Histopathology of the Male Reproductive System I: Techniques

Effective evaluation of the male reproductive system relies on adequate sampling of the various parts of the reproductive tract as well as appropriate fixation, orientation, processing, embedding, and staining of the tissues. This unit describes protocols for these various aspects of tissue preparation. The presence or absence, as well as the anatomical arrangement, of various components of the reproductive tract varies with species. These differences must be appreciated to ensure appropriate sampling. Basic Protocol 1 and Support Protocol 1 describe the identification and dissection procedures for the reproductive organs from the rodent, dog, and primate, which are the animals most frequently used in toxicological research. Basic Protocol 1 describes the routine immersion fixation of these tissues followed by processing and embedding in paraffin wax. For a variety of reasons, the testis presents a problem for good fixation. Conventional immersion fixation in formalin generally results in poor preservation of the seminiferous tubules, and the use of an alternative fixative, such as Bouins, is recommended in most regulatory guidelines. Although Bouins provides good morphology suitable for detecting subtle disturbances in spermatogenesis, it suffers from a number of increasingly important safety and disposal problems and also results in significant shrinkage of the tubules. This protocol recommends the use of a novel, alcohol-based fixative (modified Davidsons fixative), which provides comparable cellular and nuclear preservation to that achieved with Bouins but with less tubular shrinkage and none of its safety and disposal hazards. Once the tissues are fixed, sampling of specific structures and tissue orientation are very important to ensure that critical regions of the reproductive tract are presented for examination. Support Protocol 1 provides guidance on how to trim and sample tissues from rodents, dogs, and primates. Basic Protocol 2 describes the methodology used to obtain excellent preservation of the rodent testis by whole-body perfusion via the heart. The testes of large animals can also be fixed by perfusion, but to reduce the volume of fixative required this is best carried out by introducing the fixative into the testicular artery. Support Protocols 2 and 3 describe the processing procedures required for embedding in epoxy and glycol methacrylate resin, respectively. Perfusion fixation followed by embedding in resin is essential for proper evaluation of the testis by electron microscopy. The use of epoxy resin is necessary to maintain stability of the section under the electron beam; however, epoxy-embedded tissue can only be stained with toluidine blue for light microscopic examination. Glycol methacrylate resin is a water-soluble resin that can be used to prepare semithin (2 m) sections that can be stained with conventional histologic stains for high-resolution light microscopy. A compromise between the convenience of immersion fixation followed by paraffin embedding and the labor-intensive perfusion fixation followed by resin embedding can be achieved by immersion fixation followed by embedding in glycol methacrylate resin. CAUTION: Many of the chemicals used in these protocols are hazardous, including formalin, glutaraldehyde, propylene oxide, epoxy and glycol methacrylate resins, sodium cacodylate, and osmium tetroxide. Refer to material safety data sheets for appropriate handling, storage, and disposal.

UNIT 16.3

Male Reproductive Toxicology Contributed by Dianne M. Creasy

Current Protocols in Toxicology (2002) 16.3.1-16.3.18 Copyright 2002 by John Wiley & Sons, Inc.

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NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must conform to governmental regulations regarding the care and use of laboratory animals.
BASIC PROTOCOL 1

DISSECTION, IMMERSION FIXATION, AND PARAFFIN EMBEDDING OF MALE REPRODUCTIVE-TRACT TISSUES This procedure is suitable for routine screening of tissues for toxicological changes. It involves dissection of the major reproductive tissues, measurement of organ weights, and immersion fixation of the testes using a special fixative (the remaining tissues can be fixed in the same fixative or in neutral buffered formalin). This is followed by careful trimming of the tissues to include specific structures and conventional processing through an ascending series of alcohols and clearing agents, followed by embedding in paraffin wax. Sections are then cut and stained with periodic acid Schiffs stain/hematoxylin (PAS-H; for testes and epididymides) and hematoxylin and eosin (for remaining tissues). Materials Male rodent, dog, or primate Modified Davidsons 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 paraffin sectioning 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 sodium pentobarbital (e.g., Sleepaway).

2. Depending on the species under investigation (Table 16.3.1), use surgical instruments to dissect out the testes, epididymides, and accessory sex glands (i.e., seminal vesicles with coagulating glands and prostate; see Support Protocol 1).
Standard dissection guides, such as Feldman and Seeley (1988) and Evans (2000), contain detailed dissection procedures. It is important to minimize squeezing of the testes during handling to prevent artifactual sloughing of the germ cells from the seminiferous epithelium. When trimming the epididymis from the testes, take care not to cut the testicular capsule, which will cause extrusion of the seminiferous tubules through the cut and consequent disruption of tissue architecture.

