been made to the BEST system to adapt it for use. For
45
-------
example, there is currently no provision in the system for gonads that are comprised
entirely of spermatogonia or oogonia. Although it is intended that reproductively mature
fish are used, it is possible that an occasional animal may not attain sexual maturity by
the time the experiment is terminated, or that certain test compounds might cause
reversion of the gonads to a juvenile phenotype. Therefore, as one modification of the
BEST system, a pre-staging category called "juvenile" has been added for both male and
female fish. Another modification to the system involves an apparent discrepancy
between the BEST system and Goodbred et al. concerning the thickness of the testicular
germinal epithelium as a staging criterion. As indicated by Goodbred et al., the germinal
epithelium becomes thinner as the testis stage increases, whereas, the reverse occurs
according to the BEST system (as presented in McDonald et al.). Although it is difficult
to find corroborating statements in the scientific literature, empirical evidence indicates
that Goodbred et al. is correct on this point. A third modification to the system is the
option to subdivide a stage into two subordinate stages (e.g., Stages 3A and 3B) if the
pathologist believes that this tactic would reveal a subtle, compound-related effect that
might otherwise be missed. Other modifications to the system are relatively minor and
primarily involve rewording for clarification.
The cell distribution pattern is likely to vary throughout a given tissue section, the gonad
should be staged according to the predominant pattern in that section. Both gonads
should be staged as a single organ according to the predominant pattern. Gonads that
cannot be reasonably staged for various reasons (e.g., insufficient tissue, or extensive
necrosis, inflammation, or artifact) should be recorded as UTS (unable to stage).
Criteria for Staging Testes
The following are morphologic criteria for staging male fish:
• Juvenile: gonad consists of spermatogonia exclusively; it may be difficult or
impossible to confirm the sex of these individuals.
• Stage 0 - Undeveloped: entirely immature phases (spermatogonia to
spermatids) with no spermatozoa.
• Stage 1 - Early spermatogenic: immature phases predominate, but
spermatozoa may also be observed; the germinal epithelium is thinner than it
is during Stage 2.
• Stage 2 - Mid-spermatogenic: spermatocytes, spermatids, and spermatozoa
are present in roughly equal proportions; the germinal epithelium is thinner
than Stage 1 but thicker than Stage 3.
• Stage 3 - Late spermatogenic: all stages may be observed, however, mature
sperm predominate; the germinal epithelium is thinner than it is during Stage
2.
• Stage 4 - Spent: loose connective tissue with some remnant sperm.
46
-------
" S-?';1\ $- f^|*V?5^£- •:•>••'
':2ii^Pfill
Examples of staging system applied to the FHM testis. Testes from four different adult male FHM.
There is progressive thinning of the germinal epithelium and expansion of the lobular lumen with each
increase in stage. Note that no spermatozoa are present at in the Stage 0 image (GMA, H&E).
47
-------
Criteria for Staging Ovaries
The following are morphologic criteria for staging female fish:
• Juvenile: gonad consists of oogonia exclusively; it may be difficult or impossible
to confirm the sex of these individuals.
• Stage 0 - Undeveloped: entirely immature phases (oogonia to perinucleolar
oocytes); no cortical alveoli.
• Stage 1 - Early development: vast majority (e.g., >90%) are pre-vitellogenic
follicles, predominantly perinucleolar through cortical alveolar.
• Stage 2 - Mid-development: at least half of observed follicles are early and mid-
vitellogenic.
• Stage 3 - Late development: majority of developing follicles are late
vitellogenic.
• Stage 4 - Late development/hydrated: majority are late vitellogenic and mature
/ spawning follicles; follicles are larger as compared to Stage 3.
• Stage 5 - Post-ovulatory: predominately spent follicles, remnants of theca
externa and granulosa.
QW^ u m -. W ^Q vVff^W^
ho^O*tQ*«O<|Q$ ip<$5- *'
^w^^Pte^'.;
i A fir>*rftMo° .'IW1;,. • S^; •
, O '<»
*','/ ^n Stage 1
,r. • StageO D| . %
Examples of staging system applied to the FHM ovary. Ovaries from four adult female FHM. Due its
transient nature in FHM, Stage 4 is not often observed (paraffin, H&E).
