current protocols in toxicology i

UNIT 16.3Histopathology of the Male ReproductiveSystem I: Techniques

Effective evaluation of the male reproductive system relies on adequate sampling of thevarious parts of the reproductive tract as well as appropriate fixation, orientation,processing, embedding, and staining of the tissues. This unit describes protocols for thesevarious aspects of tissue preparation.

The presence or absence, as well as the anatomical arrangement, of various componentsof the reproductive tract varies with species. These differences must be appreciated toensure appropriate sampling. Basic Protocol 1 and Support Protocol 1 describe theidentification and dissection procedures for the reproductive organs from the rodent, dog,and primate, which are the animals most frequently used in toxicological research. BasicProtocol 1 describes the routine immersion fixation of these tissues followed by process-ing and embedding in paraffin wax. For a variety of reasons, the testis presents a problemfor good fixation. Conventional immersion fixation in formalin generally results in poorpreservation of the seminiferous tubules, and the use of an alternative fixative, such asBouin’s, is recommended in most regulatory guidelines. Although Bouin’s provides goodmorphology suitable for detecting subtle disturbances in spermatogenesis, it suffers froma number of increasingly important safety and disposal problems and also results insignificant shrinkage of the tubules. This protocol recommends the use of a novel,alcohol-based fixative (modified Davidson’s fixative), which provides comparable cellu-lar and nuclear preservation to that achieved with Bouin’s but with less tubular shrinkageand none of its safety and disposal hazards.

Once the tissues are fixed, sampling of specific structures and tissue orientation are veryimportant to ensure that critical regions of the reproductive tract are presented forexamination. Support Protocol 1 provides guidance on how to trim and sample tissuesfrom rodents, dogs, and primates.

Basic Protocol 2 describes the methodology used to obtain excellent preservation of therodent testis by whole-body perfusion via the heart. The testes of large animals can alsobe fixed by perfusion, but to reduce the volume of fixative required this is best carried outby introducing the fixative into the testicular artery. Support Protocols 2 and 3 describethe processing procedures required for embedding in epoxy and glycol methacrylate resin,respectively. Perfusion fixation followed by embedding in resin is essential for properevaluation of the testis by electron microscopy. The use of epoxy resin is necessary tomaintain stability of the section under the electron beam; however, epoxy-embeddedtissue can only be stained with toluidine blue for light microscopic examination. Glycolmethacrylate resin is a water-soluble resin that can be used to prepare semithin (2 µm)sections that can be stained with conventional histologic stains for high-resolution lightmicroscopy. A compromise between the convenience of immersion fixation followed byparaffin embedding and the labor-intensive perfusion fixation followed by resin embed-ding can be achieved by immersion fixation followed by embedding in glycol methacry-late resin.

CAUTION: Many of the chemicals used in these protocols are hazardous, includingformalin, glutaraldehyde, propylene oxide, epoxy and glycol methacrylate resins, sodiumcacodylate, and osmium tetroxide. Refer to material safety data sheets for appropriatehandling, storage, and disposal.

Supplement 12

Contributed by Dianne M. CreasyCurrent Protocols in Toxicology (2002) 16.3.1-16.3.18Copyright © 2002 by John Wiley & Sons, Inc.

16.3.1

MaleReproductiveToxicology

NOTE: All protocols using live animals must first be reviewed and approved by anInstitutional Animal Care and Use Committee (IACUC) and must conform to governmen-tal regulations regarding the care and use of laboratory animals.

BASICPROTOCOL 1

DISSECTION, IMMERSION FIXATION, AND PARAFFIN EMBEDDING OFMALE REPRODUCTIVE-TRACT TISSUES

This procedure is suitable for routine screening of tissues for toxicological changes. Itinvolves dissection of the major reproductive tissues, measurement of organ weights, andimmersion fixation of the testes using a special fixative (the remaining tissues can be fixedin the same fixative or in neutral buffered formalin). This is followed by careful trimmingof the tissues to include specific structures and conventional processing through anascending series of alcohols and clearing agents, followed by embedding in paraffin wax.Sections are then cut and stained with periodic acid Schiff’s stain/hematoxylin (PAS-H;for testes and epididymides) and hematoxylin and eosin (for remaining tissues).

Materials

Male rodent, dog, or primateModified Davidson’s fixative (see recipe)10% neutral buffered formalin (∼4% [w/v] formaldehyde; Sigma or standard

recipe, see Bancroft et al., 1990)Surgical instruments for dissection

Additional reagents and equipment for euthanization; dissecting the testes,epididymides, and accessory sex glands (see Support Protocol 1); dehydration,clearing, and infiltration with paraffin wax (Bancroft et al., 1990); and paraffinsectioning and staining (Bancroft et al., 1990)

1. Euthanize a male rodent, dog, or primate by an approved method.

Some examples of euthanization methods include carbon dioxide (for rodents) and sodiumpentobarbital (e.g., Sleepaway).

2. Depending on the species under investigation (Table 16.3.1), use surgical instrumentsto dissect out the testes, epididymides, and accessory sex glands (i.e., seminal vesicleswith coagulating glands and prostate; see Support Protocol 1).

Standard dissection guides, such as Feldman and Seeley (1988) and Evans (2000), containdetailed dissection procedures.

It is important to minimize squeezing of the testes during handling to prevent artifactualsloughing of the germ cells from the seminiferous epithelium. When trimming theepididymis from the testes, take care not to cut the testicular capsule, which will causeextrusion of the seminiferous tubules through the cut and consequent disruption of tissuearchitecture.

3. Weigh each organ and record the weight to the nearest 1 mg (for rodents) or 100 mg(for dogs or primates).

For paired organs, individual weights provide valuable information to support unilateralmicroscopic findings. Individual organ weights are also required where one testis and oneepididymis are used for quantifying sperm parameters (Table 16.3.2).

When weighing seminal vesicles and the prostate, it is important that the fluid be includedin the weight because its volume reflects the functional activity of the glands. For someregulatory guidelines, the combined weight of seminal vesicles and prostate is recom-mended (Table 16.3.2). If separate weights are desired, dissect out the prostate and seminalvesicles as a unit, being careful to retain all fluids, and then separate the seminal vesiclesand coagulating glands from the prostate over a weigh boat, so that any fluid leakage fromthe seminal vesicles is caught and included for weighing.