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 unilateral microscopic findings. Individual organ weights are also required where one testis and one epididymis are used for quantifying sperm parameters (Table 16.3.2). When weighing seminal vesicles and the prostate, it is important that the fluid be included in the weight because its volume reflects the functional activity of the glands. For some regulatory guidelines, the combined weight of seminal vesicles and prostate is recommended (Table 16.3.2). If separate weights are desired, dissect out the prostate and seminal vesicles as a unit, being careful to retain all fluids, and then separate the seminal vesicles and coagulating glands from the prostate over a weigh boat, so that any fluid leakage from the seminal vesicles is caught and included for weighing.

Histopathology of the Male Reproductive System I: Techniques

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Table 16.3.1

Presence of Accessory Sex Glands in Common Laboratory Species

Species Dog Mouse Primate Rabbit Rat

Ampulla ductus deferens + + + + +

Bulbourethral (Cowpers) glands + + + +

Coagulating glands + +

Preputial gland + + +

Prostate + + +a + +

Seminal vesicles + + +b +

aNo anterior lobe present. bPresent as rudimentary glandula vesicularis.

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

Guidelinea

Tissues to be weighed

Tissues to be preserved One testisb,c One epididymisb

Tissues to be examined Testis Epididymis Seminal vesicles

OECD 416: Two-generation Testes reproduction toxicity study Epididymides (total and (OECD, 2001) cauda)

Seminal vesicles with Seminal vesicles coagulating glands and their fluids and prostate (as one unit) Coagulating glands Prostate Testes OECD 421: Reproduction/developmental toxicity Epididymides screening test (OECD, 1995) Testes Testesc Epididymides Seminal vesicles Coagulating glands Prostate Right testisb,c epididymisb

Coagulating glands Prostate Epididymides

OPPTS 870.3800: Reproduction and fertility effects (EPA, 1998)

Testes

Testis Epididymis Seminal vesicles

Epididymides (total and Right cauda) Seminal vesicles Seminal vesicles with coagulating glands and their fluids Coagulating glands Prostate None Prostate Testesc and epididymides

Coagulating glands Prostate Testes and epididymides if indicated by altered fertility indices

ICH S5A and S5B: Detection of toxicity to reproduction for medicinal products (ICH, 1994, 1996d)

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 Bouins or comparable fixative. dAn addendum on male fertility studies.

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4. Place the testes into modified Davidsons fixative for 48 hr at room temperature and then transfer to 10% neutral buffered formalin for storage. Place the remaining tissues into 10% neutral buffered formalin for 48 hr at room temperature until required for processing.
Speed of penetration of the fixative can be improved by pricking or nicking the capsule before immersion and also by slicing the testis following several hours in fixative (see Critical Parameters and Troubleshooting, discussion of choice of fixative). The tissues can be stored in 10% neutral buffered formalin for up to 10 years at room temperature. For convenience, the epididymides and secondary sex organs can also be fixed in Davidsons fixative, but the cytological and staining characteristics are slightly inferior to those of formalins.

5. Trim the tissues according to Support Protocol 1 and process through standard histology 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 glycol methacrylate resin following immersion fixation (see Support Protocol 3).

6. Prepare sections at 5 to 6 m.
Standard paraffin sectioning is described in Bancroft et al. (1990).

7. Stain testes and epididymides with PAS-H stain and the remaining tissues with hematoxylin 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 spermatid acrosomic granule or acrosome cap. These are very small structures, which can be difficult to visualize if the stain is not optimal. It is important that the periodic acid and the Schiff reagent are relatively fresh solutions. In dog testes, the acrosome does not stain with PAS and cannot be adequately distinguished in paraffin-embedded sections (see Anticipated Results).

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

Solutiona Neutral buffered formalin 70% ethanol 80% ethanol 90% ethanol 100% ethanol 100% ethanol 100% ethanol Xylene Xylene Xylene Wax Wax Wax

Temperature (C) 37 37 37 37 37 37 37 37 37 37 60 60 60

Time (hr) Rodent 0.5 1.0 1.5 1.0 1.0 1.5 2.0 0.5 1.0 1.5 1.0 1.0 2.0 Dog/primate 0.5 1.0 2.0 1.5 1.0 1.5 2.0 0.5 1.5 2.0 1.0 1.5 2.0

Histopathology of the Male Reproductive System I: Techniques

aTissues should be agitated in each solution.