48
-------
APPENDIX A: HISTOLOGY FIGURES
Bouin's Fixative
Modified Davidson's Fixative
m
%£& "•••• .--V™
mi$lffiz$i?i&$
i£*«S SSPvMr.v;<
Fig. 1. Fathead Minnows, Testis (A&B) and Ovary (C&D): Gonads fixed in Bouin's fixative (A&C)
and modified Davidson's fixative (B&D). Color contrast was slightly superior in testes fixed with
Davidson's fixative and was clearly superior in ovaries fixed with Bouin's fixative. Either fixative is
satisfactory for diagnostic purposes; however, Davidson's fixative was selected for the Phase IB assay.
49
-------
Fig. 2. Fathead Minnow, Male: Excision of the testes during necropsy. A. The abdominal wall is
pinned laterally. B. The terminal intestine is severed and retracted prior to removal. C. The testes are
grasped near the spermatic ducts. D. Removal of the testes is complete.
50
-------
Fig. 3. Fathead Minnow, Female: Excision of the ovaries during necropsy. A. The abdominal wall is
pinned laterally. B. The terminal intestine is severed and retracted prior to removal. C. The ovaries are
grasped near the oviducts. D. Removal of the ovaries is complete.
51
-------
APPENDIX B: SCHEDULES
Schedule 1. Tissue Processing
Station
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Reagent
10% NBFa
70% ethyl alcohol
80% ethyl alcohol
95% ethyl alcohol
95% ethyl alcohol
100% ethyl alcohol
100% ethyl alcohol
100% ethyl alcohol
Clear Rite 3
Clear Rite 3
Paraffin
Paraffin
Paraffin
Paraffin
Pressure/
Vacuum
Cycle
On
On
On
On
On
On
On
On
On
On
On
On
On
On
Heat (°C)
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
60
60
60
60
GONAD
PROGRAM
(minutes)
40
40
40
40
40
40
40
40
60
60
45(60b)
45(60b)
45(60b)
45
WHOLE-FISH
PROGRAM
(minutes)
60
60
60
60
60
60
60
60
80
80
75(100b)
75(100b)
75(100b)
75
Drain and Clean Cycle0
a Neutral buffered formalin.
b Times are increased for processors that have three (versus four) final stations
c Automatic cleaning cycle to be run after removal of tissues from the processor. Time, temperature, and vacuum are preset by the
manufacturer.
52
-------
Schedule 2. Hematoxylin and Eosin Staining
Reagent
Xylene
Absolute Alcohol
80% Alcohol
Water
Hematoxylin
Water
Clarifier
Water
Bluing
Water
95% Alcohol
Eosin
Absolute Alcohol
Xylene
Minutes in Reagent
4
2
1
1
3
2
1
1
1
2
1
1
4
3
Reagent Maintenance
After 1st Run
Remove
Remove
Renew
—
—
—
Renew
—
Renew
—
Renew
—
Remove
Remove
After 2nd Run
Remove
Remove
Renew
—
Remove
—
Renew
—
Renew
—
Renew
Renew
Remove
Remove
53
-------
APPENDIX C: FORMULARY
Euthanasia Solution
Tricaine methanesulfonate 100 mg
Sodium bicarbonate 200 mg
Tank or reservoir water 1 L
Davidson's Fixative (Fournie etal., 2000)
Formaldehyde (37-40%) 200 ml
Glycerol 100ml
Glacial acetic acid 100 ml
Absolute alcohol 300 ml
Distilled water 300 ml
Modified Davidson's Fixative
Formaldehyde (37-40%) 220 ml
Glacial acetic acid 115 ml
95% Ethyl alcohol 330 ml
Distilled water 335 ml
It is recommended that hematoxylin and eosin be purchased as premixed solutions.
Examples are the Hematoxylin-2 (Gill hematoxylin) and Eosin Y solutions that are
manufactured by Richard-Allan Medical Industries (Appendix D).