Supplement 12 Current Protocols in Toxicology

16.3.2

Histopathology ofthe Male

ReproductiveSystem I:

Techniques

Table 16.3.2 Recommendations from Recently Revised Regulatory Guidelines for Reproductive Studies Relating toSampling and Fixation of Male Reproductive Tissues

Guidelinea Tissues to be weighed Tissues to be preserved Tissues to be examined

OECD 416: Two-generationreproduction toxicity study(OECD, 2001)

Testes One testisb,c Testis

Epididymides (total andcauda)

One epididymisb Epididymis

Seminal vesicles withcoagulating glands and theirfluids and prostate (as oneunit)

Seminal vesicles Seminal vesicles

Coagulating glands Coagulating glands

Prostate Prostate

OECD 421: Reproduc-tion/developmental toxicityscreening test (OECD, 1995)

Testes Testes Testesc

Epididymides Epididymides Epididymides

Seminal vesicles

Coagulating glands

Prostate

OPPTS 870.3800: Repro-duction and fertility effects(EPA, 1998)

Testes Right testisb,c Testis

Epididymides (total andcauda)

Right epididymisb Epididymis

Seminal vesicles withcoagulating glands and theirfluids

Seminal vesicles Seminal vesicles

Coagulating glands Coagulating glands

Prostate Prostate Prostate

ICH S5A and S5B:Detection of toxicity toreproduction for medicinalproducts (ICH, 1994, 1996d)

None Testesc and epididymides Testes and epididymides ifindicated by altered fertilityindices

aAbbreviations: ICH, International Conference on Harmonization of Technical Requirements of Registration of Pharmaceuticals for Human Use; OECD,Organization for Economic Cooperation and Development; OPPTS, Office of Prevention, Pesticides, and Toxic Substances.bRemaining testis or epididymis is retained for assessment of sperm parameters (UNITS 16.1 & 16.2).cPreserved in Bouin’s or comparable fixative.dAn addendum on male fertility studies.

Table 16.3.1 Presence of Accessory Sex Glands in Common Laboratory Species

Species Ampulla ductusdeferens

Bulbourethral(Cowper’s) glands

Coagulatingglands

Preputialgland Prostate Seminal

vesicles

Dog + − − − + −Mouse + + + + + +Primate + + − − +a +Rabbit + + − + + +b

Rat + + + + + +aNo anterior lobe present.bPresent as rudimentary glandula vesicularis.

Current Protocols in Toxicology Supplement 12

16.3.3

4. Place the testes into modified Davidson’s fixative for ≥48 hr at room temperature andthen transfer to 10% neutral buffered formalin for storage. Place the remaining tissuesinto 10% neutral buffered formalin for ≥48 hr at room temperature until required forprocessing.

Speed of penetration of the fixative can be improved by pricking or nicking the capsulebefore immersion and also by slicing the testis following several hours in fixative (seeCritical Parameters and Troubleshooting, discussion of choice of fixative).

The tissues can be stored in 10% neutral buffered formalin for up to 10 years at roomtemperature.

For convenience, the epididymides and secondary sex organs can also be fixed in David-son’s fixative, but the cytological and staining characteristics are slightly inferior to thoseof formalins.

5. Trim the tissues according to Support Protocol 1 and process through standardhistology procedures for dehydration, clearing, and infiltration with paraffin wax.Embed in paraffin wax (Table 16.3.3).

Standard histological processing methodology can be found in Bancroft et al. (1990).

Alternatively, for high resolution light microscopy, the tissues can be embedded in glycolmethacrylate resin following immersion fixation (see Support Protocol 3).

6. Prepare sections at 5 to 6 µm.

Standard paraffin sectioning is described in Bancroft et al. (1990).

7. Stain testes and epididymides with PAS-H stain and the remaining tissues withhematoxylin and eosin.

Standard histological staining methodology can be found in Bancroft et al. (1990).

PAS-H staining in the testis will stain the glycoprotein component of the spermatidacrosomic granule or acrosome cap. These are very small structures, which can be difficultto visualize if the stain is not optimal. It is important that the periodic acid and the Schiffreagent are relatively fresh solutions. In dog testes, the acrosome does not stain with PASand cannot be adequately distinguished in paraffin-embedded sections (see AnticipatedResults).

Table 16.3.3 Schedule for Processing Fixed Tissues From Rodent, Dog, orPrimate into Paraffin Wax

Solutiona Temperature (°C)Time (hr)

Rodent Dog/primate

Neutral buffered formalin 37 0.5 0.5

70% ethanol 37 1.0 1.0

80% ethanol 37 1.5 2.0

90% ethanol 37 1.0 1.5

100% ethanol 37 1.0 1.0

100% ethanol 37 1.5 1.5

100% ethanol 37 2.0 2.0

Xylene 37 0.5 0.5

Xylene 37 1.0 1.5

Xylene 37 1.5 2.0

Wax 60 1.0 1.0

Wax 60 1.0 1.5

Wax 60 2.0 2.0aTissues should be agitated in each solution.


16.3.4



Techniques

BASICPROTOCOL 2

PERFUSION FIXATION OF THE MALE RODENT REPRODUCTIVE TRACTBY CARDIAC PERFUSION AND RESIN EMBEDDING OF TISSUES

This method is used when high-resolution light microscopy or electron microscopy isrequired. The methodology is time consuming and labor intensive and is more suited toinvestigative studies than to large-scale screening studies. The procedure involves anes-thetizing the animal, cannulating the thoracic aorta through the heart, and flushing outthe systemic vasculature with Ringer’s solution followed by systemic perfusion with abuffered paraformaldehyde and glutaraldehyde fixative. The method described is awhole-body perfusion using a peristaltic pump to maintain a constant pressure duringperfusion. Following fixation, the tissue may be embedded in glycol methacrylate orepoxy resin for light microscopy. Electron microscopy requires the use of epoxy resin.Whole-body perfusion is not suitable for large animals such as primates and dogs becauseof the large volume of fixative required. An alternative technique introduces the fixativeinto the testicular artery (Frederick and Doorn, 1973; Russell et al., 1990).

Materials

Krebs Ringer’s solution (see recipe)Karnovsky’s fixative (see recipe)Male rodent65 mg/ml sodium pentobarbital solution (e.g., Sleepaway; Fort Dodge Animal

Health) or equivalent for anesthesia0.1 M sodium cacodylate buffer (see recipe)Osmium tetroxide/potassium ferrocyanide fixative (see recipe)

Perfusion setup (Fig. 16.3.1), including: Two 1-liter stoppered bottles, each connected to a length of silastic tubing (∼3.0

mm internal diameter) and each vented to air Silastic tubing, ∼3.0 mm internal diameter (e.g., Masterflex L/S 16 pump

tubing; Cole-Parmer Instrument) Three-way stopcock Variable-speed peristaltic pump (e.g., Masterflex; Cole-Parmer Instrument) Manometer Dosing needle: stainless steel intubation needle with bulbous end, 16 to 18G

(depending on size of animal), attached to a cut-off 1-ml tuberculin syringe Dissection board suspended in a tray Vacuum pump connected to 1-liter bottle trapRubber bandsDissecting tools, including: Scalpel Hemostats Forceps5-cm (2-in) Dieffenbach serrefine (bulldog) clampSingle-edged blades

Additional reagents and equipment for dehydrating, infiltrating, and embedding inepoxy resin (see Support Protocol 2); sectioning and staining for electronmicroscopy or dehydrating, infiltrating, and embedding in glycol methacrylate(see Support Protocol 3); sectioning and staining for high-resolution lightmicroscopy

Set up perfusion apparatus1. Set up a perfusion apparatus as shown in Figure 16.3.1.