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PERFUSION FIXATION OF THE MALE RODENT REPRODUCTIVE TRACT BY CARDIAC PERFUSION AND RESIN EMBEDDING OF TISSUES This method is used when high-resolution light microscopy or electron microscopy is required. The methodology is time consuming and labor intensive and is more suited to investigative studies than to large-scale screening studies. The procedure involves anesthetizing the animal, cannulating the thoracic aorta through the heart, and flushing out the systemic vasculature with Ringers solution followed by systemic perfusion with a buffered paraformaldehyde and glutaraldehyde fixative. The method described is a whole-body perfusion using a peristaltic pump to maintain a constant pressure during perfusion. Following fixation, the tissue may be embedded in glycol methacrylate or epoxy 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 because of the large volume of fixative required. An alternative technique introduces the fixative into the testicular artery (Frederick and Doorn, 1973; Russell et al., 1990). Materials Krebs Ringers solution (see recipe) Karnovskys fixative (see recipe) Male rodent 65 mg/ml sodium pentobarbital solution (e.g., Sleepaway; Fort Dodge Animal Health) or equivalent for anesthesia 0.1 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 trap Rubber bands Dissecting tools, including: Scalpel Hemostats Forceps 5-cm (2-in) Dieffenbach serrefine (bulldog) clamp Single-edged blades Additional reagents and equipment for dehydrating, infiltrating, and embedding in epoxy resin (see Support Protocol 2); sectioning and staining for electron microscopy or dehydrating, infiltrating, and embedding in glycol methacrylate (see Support Protocol 3); sectioning and staining for high-resolution light microscopy Set up perfusion apparatus 1. 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.

BASIC PROTOCOL 2

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vacuum pump

manometer waste saline fixative dosing needle

three-way stopcock peristaltic pump dissection board

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

2. Fill one 1-liter bottle with Krebs Ringers solution and a second one with Karnovskys fixative. Connect these with silastic tubing and a three-way stopcock to a variablespeed peristaltic pump. Connect a manometer in-line and attach a dosing needle to the end of the tubing. 3. Open the stopcock and flush the tubing with Krebs Ringers solution. Ensure that the tubing 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 110 to 130 l/min. This should be equivalent to a perfusion pressure of 100 to 120 mm Hg. During the perfusion the pressures should not be allowed to rise above 150 mm Hg. 5. Set up a dissection board suspended in a tray and a vacuum pump connected to a 1-liter bottle trap to drain used fluids. Perfuse animal 6. Anesthetize a male rodent with 0.8 to 1.0 l/Hg of 65 mg/ml sodium pentobarbital solution.
It is important to achieve a level of anesthesia that results in maintenance of shallow breathing and adequate cardiac output and yet produces a sufficient level of surgical anesthesia. Speed is critical to the success of the technique. Once the animal is adequately anesthetized, dissection and introduction of the Krebs Ringers solution must be accomplished as efficiently and quickly as possible.
Histopathology of the Male Reproductive System I: Techniques

7. Secure the animal on its back to the dissection board using rubber bands around its paws.
The dissection board should be located over a draining vessel to catch the perfusion fluids.

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8. Make a midline incision with a scalpel to open the abdomen and then cut through the rib cage in a V shape from the xiphoid process to the axilla. Avoid cutting any major blood vessels in the axilla or along the sternum. 9. Clamp the xiphoid process with a pair of hemostats and displace the flap of thoracic tissue 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 Ringers solution through the perfusion apparatus at 110 to 120 ml/min. 12. Grasping the ventricular apex with a pair of forceps, rapidly make an incision in the right ventricle to allow fluid efflux. Then make a small incision in the left ventricle and insert the dosing needle through the ventricle and into the aorta so that it is just visible through the vessel wall. Clamp it in place using a Dieffenbach serrefine clamp on the proximal portion of the aortic arch.
Krebs Ringers solution should be flowing through the apparatus when the dosing needle is inserted. The needle must not be pushed too far into the aorta, otherwise it will press against the aortic arch and block the flow. Once the perfusion is underway, the angle that the needle enters the heart and aorta needs to be maintained at a slight elevation so that the outflow of the fluid is not obstructed. This can be accomplished using clamps or supports.