Gill Hematoxylin Solution (Gill etal. 1974)
Distilled water 730 ml
Ethylene glycol 250 ml
Hematoxylin, anhydrous 2 g
Sodium iodate 0.2 g
Aluminum sulfate 17.6 g
Glacial acetic acid 20 ml
Eosin Solution
Eosin Y (1% aqueous solution) 100 ml
Ethyl alcohol, 95% 600 ml
Glacial acetic acid 4 ml
C-l
-------
APPENDIX D: EXAMPLE PRODUCT GUIDE
Example Product
Clear Rite-3™
Coverglass, 24x50 premier nonstick
Thinness: 0.13mm-0.17mm
Davidson's Fixative
Decalcifier: Formical-2000®
Eosin Y (for H&E Stain)
Eosin-Y
Reagent Alcohol
Deionized Water
Glacial Acetic Acid
Hematoxylin 2 (for H&E Stain)
Hematoxylin
Aluminum Sulfate
Sodium lodate
Ethylene Glycol
Deionized Water
Glacial Acetic Acid
MS-222 Fenquel™ ( Tricaine
Methanesulfonate)
Paraplast® (CSMP)
Kendall Paraplast Tissue Embedding
Medium 8889 50 1006
Permount® Mounting Media
Toluene 55%
BHT < 1%
Polymer Alpha pinene &
Betapinene 45%
Slide, Single Frosted, ground edge
Crystal Line Premier Brand
Catalogue #
6901
00145-ACS
S2250
1354
7111
7231
C-FINQ-UE
SHM8889-501006
SP15-500
8105
Supplier/Manufacturer
Richard Allen Medical Industries
8850 M89 Box 351
Richland, MI 49083
800-522-7270
http://www.rallansci.com.
Surgipath Medical Industries, Inc.
P. O. Box 528
Richmond, IL 60071
800-225-3035
Poly Scientific R&D Corp.
70 Cleveland Avenue
Bay Shore, NY 11706
631-586-0400
Decal Chemical Corp.
PO Box 9 16
Tallman, NY 10982-0916
800-428-5856
Richard Allen Medical Industries
8850 M89 Box 351
Richland, MI 49083
800-522-7270
http://www.rallansci.com.
Richard Allen Medical Industries
8850 M89 Box 351
Richland, MI 49083
800-522-7270
http://www.rallansci.com.
Argent Chemical Laboratories,
Redmond, WA 98052, USA.
Supplier: Laboratory Supply Co.
800-888-9004
Manufacturer:
Sherwood Services AG
Tyco Healthcare Group L
15 Hampshire Street
Mansfield, MA 02048
Fisher HealthCare
800-640-0640
C&A Scientific Co., Inc.
7241 Gabe Court
Manassas, VA 20109
703-330-1413
D-l
-------
REFERENCES
Ankley, G. T., Jensen, K. M, Kahl, M. D., Korte, J. J., Makynen, M. E. (2001). Description and
evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas).
Environ Toxicol Chem 20, 1276-1290.
Ankley, G. T., Jensen, K. M., Makynen, E. A., Kahl, M. D., Korte, J. J., Hornung, M. W., Henry, T. R.,
Denny, J. S., Leino, R. L., Wilson, V. S., Cardon, M. C., Hartig, P. C, Gray, E. L. (2003). Effects
of the androgenic growth promoter 17-p-trenbolone on fecundity and reproductive endocrinology of
the fathead minnow. Environ Toxicol Chem 22, 1350-1360.
Ankley, G. T., Kahl, M. D., Jensen, K. M., Hornung, M. W., Korte, J. J., Makynen, E. A., and Leino, R.
L. (2002). "Evaluation of the Aromatase Inhibitor Fadrozole in a Short-Term Reproduction Assay
with the Fathead Minnow (Pimephales promelas}" Toxicol Sci 67, 121-130.
Blazer, V.S., Wolke, R.E., Brown, J., Powell, C.A. (1987). "Piscine macrophage aggregate parameters
as health monitors: effect of age, sex, relative weight, season and site quality in largemouth bass
(Micropterus salmoides). Aquat. Toxicol. 10:199-215.
Crissman JW et al. "Best Practices Guideline: Toxicologic Pathology." Tox Path 32 (2004): 126-31.