The perfusion needs to be carried out under a ventilation hood or on a ventilated table.


16.3.5


2. Fill one 1-liter bottle with Krebs Ringer’s solution and a second one with Karnovsky’sfixative. Connect these with silastic tubing and a three-way stopcock to a variable-speed peristaltic pump. Connect a manometer in-line and attach a dosing needle tothe end of the tubing.

3. Open the stopcock and flush the tubing with Krebs Ringer’s solution. Ensure that thetubing of the perfusion apparatus is filled with fluid and free of air bubbles.

4. Using the manometer, calibrate the speed of the peristaltic pump to a flow rate of 110to 130 µl/min. This should be equivalent to a perfusion pressure of 100 to 120 mmHg. During the perfusion the pressures should not be allowed to rise above 150 mmHg.

5. Set up a dissection board suspended in a tray and a vacuum pump connected to a1-liter bottle trap to drain used fluids.

Perfuse animal6. Anesthetize a male rodent with 0.8 to 1.0 µl/Hg of 65 mg/ml sodium pentobarbital

solution.

It is important to achieve a level of anesthesia that results in maintenance of shallowbreathing and adequate cardiac output and yet produces a sufficient level of surgicalanesthesia.

Speed is critical to the success of the technique. Once the animal is adequately anesthetized,dissection and introduction of the Krebs Ringer’s solution must be accomplished asefficiently and quickly as possible.

7. Secure the animal on its back to the dissection board using rubber bands around itspaws.

The dissection board should be located over a draining vessel to catch the perfusion fluids.

manometer

waste

three-waystopcock

fixative

peristaltic pump

vacuumpump

dosing needle

dissection board

saline

Figure 16.3.1 Apparatus for perfusion fixation of rodents. Two bottles, one containing KrebsRinger’s saline and the other containing fixative are connected via a three-way stopcock to tubingterminating in a blunt-ended gavage dosing needle. Perfusion pressure is generated using aperistaltic pump and monitored using an in-line manometer.


16.3.6



Techniques

8. Make a midline incision with a scalpel to open the abdomen and then cut through therib cage in a V shape from the xiphoid process to the axilla. Avoid cutting any majorblood vessels in the axilla or along the sternum.

9. Clamp the xiphoid process with a pair of hemostats and displace the flap of thoracictissue cranially.

10. Cut through the pericardium and reflect it back from the heart and aorta.

11. Turn on perfusion pump and begin flow of Krebs Ringer’s solution through theperfusion apparatus at 110 to 120 ml/min.

12. Grasping the ventricular apex with a pair of forceps, rapidly make an incision in theright ventricle to allow fluid efflux. Then make a small incision in the left ventricleand insert the dosing needle through the ventricle and into the aorta so that it is justvisible through the vessel wall. Clamp it in place using a Dieffenbach serrefine clampon the proximal portion of the aortic arch.

Krebs Ringer’s solution should be flowing through the apparatus when the dosing needleis inserted. The needle must not be pushed too far into the aorta, otherwise it will pressagainst the aortic arch and block the flow. Once the perfusion is underway, the angle thatthe needle enters the heart and aorta needs to be maintained at a slight elevation so thatthe outflow of the fluid is not obstructed. This can be accomplished using clamps orsupports.

13. Perfuse the animal at a flow rate of 110 to 120 ml/min until the blood has clearedfrom the testes (∼1 to 2 min).

The blood will gradually be replaced by the Krebs Ringer’s solution.

14. During the perfusion, expose the testes by cutting through the scrotum. Leave thetestes in situ and watch for the coiled, capsular testicular artery to clear of blood andfor the testicular tissue to become uniformly pale.

Fix tissue15. Once the testes have become pale and the effusate from the right ventricle is clear

(∼1 to 2 min), turn the stopcock to change over to fixative. Infuse the fixative for ≥30min for electron microscopy and ≥20 min for light microscopy only. After the first 2to 3 min, slow the rate of perfusion to ∼10 ml/min.

Duration of perfusion is more important than the volume of fluid perfused. The initialpressure and flow rate is important to introduce fixative rapidly while minimizing pressureartifacts. Once the vasculature and basic architecture have been stabilized, the perfusionpressure can be reduced to conserve fixative. A volume of ∼300 ml is appropriate for a300-g animal.

As described, the technique will provide excellent fixation for ultrastructural studies withminimal fixation and handling artifacts. If the tissue is only intended for high-resolutionlight microscopy, then the perfusion time with fixative can be reduced. With this shortertime, the tissue may be subject to some minor handling artifacts.

16. Between perfusions, flush the perfusion line thoroughly with Krebs Ringer’s solutionto remove all traces of fixative.

Remove, trim, and process tissue

For electron microscopy:17a. Remove the testes or tissue of interest and dice into cubes of ≤1 mm using a sharp

single-edged blade. Place these into fresh fixative for ∼1.5 hr at 4°C.


16.3.7


18a. Wash in three changes of 0.1 M sodium cacodylate buffer at 4°C, with one washbeing overnight and other washes ≥15 min.

19a. Postfix 1 hr in osmium tetroxide/potassium ferrocyanide fixative at room tempera-ture.

20a. Dehydrate, infiltrate, and embed tissues in epoxy resin as described (see SupportProtocol 2).

21a. Section embedded tissue at 1 to 2 µm, float sections onto a hot water bath at ∼60°C,and pick up onto grease-free glass microscope slides.

22a. Stain sections and examine with a light microscope.

A solution of 1% (w/v) toluidine blue at 60°C can be used for staining. Bancroft et al. (1990)describes resin sectioning and staining techniques.

23a. Section on an ultramicrotome (silver or gold interface colors), float sections ontowater at room temperature, and pick up onto electron microscopy grids.

24a. Stain sections with uranyl acetate and lead citrate and examine under an electronmicroscope.

Preparation of tissues for examination by electron microscopy requires specialized tech-niques and equipment. Detailed instructions can be found in Hayat (1989).

For high-resolution light microscopy:17b. Remove the testes or tissue of interest and trim with a single-edged blade to provide

samples ∼1 cm × 1 cm × 1 mm thick.

18b. Dehydrate, infiltrate, and embed samples in glycol methacrylate as described (seeSupport Protocol 3).

19b. Section at ∼2 µm using a purpose-designed motorized, retracting microtome and atungsten-carbide or large-area glass (Ralph) knife.

Examples of appropriate microtomes include the Reichert-Jung Supercut and LKB His-torange.

When the resin blocks are trimmed to reach the tissue, the use of 70% (v/v) ethanol on thesurface of the block will soften the resin and allow thicker sections to be taken withoutshattering the resin.

20b. Float the sections on a water bath at room temperature until the resin has expandedmaximally (∼1 to 2 min).

21b. Pick up sections on a grease-free glass slide and dry on a hot plate or in an oven at60°C for ≥30 min before staining.