13. Perfuse the animal at a flow rate of 110 to 120 ml/min until the blood has cleared from the testes (1 to 2 min).
The blood will gradually be replaced by the Krebs Ringers solution.

14. During the perfusion, expose the testes by cutting through the scrotum. Leave the testes in situ and watch for the coiled, capsular testicular artery to clear of blood and for the testicular tissue to become uniformly pale. Fix tissue 15. 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 30 min for electron microscopy and 20 min for light microscopy only. After the first 2 to 3 min, slow the rate of perfusion to 10 ml/min.
Duration of perfusion is more important than the volume of fluid perfused. The initial pressure and flow rate is important to introduce fixative rapidly while minimizing pressure artifacts. Once the vasculature and basic architecture have been stabilized, the perfusion pressure can be reduced to conserve fixative. A volume of 300 ml is appropriate for a 300-g animal. As described, the technique will provide excellent fixation for ultrastructural studies with minimal fixation and handling artifacts. If the tissue is only intended for high-resolution light microscopy, then the perfusion time with fixative can be reduced. With this shorter time, the tissue may be subject to some minor handling artifacts.

16. Between perfusions, flush the perfusion line thoroughly with Krebs Ringers solution to 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 4C.

Male Reproductive Toxicology

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18a. Wash in three changes of 0.1 M sodium cacodylate buffer at 4C, with one wash being overnight and other washes 15 min. 19a. Postfix 1 hr in osmium tetroxide/potassium ferrocyanide fixative at room temperature. 20a. Dehydrate, infiltrate, and embed tissues in epoxy resin as described (see Support Protocol 2). 21a. Section embedded tissue at 1 to 2 m, float sections onto a hot water bath at 60C, 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 60C 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 onto water at room temperature, and pick up onto electron microscopy grids. 24a. Stain sections with uranyl acetate and lead citrate and examine under an electron microscope.
Preparation of tissues for examination by electron microscopy requires specialized techniques 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 (see Support Protocol 3). 19b. Section at 2 m using a purpose-designed motorized, retracting microtome and a tungsten-carbide or large-area glass (Ralph) knife.
Examples of appropriate microtomes include the Reichert-Jung Supercut and LKB Historange. When the resin blocks are trimmed to reach the tissue, the use of 70% (v/v) ethanol on the surface of the block will soften the resin and allow thicker sections to be taken without shattering the resin.

20b. Float the sections on a water bath at room temperature until the resin has expanded maximally (1 to 2 min). 21b. Pick up sections on a grease-free glass slide and dry on a hot plate or in an oven at 60C for 30 min before staining.
Flattening and picking up the section is aided if the water bath is previously wiped with 70% (v/v) ethanol to reduce surface tension.

22b. Stain with periodic acid Schiffs (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 standard histological staining methodology.
Histopathology of the Male Reproductive System I: Techniques

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

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REMOVAL, TRIMMING, AND ORIENTATION OF MALE TESTES, EPIDIDYMIDES, AND ACCESSORY SEX GLANDS This protocol describes the dissection of testes, epididymides, and accessory sex glands for male rodents, dogs, and primates that have been euthanized as described in Basic Protocol 1. Specific directions for fixing and sectioning the samples are described in Basic Protocol 1. Rodent For a male rodent, remove the testes and epididymides as a unit. Separate the epididymides from the testes and trim to remove the surrounding adipose tissue. Weigh the testes and epididymides separately. After fixation, take a 5-mm transverse section through the testis slightly cranial to the midline to include a small portion of the rete testis (which is subcapsular and originates at the point where the epididymis is attached and is where all the capsular blood vessels converge at the cranial pole). Alternatively, take a longitudinal section through the plane of the rete testis. (For a discussion of the advantages and disadvantages of the plane of section, see Critical Parameters and Troubleshooting, discussion of sampling and orientation of tissues). Take a longitudinal section through the epididymis to include the caput (head), corpus (body), and cauda (tail). To achieve this, trim a thin slice of tissue from the caput and the cauda so that the length of the epididymis 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). Carefully remove the bladder and weigh and fix the seminal vesicles, coagulating glands, and prostate 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 to catch 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-