Dorland's Illustrated Medical Dictionary. 26th Edition (1981). W.B. Saunders Company, Philadelphia,
pp 1485.
Fournie, J. W., Krol, R. M., and Hawkins, W. E. (2000). Fixation of Fish Tissues. In: The Laboratory
Fish, (GK Ostrander, ed.), pp. 569-578, Academic Press, San Diego.
Gill, G. W., Frost, J. K., Miller, K. A. (1974). A new formula for a half-oxidized hematoxylin solution
that neither overstains nor requires differentiation. Acta Cytol 18, 300.
Goodbred, S. L., Gilliom, R. J., Gross, T. S., Denslow, N. P., Bryant, W. L., Schoeb,
T. R. (1997). "Reconnaissance of 17p-estradiol, 11-ketotestosterone, Vitellogenin, and Gonad
Histopathology in Common Carp of United States Streams: Potential for Contaminant-induced
Endocrine Disruption." U.S. Geological Survey, Open-File Report 96-627, pp. 47.
Harries, J. E., Runnalls, T., Hill, E., Harris, C. A., Maddix, S., Sumpter, J. P., Tyler,
C. R. (2000). Development of a reproductive performance test for endocrine disrupting chemicals
using pair-breeding fathead minnows (Pimephalespromelas). Environ Sci Technol 34, 3003-3011.
Herman, R. L., Kincaid, H. L. (1988). "Pathological Effects of Orally Administered Estradiol to
Rainbow Trout." Aquaculture 72, 165-172
Illustrated Stedman's Medical Dictionary. 24th Edition (1982). Williams & Wilkins, Baltimore, pp.
1678.
Jensen, K. M., Korte, J. J., Kahl, M. D., Pasha, M. S., and Ankley, G. T. (2001). "Aspects of Basic
Reproductive Biology and Endocrinology in the Fathead Minnow (Pimephales promelas)" Comp
Biochem Physiol C Toxicol Pharmacol. 128,
127-141.
Kahl, M. D., Jensen, K. M., Korte, J. J., Ankley, G. T. (2001). Effects of handling on endocrinology
and reproductive performance of the fathead minnow. J Fish Biol 59, 515-523.
Kinnberg, K., Korsgaard, B., Bjerregaard, P., and Jespersen, A. (2000) "Effects of Nonylphenol and
17b-estradiol on Vitellogenin Synthesis and Testis Morphology in Male Platyfish Xiphophorus
maculatus" The Journal of Experimental Biology 203, 171-181.
McDonald, K. K, Gross, T. S., Denslow, N. D., Blazer, V. S. (2000). "Reproductive Indicators". In:
Biomonitoring of Environmental Status and Trends (BEST) Program: selected methods for
monitoring chemical contaminants and their effects in aquatic ecosystems. (C. J Schmitt and G. M.
Dethloff, eds.) U.S. Geological Survey, Biological Resources Division, Columbia, (MO):
G-l
-------
Information and Technology Report USGS/BRD-2000-0005, pp. 30-42.
Miles-Richardson, S. R., Kramer, V. J., Fitzgerald, S. D., Render, J. A., Yamini, B., Barbee, S. J., and
Giesy, J. P. (1999a). "Effects of Waterborne Exposure of 17p-estradiol on Secondary Sex
Characteristics and Gonads of Fathead Minnows (Pimephales promelas)" Aquat Toxicol 41, 129-
145.
Miles-Richardson, S. R., Pierens, S. L., Nichols, K. M., Kramer, V. J., Snyder, E. M., Snyder, S. A.,
Render, J. A., Fitzgerald, S. D., Giesy, J. P. (1999b) "Effects of Waterborne Exposure to 4-
nonylphenol and Nonylphenol Ethoxylate on Secondary Sex Characteristics and Gonads of Fathead
Minnows (Pimephales promelas}" Environmental Research. Section A. 80,8122-8137.
Nagahama, Y. (1983). "The Functional Morphology of Teleost Gonads." Fish Physiology. (W. S.
Hoar, D. J. Randall, E. M. Donaldson, eds.), San Diego, California, USA, Academic Press, pp. 223-
275.