Flattening and picking up the section is aided if the water bath is previously wiped with70% (v/v) ethanol to reduce surface tension.

22b. Stain with periodic acid Schiff’s (PAS) stain with a light counterstain of hematoxylin(i.e., PAS-H) or with hematoxylin and eosin.

Staining times will depend on the solutions used. Bancroft et al. (1990) describe standardhistological staining methodology.

PAS-H on the testis will stain the glycoprotein component of the spermatid acrosomicgranule or acrosome cap. These are very small structures, which can be difficult to visualizeif the stain is not optimal. It is important that the periodic acid and the Schiff reagent arerelatively fresh solutions.


16.3.8



Techniques

SUPPORTPROTOCOL 1

REMOVAL, TRIMMING, AND ORIENTATION OF MALE TESTES,EPIDIDYMIDES, AND ACCESSORY SEX GLANDS

This protocol describes the dissection of testes, epididymides, and accessory sex glandsfor male rodents, dogs, and primates that have been euthanized as described in BasicProtocol 1. Specific directions for fixing and sectioning the samples are described in BasicProtocol 1.

Rodent

For a male rodent, remove the testes and epididymides as a unit. Separate the epididymidesfrom the testes and trim to remove the surrounding adipose tissue. Weigh the testes andepididymides separately. After fixation, take a 5-mm transverse section through the testisslightly cranial to the midline to include a small portion of the rete testis (which issubcapsular and originates at the point where the epididymis is attached and is where allthe capsular blood vessels converge at the cranial pole). Alternatively, take a longitudinalsection through the plane of the rete testis. (For a discussion of the advantages anddisadvantages of the plane of section, see Critical Parameters and Troubleshooting,discussion of sampling and orientation of tissues). Take a longitudinal section throughthe epididymis to include the caput (head), corpus (body), and cauda (tail). To achievethis, trim a thin slice of tissue from the caput and the cauda so that the length of theepididymis can be embedded flat.

Remove the seminal vesicles, coagulating glands, prostate, and bladder as a unit (Fig.16.3.2A). For detailed dissection guidance, see Feldman and Seeley (1988). Carefullyremove the bladder and weigh and fix the seminal vesicles, coagulating glands, andprostate as a unit. If individual weights of seminal vesicles and prostate are required,carefully separate the glands, minimizing egress of fluid. Do this over a weigh boat tocatch any fluid. After fixation, separate the seminal vesicles (with the coagulating glands)from the prostate. Take a transverse section through each seminal vesicle and its coagu-

DP

A

SV

CG

VD

AG

B

BG

PG

LP

VP

dorsal lobe

B

lateral lobe

vas deferensampullary gland

ventral lobe

urethra

Figure 16.3.2 Accessory male reproductive organs in the rat. (A) Anatomic layout. (B) Histologicappearance of a longitudinal section of the prostate. In addition to the dorsal, lateral, and ventrallobes, sections through the urethra as well as the vas deferens (VD), with varying amounts ofassociated ampullary gland (AG), may be included. B, bladder; BG, bulbourethral gland; CG,coagulating gland; DP, dorsal prostrate; LP, lateral prostate; PG, preputial gland; SV, seminalvesicles; Ventral prostate. Figure drawn by Ann Hoffenberg.


16.3.9


lating gland. Take a midtransverse section through the prostate to include dorsal, lateral,and ventral lobes (Fig. 16.3.2B; Suwa et al., 2001).

Dog

For a male dog, remove each testis and epididymis as a unit. Carefully separate theepididymis from the testis and trim away any adipose tissue. Weigh the testes andepididymides separately. After fixation, take a 5-mm transverse slice through the midlineof the testis. Depending on the size of the testis, it may be necessary to trim this furtherto fit into a standard histology processing cassette. If so, ensure that the rete testis (whichis enclosed in the mediastinum and runs as a longitudinal central core through the middleof the testis) is included. Bisect the epididymis transversely through the corpus so thathalf remains with the caput and half with the cauda. Trim off a thin, longitudinal slice oftissue from the caput and the cauda to allow the tissue to be embedded flat. This willprovide a longitudinal section through the caput, corpus, and cauda.

Remove the prostate and bladder as a unit (Fig. 16.3.3A). For detailed dissection guidance,see Evans (2000). Carefully remove the bladder. Weigh and fix the prostate. Take atransverse section through the prostate and the central urethra (Fig. 16.3.3B).

Primate

For a male primate, remove each testis and epididymis as a unit. Carefully separate theepididymis from the testis and trim away any adipose tissue. Weigh the testes andepididymides separately. After fixation, take a 5-mm transverse slice through the midlineof the testis. Depending on the size of the testis, it may be necessary to trim this furtherto fit into a standard histology processing cassette. In primates, the rete testis lies in asubcapsular location along the epididymal edge. Ensure that this portion of the testis issampled. Bisect the epididymis transversely through the corpus so that half remains withthe caput and half with the cauda. Trim off a thin, longitudinal slice of tissue from thecaput and the cauda to allow the tissue to be embedded flat. This will provide a longitudinalsection through the caput, corpus, and cauda.

VD

B

A

AG

P

urethra

B

lobule of acini

Figure 16.3.3 Accessory male reproductive organs in the dog. (A) Anatomic layout. (B) Trans-verse section through the prostate with lobules of acini separated by connective tissue trabeculaesurrounding the central urethra. AG, ampullary gland; B, bladder; P, prostate; VD, vas deferens.Figure drawn by Ann Hoffenberg.


16.3.10



Techniques

Remove the seminal vesicles, prostate, and bladder as a unit (Fig. 16.3.4A). Carefullyremove the bladder. Weigh and fix the seminal vesicles and prostate as a unit. If individualweights of seminal vesicles and prostate are required, carefully separate the glands,minimizing egress of fluid. Do this over a weigh boat to catch any fluid. After fixation,separate the seminal vesicles from the prostate. Take a transverse section through eachseminal vesicle and take a transverse section through the prostate and the prostatic urethra(Fig. 16.3.4B).

SUPPORTPROTOCOL 2

PROCESSING AND EMBEDDING OF TISSUES IN EPOXY RESIN

Epoxy resin embedding is used for fixed tissues to be examined by electron microscopy.

Materials

Fixed tissue sample postfixed in 1% osmium tetroxide/1.25% potassiumferrocyanide (see Basic Protocol 2)

30%, 50%, 70%, 85%, 95%, and 100% (v/v) ethanolPropylene oxide (e.g., Electron Microscopy Sciences)1:1 propylene oxide/epoxy resinEpoxy resin (e.g., Araldite 502 or 506 lufts; Electron Microscopy Sciences)Mold suitable for electron microscopy blocks (e.g., Electron Microscopy Sciences)

NOTE: During processing, all steps should be carried out with agitation.