SUPPORT PROTOCOL 1

A
SV CG VD DP LP VP BG AG B

B
dorsal lobe vas deferens ampullary gland lateral lobe urethra

ventral lobe

PG

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

Male Reproductive Toxicology

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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 the epididymis from the testis and trim away any adipose tissue. Weigh the testes and epididymides separately. After fixation, take a 5-mm transverse slice through the midline of the testis. Depending on the size of the testis, it may be necessary to trim this further to fit into a standard histology processing cassette. If so, ensure that the rete testis (which is enclosed in the mediastinum and runs as a longitudinal central core through the middle of the testis) is included. Bisect the epididymis transversely through the corpus so that half remains with the caput and half with the cauda. Trim off a thin, longitudinal slice of tissue from the caput and the cauda to allow the tissue to be embedded flat. This will provide 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 a transverse 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 the epididymis from the testis and trim away any adipose tissue. Weigh the testes and epididymides separately. After fixation, take a 5-mm transverse slice through the midline of the testis. Depending on the size of the testis, it may be necessary to trim this further to fit into a standard histology processing cassette. In primates, the rete testis lies in a subcapsular location along the epididymal edge. Ensure that this portion of the testis is sampled. Bisect the epididymis transversely through the corpus so that half remains with the caput and half with the cauda. Trim off a thin, longitudinal slice of tissue from the caput and the cauda to allow the tissue to be embedded flat. This will provide a longitudinal section through the caput, corpus, and cauda.

A
B

urethra VD AG P lobule of acini

Histopathology of the Male Reproductive System I: Techniques

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

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A
VD B AG SV P

B
urethra

BG

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 absent from the anterior aspect. AG, ampullary gland; B, bladder; BG, bulbourethral gland; P , prostate; SV, seminal vesicles; VD, vas deferens. Figure drawn by Ann Hoffenberg.

Remove the seminal vesicles, prostate, and bladder as a unit (Fig. 16.3.4A). Carefully remove the bladder. Weigh and fix the seminal vesicles and prostate 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 to catch any fluid. After fixation, separate the seminal vesicles from the prostate. Take a transverse section through each seminal vesicle and take a transverse section through the prostate and the prostatic urethra (Fig. 16.3.4B). 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% potassium ferrocyanide (see Basic Protocol 2) 30%, 50%, 70%, 85%, 95%, and 100% (v/v) ethanol Propylene oxide (e.g., Electron Microscopy Sciences) 1:1 propylene oxide/epoxy resin Epoxy 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% potassium ferrocyanide 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 manufacturers instructions for specific resin for details. SUPPORT PROTOCOL 2

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 60C.

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SUPPORT PROTOCOL 3

PROCESSING AND EMBEDDING OF TISSUES IN GLYCOL METHACRYLATE Fixed tissues are postfixed (optional) and embedded in glycol methacrylate for high-resolution 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), optional Osmium tetroxide/potassium ferrocyanide fixative (see recipe), optional 50%, 70%, 80%, 95%, and 100% (v/v) ethanol JB4 glycol methacrylate embedding kit (e.g., Polysciences), including: Solution A (acrylic monomer) Catalyst (organic peroxide) Solution B (accelerator) Ice bath Glycol 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 buffer and postfix 1 hr in 1% osmium tetroxide/1.25% potassium ferrocyanide at room temperature. Wash again in two changes, 15 min each, 0.1 M sodium cacodylate buffer.
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 instructions by adding 100 ml solution A/1 g catalyst. Stir at room temperature to dissolve and store at 4C for use in steps 4 to 7.
Polymerization of the resin is an exothermic reaction, and the heat generated can cause artifacts in the tissue. A smaller proportion of catalyst can be used (0.7 g/100 ml solution A) to slow polymerization time and reduce heat artifacts. This is particularly important when larger blocks (e.g., 1.5 cm2) are produced.

4. Infiltrate tissue sample with a 1:1 mixture of 100% ethanol/infiltration solution for 2 hr at 4C. 5. Infiltrate overnight in full-strength infiltration solution at 4C. 6. Infiltrate 2 hr in a second change of infiltration solution at 4C. 7. Prepare the embedding solution by adding 1 ml solution B to 15 ml infiltration solution. Use immediately and keep solution in an ice bath while embedding. 8. Orient sample in a glycol methacrylate embedding mold and fill mold with embedding solution. Polymerize overnight at 4C.
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 safety measure polymerization may be carried out in a vacuum desiccator or in a nitrogen-filled desiccator.