Nichols, K.M., Miles-Richardson, S. R., Snyder, E. M., and Giesy, J. P. (2001). "Effects of Exposure to
Municipal Wastewater in Situ on the Reproductive Physiology of the Fathead Minnow (Pimephales
promelas)" Environ Toxicol and Chem 18, 2001-2012.
Noga, E. J., Dykstra, M. J., Wright, J. F. (1989). "Chronic Inflammatory Cells with Epithelial Cell
Characteristics in Teleost Fishes." VetPathol. 26(5): 429-37.
Norberg, B., Kjesbu, 0. S., Taranger, G. L., Andersson, E., Stefansson, S. 0. (1999). "The Teleost
Germinal Epithelium: A Unifying Concept." Proceedings of the 6th International Symposium on
the Reproductive Physiology of Fish, pp. 233-236.
Palace, V. P., Evans, R. E., Wautier, K., Baron, C., Vandenbyllardt, L., Vandersteen, W., Kidd, K.
(2002). "Induction of Vitellogenin and Histological Effects in Wild Fathead Minnows from a Lake
Experimentally Treated with the Synthetic Estrogen, Ethynylestradiol." Water Qual. Res. J.
Canada, 37(3): 637-650.
Panter, G. H., Thompson, R. S., Sumpter, J. P. (1998). Adverse reproductive effects in
male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations
of the natural oestrogens oestradiol and oestrone. Aquatic Toxicol 42, 243-253.
Parenti, L. R., Grier, H. J. (2003). "Patterns and Processes in the Evolution of Fishes". Annual
Meeting of the Society of Integrative and Comparative Biology, Toronto, Canada, p. 8.
Rodriguez, J. N., Oteme, Z. J., Hem, S. (1995). "Comparative study of vitellogenesis of two African
catfish species Chrysichthys nigrodigitatus(Claroteidae) and Heterobraanchus longifilis(Clarudae)"
Aquat. Living Resour. 8, 291-296.
Saidapur, S. K. (1982). "Structure and Function of Postovulatory Follicles (Corpora lutea) in the Ovary
of Nonmammalian Vertebrates" International Review of Cytology 75, 243-283.
Selman, K., Wallace, R. A. (1989). "Cellular aspects of oocyte growth in teleosts" Zoo/. Sci.. 6, 211-
231.
Shackelford, C. C, Long, G., Wolf, J., Okerberg, C., and Herbert, R. (2002). "Qualitative and
Quantitative Analysis of Nonneoplastic Lesions in Toxicology Studies. Toxicol Pathol 30, 93-96.
Smith, R. J. F. (1974). Effects of 17a-methyltestosterone on the dorsal pad and tubercles of fathead
minnows (Pimephalespromelas). Can JZool52, 1031-1038.
Treasurer, J. W. and Holliday, F. G. T. (1981). "Some Aspects of the Reproductive Biology of Perch
Percaflaviatilis L.: A Histological Description of the Reproductive Cycle." J. Fish Biol. 18, 359-
76.
Tyler, C. R. and Sumpter, J. P. (1996). "Oocyte Growth and Development in Teleosts." Rev Fish Biol
Fisheries 6, 287-318.
G-2
-------
U.S. Environmental Protection Agency (2002). A Short-term Test Method for Assessing the
Reproductive Toxicity of Endocrine-Disrupting Chemicals Using the Fathead Minnow (Pimephales
promelas). EPA 600/R-01-067, pp. 141,144.
van derVen, L., Wester, P.W. (2004). Toxicological Pathology Atlas of Small Laboratory Fish.
Laboratory of Pathology and Immunobiology, National Institute of Public Health and the
Environment, RIVM, Bilthoven, the Netherlands, http://www.rivm.nl.fishtoxpat.
Zillioux, E. J., Johnson, I. C., Kiparissis, Y., Metcalfe, C. D., Wheat, J. V., Ward, S. G., Liu, H. (2001).
"The Sheepshead Minnow as an In Vivo model for endocrine disruption in marine teleosts: A
Partial Life Cycle Test with 17a-ethynylestradiol." Toxicol. Chem. 20, 1968-1978.
G-3
------- |