1. Dehydrate a fixed tissue sample postfixed in 1% osmium tetroxide/1.25% potassiumferrocyanide using an alcohol series consisting of 15-min steps in 30%, 50%, 70%,85%, 95%, and 100% ethanol.

2. Transfer sample to propylene oxide and incubate 5 min without agitation.

3. Incubate 1 hr in 1:1 propylene oxide/epoxy resin at room temperature.

Incubation times here and in step 4 will vary. Refer to manufacturer’s instructions forspecific resin for details.

4. Incubate in epoxy resin, two changes, 1.5 hr each at room temperature.

5. Orient sample in a mold and embed in fresh resin. Polymerize ≥48 hr at 60°C.

VDB

A

AG

SV

P

BG

urethra

B

Figure 16.3.4 Accessory male reproductive organs in the primate. (A) Anatomic layout. (B)Transverse section through cynomologous monkey prostate, in which glandular acini are absentfrom the anterior aspect. AG, ampullary gland; B, bladder; BG, bulbourethral gland; P, prostate; SV,seminal vesicles; VD, vas deferens. Figure drawn by Ann Hoffenberg.


16.3.11


SUPPORTPROTOCOL 3

PROCESSING AND EMBEDDING OF TISSUES IN GLYCOLMETHACRYLATE

Fixed tissues are postfixed (optional) and embedded in glycol methacrylate for high-reso-lution light microscopy.

Materials

Fixed tissue sample, 3 to 5 mm thick (see Basic Protocol 2)0.1 M sodium cacodylate buffer (Electron Microscopy Sciences or see recipe),

optionalOsmium tetroxide/potassium ferrocyanide fixative (see recipe), optional50%, 70%, 80%, 95%, and 100% (v/v) ethanolJB4 glycol methacrylate embedding kit (e.g., Polysciences), including: Solution A (acrylic monomer) Catalyst (organic peroxide) Solution B (accelerator)

Ice bathGlycol methocrylate embedding molds, available as block-mold system that allows

polymerization directly onto a metal or plastic microtome chuck (Polysciences)

1. Optional: Wash the fixed tissue sample overnight in 0.1 M sodium cacodylate bufferand postfix 1 hr in 1% osmium tetroxide/1.25% potassium ferrocyanide at roomtemperature. Wash again in two changes, 15 min each, 0.1 M sodium cacodylatebuffer.

This step will provide improved fixation quality, but is optional.

2. Dehydrate tissue using an alcohol series consisting of 30-min steps in 50%, 70%,80%, 95%, and 100% ethanol.

NOTE: Sample should be agitated slowly during all dehydration and infiltration steps.

3. Prepare the glycol methacrylate infiltration solution according to the kit instructionsby adding 100 ml solution A/1 g catalyst. Stir at room temperature to dissolve andstore at 4°C for use in steps 4 to 7.

Polymerization of the resin is an exothermic reaction, and the heat generated can causeartifacts in the tissue. A smaller proportion of catalyst can be used (0.7 g/100 ml solutionA) to slow polymerization time and reduce heat artifacts. This is particularly importantwhen larger blocks (e.g., 1.5 cm2) are produced.

4. Infiltrate tissue sample with a 1:1 mixture of 100% ethanol/infiltration solution for 2hr at 4°C.

5. Infiltrate overnight in full-strength infiltration solution at 4°C.

6. Infiltrate 2 hr in a second change of infiltration solution at 4°C.

7. Prepare the embedding solution by adding 1 ml solution B to 15 ml infiltrationsolution. Use immediately and keep solution in an ice bath while embedding.

8. Orient sample in a glycol methacrylate embedding mold and fill mold with embed-ding solution. Polymerize overnight at 4°C.

Polymerization needs to be carried out in the absence of oxygen and in a dry atmosphere.The design of the molds largely excludes contact with air, but as an additional safetymeasure polymerization may be carried out in a vacuum desiccator or in a nitrogen-filleddesiccator.


16.3.12



Techniques

The use of glycol methacrylate resin as an embedding medium is technically demandingand potentially subject to numerous problems, including difficulties obtaining satisfactorypolymerization (leading to sectioning difficulties) as well as morphological artifacts anddifficulties with staining (Hess and Moore, 1993). It is advisable to experiment with thetechnique prior to its use in a study.

REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions,see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Karnovsky’s fixative10 ml 10% (w/v) paraformaldehyde (see recipe; 1% final)35 ml 0.2 M sodium cacodylate (0.07 M final)12 ml 25% (w/v) glutaraldehyde (e.g., Electron Microscopy Sciences; 3% final)43 ml deionized H2OAdjust to pH 7.4 with 0.1 N hydrochloric acidStore up to 1 week at 4°C.

Krebs Ringer’s solutionDissolve 6.6 g sodium chloride, 0.15 g potassium chloride, and 0.15 g calciumchloride in 1 liter deionized water. Adjust to pH 7.6 with 5% (w/v) sodiumbicarbonate. Store up to 1 month at 4°C. Immediately before use add 5 g procainehydrochloride and 1 ml heparin (10,000 IU).

Modified Davidson’s fixative140 ml ethanol62.5 ml glacial acetic acid375 ml 37% (w/v) formaldehyde422.5 ml distilled H2OStore up to 3 months at room temperature.

Osmium tetroxide/potassium ferrocyanide fixativePrepare a 2% (w/v) aqueous solution of osmium tetroxide and a 2.5% (w/v) aqueoussolution of potassium ferrocyanide. Mix equal volumes together immediately beforeuse (1% osmium tetroxide and 1.25% potassium ferrocyanide, final concentrations).

Paraformaldehyde, 10%Dissolve 2 g paraformaldehyde in 20 ml deionized water. Stir 20 min at 60°C. Adda few drops of 1 M sodium hydroxide to clear the solution. Cool and filter throughWhatman no. 5 filter paper. Store up to 1 month at 4°C.

Sodium cacodylate buffer, 0.1 MDissolve 10.7 g sodium cacodylate in 500 ml deionized water. Store up to 1 monthat room temperature.

This buffer is also commercially available from Electron Microscopy Sciences.

COMMENTARY

Background InformationThe procedures used for reproductive-tract

histology will largely depend on the objectivesof the study. For example, a regulatory screen-ing study to detect toxicity will generally weighand sample the tissues according to the appro-priate regulatory guidelines (Table 16.3.2) anduse routine immersion fixation followed by

paraffin embedding. An investigative study tocharacterize toxicity may concentrate on targettissues of interest and use perfusion fixationfollowed by resin embedding. The latter proce-dure will provide enhanced resolution for lightmicroscopy and, if epoxy resin is used, allowfor ultrastructural examination with the elec-tron microscope.