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The use of glycol methacrylate resin as an embedding medium is technically demanding and potentially subject to numerous problems, including difficulties obtaining satisfactory polymerization (leading to sectioning difficulties) as well as morphological artifacts and difficulties with staining (Hess and Moore, 1993). It is advisable to experiment with the technique 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.

Karnovskys fixative 10 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 H2O Adjust to pH 7.4 with 0.1 N hydrochloric acid Store up to 1 week at 4C. Krebs Ringers solution Dissolve 6.6 g sodium chloride, 0.15 g potassium chloride, and 0.15 g calcium chloride in 1 liter deionized water. Adjust to pH 7.6 with 5% (w/v) sodium bicarbonate. Store up to 1 month at 4C. Immediately before use add 5 g procaine hydrochloride and 1 ml heparin (10,000 IU). Modified Davidsons fixative 140 ml ethanol 62.5 ml glacial acetic acid 375 ml 37% (w/v) formaldehyde 422.5 ml distilled H2O Store up to 3 months at room temperature. Osmium tetroxide/potassium ferrocyanide fixative Prepare a 2% (w/v) aqueous solution of osmium tetroxide and a 2.5% (w/v) aqueous solution of potassium ferrocyanide. Mix equal volumes together immediately before use (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 60C. Add a few drops of 1 M sodium hydroxide to clear the solution. Cool and filter through Whatman no. 5 filter paper. Store up to 1 month at 4C. Sodium cacodylate buffer, 0.1 M Dissolve 10.7 g sodium cacodylate in 500 ml deionized water. Store up to 1 month at room temperature.
This buffer is also commercially available from Electron Microscopy Sciences.

COMMENTARY Background Information

The procedures used for reproductive-tract histology will largely depend on the objectives of the study. For example, a regulatory screening study to detect toxicity will generally weigh and sample the tissues according to the appropriate regulatory guidelines (Table 16.3.2) and use routine immersion fixation followed by paraffin embedding. An investigative study to characterize toxicity may concentrate on target tissues of interest and use perfusion fixation followed by resin embedding. The latter procedure will provide enhanced resolution for light microscopy and, if epoxy resin is used, allow for ultrastructural examination with the electron microscope.

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Critical Parameters and Troubleshooting

Sampling and orientation of tissues Testes. There are a number of factors to consider when deciding how to trim the various tissues of the reproductive tract. In the case of the testes, a longitudinal or transverse section can be taken. The advantage of a longitudinal section is that it provides more tissue to examine and provides frequent longitudinal sections through seminiferous tubules, many of which will include consecutive stages of the spermatogenic cycle. The advantage of a transverse section is that it provides consistent cross-sections through all the seminiferous tubules. This makes staging of the tubule easier and allows quantitation of cell numbers and tubular measurements, if required. Most technicians sample the testis by taking a midline sample. However, the testis has asymmetric structures that may be missed by a midline section. For example, in the rodent, the rete testis (the network of canals into which the seminiferous tubules empty) is located at the cranial pole of the testis and spreads down directly beneath the capsule towards the midline. The rete can provide important information regarding fluid dynamics of the seminiferous tubule fluid. In cases of obstruction of the excurrent duct system or disturbances in fluid reabsorption, it will show dilation. It can also be the site of proliferative lesions and rete testis tumors. Both ends of the seminiferous tubules empty into the rete and this often appears to be a preferential site for germ cell degeneration and depletion for both spontaneous and chemically induced lesions. It is therefore an important site to sample, and routine sectioning should include at least part of the rete complex. In the dog the rete forms a central core that extends from the cranial surface to approximately two-thirds of the way towards the caudal tip and will be adequately sectioned with a midline transverse section. In the primate it is located in a peripheral position along the medial aspect for approximately half the medial border. Again it will be adequately sampled with a midline transverse section but a longitudinal sample needs to be specifically orientated to include it. Efferent ducts. The efferent ducts link the rete testis with the ductus epididymis, which is a single, highly coiled duct, the convolutions of which form the caput, corpus, and cauda epididymis. The number and course of the efferent ducts vary with species. They are long and tortuous in rodents, extending up into the