16.3.13


Critical Parameters andTroubleshooting

Sampling and orientation of tissuesTestes. There are a number of factors to

consider when deciding how to trim the varioustissues of the reproductive tract. In the case ofthe testes, a longitudinal or transverse sectioncan be taken. The advantage of a longitudinalsection is that it provides more tissue to exam-ine and provides frequent longitudinal sectionsthrough seminiferous tubules, many of whichwill include consecutive stages of the sperma-togenic cycle. The advantage of a transversesection is that it provides consistent cross-sec-tions through all the seminiferous tubules. Thismakes staging of the tubule easier and allowsquantitation of cell numbers and tubular meas-urements, if required. Most technicians samplethe testis by taking a midline sample. However,the testis has asymmetric structures that maybe missed by a midline section. For example,in the rodent, the rete testis (the network ofcanals into which the seminiferous tubulesempty) is located at the cranial pole of the testisand spreads down directly beneath the capsuletowards the midline. The rete can provide im-portant information regarding fluid dynamicsof the seminiferous tubule fluid. In cases ofobstruction of the excurrent duct system ordisturbances in fluid reabsorption, it will showdilation. It can also be the site of proliferativelesions and rete testis tumors. Both ends of theseminiferous tubules empty into the rete andthis often appears to be a preferential site forgerm cell degeneration and depletion for bothspontaneous and chemically induced lesions. Itis therefore an important site to sample, androutine sectioning should include at least partof the rete complex. In the dog the rete forms acentral core that extends from the cranial sur-face to approximately two-thirds of the waytowards the caudal tip and will be adequatelysectioned with a midline transverse section. Inthe primate it is located in a peripheral positionalong the medial aspect for approximately halfthe medial border. Again it will be adequatelysampled with a midline transverse section buta longitudinal sample needs to be specificallyorientated to include it.

Efferent ducts. The efferent ducts link therete testis with the ductus epididymis, which isa single, highly coiled duct, the convolutions ofwhich form the caput, corpus, and caudaepididymis. The number and course of the ef-ferent ducts vary with species. They are longand tortuous in rodents, extending up into the

epididymal fat pad surrounding the caputepididymis; they are normally trimmed off anddiscarded when the epididymis is trimmed forweighing. In the dog and primate they are veryshort because the epididymis is more closelyapplied to the testis in these species; they arerarely sampled in routine studies but can be animportant site for lesions. The efferent ducts inthe dog are a frequent site for sperm granulomasbecause of the presence of numerous blind-end-ing tubules. Similarly in the rat, this may be thelocation of sperm granulomas that give rise totubular dilation in the testes and absence ordecreased sperm in the epididymis. More im-portantly, they have been shown to be a targetsite for a number of chemicals (Hess, 1998).Although it may not be practical to sample themin routine studies, their potential as a target sitefor toxicity or as a cause of secondary changesin the testes and epididymides should be appre-ciated.

Epididymides. The cellular composition andthe function of the epididymis vary with region,and it is important that the different regions aresampled and examined. Routine sampling ofthe epididymis often only includes the caudaepididymis, but this precludes important infor-mation. There are a number of chemicals thathave been shown to produce site-specific le-sions to the epididymal epithelium, some in thecaput, others in the cauda. An additional reasonfor sampling the entire epididymis is that thedensity of sperm and the presence of germ cellswithin the lumen of various segments of theepididymis reflect time-dependent events in thetestis. For example, in the rodent, dog andprimate sperm and cells present in the caudaepididymis were released from the testis ∼2weeks prior to their arrival there. The sperm inthe caput epididymis reflect the release ofsperm only days previously. Depending on theduration of the study, differential effects on thecontents of the caput and cauda can provideimportant information on the timing of a toxiceffect.

Prostate. The prostate of rodents is a hetero-geneous gland with anatomically distinct ven-tral, dorsal, and lateral lobes (Fig. 16.3.2B). Thehistologic structure and the response to toxi-cants vary between lobes such that it is impor-tant to routinely sample as much as possible. Amidtransverse section through the prostaticcomplex allows an adequate sample of all threelobes and often also includes the ampullarygland of the ductus deferens as it enters theprostatic urethra (Lee and Holland, 1987; Suwaet al., 2001). In the dog and the primate, the


16.3.14



Techniques

glandular tissue is arranged circumferentiallyaround the urethra, although in the primate it isonly present on the posterior surface of theurogenital canal. In these species, a transversesection provides an appropriate sample for ex-amination (Figs. 16.3.3B and 16.3.4B).

Seminal vesicles and coagulating gland. Inrodents, a coagulating gland lies on the anterioraspect of each seminal vesicle. Both can besampled by a cross-section. Similarly, a cross-section of the seminal vesicles in the primateprovides adequate sampling. These organs areabsent in the dog.

Choice of fixativeAll of the recently revised regulatory guide-

lines for reproductive toxicity studies specifi-cally recommend fixing the testes in Bouin’s ora comparable fixative (Table 16.3.2). Implicitin this recommendation is that the use of for-malin is avoided due to the excessive shrinkageof Sertoli cell and germ cell nuclei and cyto-plasm, which occurs when formalin-fixed tes-tes are embedded in paraffin. Although accept-able results can be obtained with formalin (Har-leman and Nolte, 1997), Bouin’s provides morereliable and consistent results. Testes fixed inBouin’s show greatly improved preservation ofthe nuclear and cytoplasmic features of Sertolicells and germ cells, with very little shrinkageof the cells away from one another. However,due to its picric acid component, Bouin’s has anumber of drawbacks. It is potentially explo-sive, mutagenic, and possibly carcinogenic,presenting significant safety and disposal prob-lems to laboratory personnel when used on alarge scale. It also results in significant shrink-age of the tubules away from the interstitialtissue, especially in the center of the testis.Testes fixed with modified Davidson’s fixative(see Basic Protocol 1) show comparable cellu-lar and nuclear resolution with very littleshrinkage of the germ cells and Sertoli cells. Inaddition, the tubules show significantly lessshrinkage from the interstitial tissue than isseen with Bouin’s. Interestingly, the problemswith formalin fixation are associated with sub-sequent processing into paraffin wax, becauseformalin fixation of rodent testes followed byembedding in glycol methacrylate results inbetter preservation of structure than withBouin’s (Chapin et al., 1984). However, thisdoes not hold true for all species and should beexperimented with prior to use. For a detailedreview of methods and the results that can beobtained using various combinations of fixa-tives, fixation methods, and embedding proce-

dures for rodent testes, see Chapin et al. (1984),Russell et al. (1990), and Hess and Moore(1993).

Many of the difficulties associated with pre-serving the testes are due to the requirement tofix the tissue whole. This is necessary so thatthe architecture of the tubules and interstitialtissue is maintained and to prevent artifactualsloughing of germ cells. If the capsule is cut orremoved, the tubules erupt and flow out of theholes. However, improved penetration of fixa-tive can be achieved by pricking the capsule ofrodent testes or making nicks in the capsule ofdog or primate testes before immersing in fixa-tive. (Large-animal testes have more fibrovas-cular stroma and are less likely to erupt thanrodent testes.) If the pricks and cuts are madesuperficially and at a location distant from thearea to be sampled, this will not disturb thearchitecture. Fixation quality can also be sig-nificantly improved if the testis is sliced afterit has been partially fixed by immersion. Allow≥4 to 5 hr (or overnight) before cutting the testisand then cut it in half or make 5-mm slices.Return to fixative to allow a total fixation timeof 48 hr. Tissues should not be left in Bouin’sor Davidson’s for very much longer than 48 hr,otherwise they will harden and become difficultto section.