Histopathology of the Male Reproductive System I: Techniques

epididymal fat pad surrounding the caput epididymis; they are normally trimmed off and discarded when the epididymis is trimmed for weighing. In the dog and primate they are very short because the epididymis is more closely applied to the testis in these species; they are rarely sampled in routine studies but can be an important site for lesions. The efferent ducts in the dog are a frequent site for sperm granulomas because of the presence of numerous blind-ending tubules. Similarly in the rat, this may be the location of sperm granulomas that give rise to tubular dilation in the testes and absence or decreased sperm in the epididymis. More importantly, they have been shown to be a target site for a number of chemicals (Hess, 1998). Although it may not be practical to sample them in routine studies, their potential as a target site for toxicity or as a cause of secondary changes in the testes and epididymides should be appreciated. Epididymides. The cellular composition and the function of the epididymis vary with region, and it is important that the different regions are sampled and examined. Routine sampling of the epididymis often only includes the cauda epididymis, but this precludes important information. There are a number of chemicals that have been shown to produce site-specific lesions to the epididymal epithelium, some in the caput, others in the cauda. An additional reason for sampling the entire epididymis is that the density of sperm and the presence of germ cells within the lumen of various segments of the epididymis reflect time-dependent events in the testis. For example, in the rodent, dog and primate sperm and cells present in the cauda epididymis were released from the testis 2 weeks prior to their arrival there. The sperm in the caput epididymis reflect the release of sperm only days previously. Depending on the duration of the study, differential effects on the contents of the caput and cauda can provide important information on the timing of a toxic effect. Prostate. The prostate of rodents is a heterogeneous gland with anatomically distinct ventral, dorsal, and lateral lobes (Fig. 16.3.2B). The histologic structure and the response to toxicants vary between lobes such that it is important to routinely sample as much as possible. A midtransverse section through the prostatic complex allows an adequate sample of all three lobes and often also includes the ampullary gland of the ductus deferens as it enters the prostatic urethra (Lee and Holland, 1987; Suwa et al., 2001). In the dog and the primate, the

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glandular tissue is arranged circumferentially around the urethra, although in the primate it is only present on the posterior surface of the urogenital canal. In these species, a transverse section provides an appropriate sample for examination (Figs. 16.3.3B and 16.3.4B). Seminal vesicles and coagulating gland. In rodents, a coagulating gland lies on the anterior aspect of each seminal vesicle. Both can be sampled by a cross-section. Similarly, a crosssection of the seminal vesicles in the primate provides adequate sampling. These organs are absent in the dog. Choice of fixative All of the recently revised regulatory guidelines for reproductive toxicity studies specifically recommend fixing the testes in Bouins or a comparable fixative (Table 16.3.2). Implicit in this recommendation is that the use of formalin is avoided due to the excessive shrinkage of Sertoli cell and germ cell nuclei and cytoplasm, which occurs when formalin-fixed testes are embedded in paraffin. Although acceptable results can be obtained with formalin (Harleman and Nolte, 1997), Bouins provides more reliable and consistent results. Testes fixed in Bouins show greatly improved preservation of the nuclear and cytoplasmic features of Sertoli cells and germ cells, with very little shrinkage of the cells away from one another. However, due to its picric acid component, Bouins has a number of drawbacks. It is potentially explosive, mutagenic, and possibly carcinogenic, presenting significant safety and disposal problems to laboratory personnel when used on a large scale. It also results in significant shrinkage of the tubules away from the interstitial tissue, especially in the center of the testis. Testes fixed with modified Davidsons fixative (see Basic Protocol 1) show comparable cellular and nuclear resolution with very little shrinkage of the germ cells and Sertoli cells. In addition, the tubules show significantly less shrinkage from the interstitial tissue than is seen with Bouins. Interestingly, the problems with formalin fixation are associated with subsequent processing into paraffin wax, because formalin fixation of rodent testes followed by embedding in glycol methacrylate results in better preservation of structure than with Bouins (Chapin et al., 1984). However, this does not hold true for all species and should be experimented with prior to use. For a detailed review of methods and the results that can be obtained using various combinations of fixatives, 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 preserving the testes are due to the requirement to fix the tissue whole. This is necessary so that the architecture of the tubules and interstitial tissue is maintained and to prevent artifactual sloughing of germ cells. If the capsule is cut or removed, the tubules erupt and flow out of the holes. However, improved penetration of fixative can be achieved by pricking the capsule of rodent testes or making nicks in the capsule of dog or primate testes before immersing in fixative. (Large-animal testes have more fibrovascular stroma and are less likely to erupt than rodent testes.) If the pricks and cuts are made superficially and at a location distant from the area to be sampled, this will not disturb the architecture. Fixation quality can also be significantly improved if the testis is sliced after it has been partially fixed by immersion. Allow 4 to 5 hr (or overnight) before cutting the testis and then cut it in half or make 5-mm slices. Return to fixative to allow a total fixation time of 48 hr. Tissues should not be left in Bouins or Davidsons for very much longer than 48 hr, otherwise they will harden and become difficult to section. The Karnovskys fixative recommended in Basic Protocol 2 and the subsequent postfixation in osmium tetroxide and potassium ferrocyanide are designed to give optimal fixation and staining of testicular structure for ultrastructural examination while minimizing osmotic artifacts. The high water content of testicular tissue and the distance of the seminiferous epithelium from the interstitial vasculature make fixation difficult and artifacts frequent. One of the most common artifacts seen is basal vacuolation of the seminiferous tubules. This is caused by the use of hyperosmotic fixative, which causes shrinkage of the cells in the basal compartment (below the level of the Sertoli cell tight junctions). Hyperosmotic fixatives will also cause artifactual condensation of Sertoli cell cytoplasm at the ultrastructural level. For ultrastructural studies it is therefore important to use approximately isotonic solutions and fixatives. The use of the osmium/ferrocyanide mixture as a postfixative is particularly good for membrane preservation, but it also has the advantage of improving the staining characteristics and resolution of Leydig cell structure at the ultrastructural level (Russell and Burguet, 1977). The choice of buffer for glutaraldehydebased fixatives is a matter of personal prefer-