The Karnovsky’s fixative recommended inBasic Protocol 2 and the subsequent postfixa-tion in osmium tetroxide and potassium ferro-cyanide are designed to give optimal fixationand staining of testicular structure for ultras-tructural examination while minimizing os-motic artifacts. The high water content of tes-ticular tissue and the distance of the seminifer-ous epithelium from the interstitial vasculaturemake fixation difficult and artifacts frequent.One of the most common artifacts seen is basalvacuolation of the seminiferous tubules. Thisis caused by the use of hyperosmotic fixative,which causes shrinkage of the cells in the basalcompartment (below the level of the Sertoli celltight junctions). Hyperosmotic fixatives willalso cause artifactual condensation of Sertolicell cytoplasm at the ultrastructural level. Forultrastructural studies it is therefore importantto use approximately isotonic solutions andfixatives. The use of the osmium/ferrocyanidemixture as a postfixative is particularly goodfor membrane preservation, but it also has theadvantage of improving the staining charac-teristics and resolution of Leydig cell structureat the ultrastructural level (Russell and Burguet,1977). The choice of buffer for glutaraldehyde-based fixatives is a matter of personal prefer-


16.3.15


ence. Cacodylate buffer has been recom-mended in this protocol, but due to its toxicityand associated disposal problems, a phosphatebuffer may be preferred as a substitute (Hessand Moore, 1993).

PerfusionThere are various techniques for perfusion

fixation of tissues. The choice is largely a matterof personal preference and resource limitations.The procedure described in Basic Protocol 2uses a peristaltic pump to perfuse the wholeanimal. This maintains a constant flow of fixa-tive for a given speed of the pump, despite theincreasing vascular resistance. A gravity-fedsystem can also be used, as described bySprando (1990). This has the advantage of notrequiring a peristaltic pump but has the disad-vantage of requiring the fixative and salinebottles to be located some distance above theanimal. With gravity-based perfusion, the flowrate also decreases as the vascular resistanceincreases.

Rather than perfuse the whole animal asdescribed in this protocol, it is also possible tolimit perfusion only to the abdominal tissues.Hess and Moore (1993) describe a method ofcannulating the abdominal aorta via the leftventricle, which bypasses perfusion of the cra-nial and thoracic tissues. This conserves fixa-tive, but the cannulation is slightly more tech-nically demanding.

A critical step in the perfusion technique isthe successful clearance of blood from the en-tire vascular bed. If vessels constrict or intravas-cular thrombi form, then the fixative will notbe uniformly distributed. Clearance of the tes-ticular vasculature appears to be particularlyproblematic. To overcome this problem, hepa-rin and/or vasodilators have been employed. Inthis protocol the incorporation of both heparinand procaine in the flushing solution (Hess andMoore, 1993) has been recommended on thebasis of a very good success rate combined withsimplicity. However, Sprando (1990) suggeststhat the preinjection of animals with 130 to 150IU heparin/kg body weight by intraperitonealinjection 15 min prior to perfusion improvesthe success rate without the need for additivesto the perfusion fluids.

Anticipated ResultsUsing Basic Protocol 1, the quality of the

paraffin sections of testes stained with periodicacid Schiff’s stain/hematoxylin (PAS-H) willbe suitable for detailed qualitative evaluationof spermatogenesis. Resolution and staining of

the spermatid acrosome in rodent testes will beadequate to identify individual stages of thespermatogenic cycle. In dog and primate testes,the acrosome does not stain with PAS andcannot be adequately distinguished in paraffin-embedded sections. Therefore staging has torely on the characteristics of the spermatocyteand round spermatid population in conjunctionwith the shape and position of the elongatingspermatid population. Cytoplasmic and nucleardetail of the germ cells, Sertoli cells, and Leydigcells will be suitable for detecting early degen-erative changes. Although quantitative proce-dures, such as cell counting and measuringtubular diameters is possible, it is better carriedout on resin-embedded tissue where sectionthickness is more consistent and nuclear chro-matin resolution is improved.

Longitudinal sect ions through theepididymis will provide information on thecellular morphology of the entire length of thecoiled epididymal duct and of the relativesperm and cell content of the ductular lumen.In the dog and primate, the longitudinal sectionof the epididymis may also include some of theefferent ducts. The transverse section throughthe rodent prostate should provide comparativemorphology of the dorsolateral lobes and ven-tral lobes of the tissue and may also includecross-sections of the vas deferens and of theassociated ampullary gland of the vas. Thesecretory products of the various lobes and ofthe ampullary gland have different affinities foreosin. The differential staining pattern can beused to identify various regions (Lee and Hol-land, 1987; Yuan et al., 1987). In the dog andthe primate, the prostatic acini should appearmore or less homogeneous with no regionalvariation.

Using Basic Protocol 2, perfusion fixationshould provide tissue with a minimum of arti-facts. All interstitial capillaries should be openand empty of erythrocytes. There should be noshrinkage of tubules away from the interstitialtissue and no shrinkage of germ cells away fromSertoli cells. Vacuoles between adjacent Sertolicells should be minimal or nonexistent andthere should be no sloughing of germ cells intothe tubular lumen. When viewed by electronmicroscopy, the morphology of the smoothendoplasmic reticulum and mitochondria in thebasal Sertoli cell cytoplasm are good indicatorsof fixation quality. Toluidine blue–stained resinsections can be used to distinguish the sperma-tid acrosome in all species including the dog,although the thinness of the section results inmany acrosomes being incompletely sectioned


16.3.16



Techniques

or out of the plane of section. Glycol methacry-late–embedded, immersion-fixed tissue gener-ally results in less tubular and cellular shrinkagethan paraffin-embedded tissue and shows im-proved resolution of nuclear and cytoplasmicfeatures. It too suffers from reduced numbersof complete acrosomes in the plane of section.

Time ConsiderationsImmersion fixation and embedding tissue in

paraffin wax is the most economical method-ology in terms of time and cost. Because all theprocedures are more or less standard routine fora histology laboratory, the processing, embed-ding, and staining is largely automated. Overalltime depends on the number of tissues sampledand weighed from each animal and the numberof animals processed. For a routine study size(e.g., two to five treatment groups, each withfive to ten animals), allowing 48 hr fixation timeplus ∼1 day processing and embedding intoparaffin and 1 day sectioning and staining,sections could be ready for evaluation within aweek.

Processing for glycol methacrylate can alsobe automated using routine, paraffin-embed-ding processors up to the point of resin infiltra-tion. Processing for epoxy resin can be auto-mated through to the point of embedding, butthis requires a purpose-designed machine tohandle the small tissue size and the viscuousresin mixtures. The alternative is to carry outthe procedure in individual bottles, carefullydraining and filling each with new solution.Embedding in resin requires special molds andspecial polymerization conditions and can takeup to 48 hr for adequate polymerization. Sec-tioning of resin blocks requires the use of aspecial, automated microtome that uses pre-pared glass or tungsten carbide knives and is atechnically demanding procedure requiring atleast 1 to 2 days training and several dayspractice to become proficient. With glycolmethacrylate, consistent polymerization of theresin and control of the relative hydration of theresin during sectioning can also add to thedifficulty and the time taken to produce accept-able sections. Overall, the size of the tissueblock and the proficiency and experience of thetechnician will determine the time taken toprepare adequate resin sections, but, as a guide,it probably takes at least twice to three timeslonger to prepare a 2- to 3-µm resin section thanit does to prepare a paraffin section.

The duration of the perfusion means that thisis also a time- and labor-intensive technique. Iflarge numbers of animals are to be fixed by this

method, multiple perfusion stations are re-quired, and animals must be staggered throughthe various procedures. With three perfusionstations and two people carrying out the perfu-sions, it is feasible to complete 20 animals in anormal working day. (Sprando, 1990).

Literature CitedBancroft, J.D., Stevens, A., and Turner, D.R. 1990.

Theory and Practice of Histological Techniques,3rd ed. Churchill Livingston, New York.

Chapin, R.E., Ross, M.D., and Lamb, J.C. 1984.Immersion fixation methods for glycolmethacrylate-embedded testes. Toxicol. Pathol.12:221-227.

Environmental Protection Agency (EPA). 1998Health Effects Test Guidelines, Report No.OPPTS 870.3800, Reproduction and FertilityEffects. pp. 1-12. EPA 712-C-98-208. Office ofPrevention, Pesticides and Toxic Substances(OPPTS), U.S. EPA, Washington, D.C.

Evans, H.E. 2000. Guide to the Dissection of theDog, 5th ed. W.B. Saunders, Philadelphia.

Feldman, D.B. and Seeley, J.C. 1988. NecropsyGuide: Rodents and the Rabbit. CRC Press, BocaRaton, Fla.

Frederick, P.M. and Doorn, L.G. 1973. A techniquefor perfusion of rat testis in situ through internalspermatic arteries. J. Reprod. Fertil. 35:117-121.

Harleman, J.H. and Nolte, T. 1997. Testicular toxic-ity: Regulatory guidelines—the end of formalinfixation? Toxicol. Pathol. 25:414-417.

Hayat, M.A. 1989. Principles and techniques ofelectron microscopy: Biological applications,3rd ed. CRC Press, Boca Raton, Fla.

Hess, R.A. 1998. Effects of environmental toxicantson the efferent ducts, epididymis and fertility. J.Reprod. Fertil. Suppl. 53:247-259.

Hess, R.A. and Moore, B.J. 1993. Histologicalmethods for evaluation of the testes. In Methodsin Toxicology, Vol. 3, Part A. Male ReproductiveToxicology (R.E. Chapin and J.J. Heindel, eds.)pp. 86-94. Academic Press, San Diego.

International Conference on Harmonization ofTechnical Requirements for Registration ofPharmaceuticals for Human Use (ICH). 1994.Tripartite Harmonized ICH Guideline S5A: Re-productive Toxicology: Detection of toxicity toreproduct ion for medicinal prod-ucts/CPMP/ICH/386/95. Fed. Regist. 59:48746-48752.

International Conference on Harmonization ofTechnical Requirements for Registration ofPharmaceuticals for Human Use (ICH). 1996.Tripartite Harmonized ICH Guideline S5B: Re-productive Toxicology: Male fertility stud-ies/CPMP/ICH/136/95. Fed. Regist. 61:15360.

Lee, C. and Holland, J.M. 1987. Anatomy, histologyand ultrastructure, prostate, rat. In Monographson Pathology of Laboratory Animals: GenitalSystem (T.C. Jones, U. Mohr, and R.D. Hint,eds.) pp. 239-251. Springer-Verlag, New York.


16.3.17


Organization for Economic Cooperation and Devel-opment (OECD). 1995. Reproduction/develop-mental toxicity screening test. In Guideline forTesting of Chemicals, No. 421 (adopted July 27,1995) pp. 1-10. OECD, Paris.

Organization for Economic Cooperation and Devel-opment (OECD). 2001. Proposal for updatingGuideline 416: Two generation reproductiontoxicity study. In Guideline for Testing ofChemicals, No. 416 (adopted January 22, 2001)pp. 1-13. OECD, Paris

Russell, L. and Burguet, S. 1977. Ultrastructure ofLeydig cells as revealed by secondary tissuetreatment with a ferrocyanide-osmium mixture.Tissue Cell 9:751-766.

Russell, L.D., Ettlin, R., Sinha Hikim, A.P., andClegg, E.D. 1990. Tissue Preparation for Evalu-ation of the Testis. In Histological and His-topathological Evaluation of the Testis (L.D.Russell, R. Ettlin, A.P. Sinha Hikim, and E.D.Clegg, eds.) pp. 195-209. Cache River Press,Clearwater, Fla.

Sprando, R.L. 1990. Perfusion of the rat testisthrough the heart using heparin. In Histologicaland Histopathological Evaluation of the Testis(L.D. Russell, R. Ettlin, A.P. Sinha Hikim, andE.D. Clegg, eds.) pp. 277-280. Cache RiverPress, Clearwater, Fla.

Suwa, R., Nyska, A., Peckham, J.C., Hanley, J.R.,Mahler, J.F., Haseman, J.K., and Maronpot, R.R.2001. A retrospective analysis of backgroundlesions and tissue accountability for male acces-

sory sex organs in Fisher-344 rats. Toxicol.Pathol. 29:467-478.

Yuan, Y.D., Ulrich, R.G., and Carlson, R.G. 1987.Histology and ultrastructure, glands of the duc-tus deferens (ampullary gland), rat. In Mono-graphs on Pathology of Laboratory Animals:Genital System. (T.C. Jones, U. Mohr, and R.D.Hint, eds.) pp. 229-234. Springer-Verlag, NewYork.

Key ReferencesHess, R.A. and Moore, B.J. 1993. See above.

Provides a detailed comparison of the resultsachieved using different combinations of fixativesand embedding procedures to examine the testes.Also provides technical details for perfusion fixationand subsequent processing and embedding in resinfor light and electron microscopy.

Russell et al., 1990. See above.

Provides various protocols for fixation, tissue proc-essing, and staining of the testis and discusses theadvantages and disadvantages of various methods.

Sprando, R.L. 1990. See above.

Provides detailed technical guidance on the art ofperfusion fixation for the testes.

Contributed by Dianne M. CreasyHuntingdon Life SciencesEast Millstone, New Jersey


16.3.18



Techniques

current protocols in toxicology i

Documents