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ence. Cacodylate buffer has been recommended in this protocol, but due to its toxicity and associated disposal problems, a phosphate buffer may be preferred as a substitute (Hess and Moore, 1993). Perfusion There are various techniques for perfusion fixation of tissues. The choice is largely a matter of personal preference and resource limitations. The procedure described in Basic Protocol 2 uses a peristaltic pump to perfuse the whole animal. This maintains a constant flow of fixative for a given speed of the pump, despite the increasing vascular resistance. A gravity-fed system can also be used, as described by Sprando (1990). This has the advantage of not requiring a peristaltic pump but has the disadvantage of requiring the fixative and saline bottles to be located some distance above the animal. With gravity-based perfusion, the flow rate also decreases as the vascular resistance increases. Rather than perfuse the whole animal as described in this protocol, it is also possible to limit perfusion only to the abdominal tissues. Hess and Moore (1993) describe a method of cannulating the abdominal aorta via the left ventricle, which bypasses perfusion of the cranial and thoracic tissues. This conserves fixative, but the cannulation is slightly more technically demanding. A critical step in the perfusion technique is the successful clearance of blood from the entire vascular bed. If vessels constrict or intravascular thrombi form, then the fixative will not be uniformly distributed. Clearance of the testicular vasculature appears to be particularly problematic. To overcome this problem, heparin and/or vasodilators have been employed. In this protocol the incorporation of both heparin and procaine in the flushing solution (Hess and Moore, 1993) has been recommended on the basis of a very good success rate combined with simplicity. However, Sprando (1990) suggests that the preinjection of animals with 130 to 150 IU heparin/kg body weight by intraperitoneal injection 15 min prior to perfusion improves the success rate without the need for additives to the perfusion fluids.

Anticipated Results
Histopathology of the Male Reproductive System I: Techniques

Using Basic Protocol 1, the quality of the paraffin sections of testes stained with periodic acid Schiffs stain/hematoxylin (PAS-H) will be suitable for detailed qualitative evaluation of spermatogenesis. Resolution and staining of

the spermatid acrosome in rodent testes will be adequate to identify individual stages of the spermatogenic cycle. In dog and primate testes, the acrosome does not stain with PAS and cannot be adequately distinguished in paraffinembedded sections. Therefore staging has to rely on the characteristics of the spermatocyte and round spermatid population in conjunction with the shape and position of the elongating spermatid population. Cytoplasmic and nuclear detail of the germ cells, Sertoli cells, and Leydig cells will be suitable for detecting early degenerative changes. Although quantitative procedures, such as cell counting and measuring tubular diameters is possible, it is better carried out on resin-embedded tissue where section thickness is more consistent and nuclear chromatin resolution is improved. Lon gitudinal sections through the epididymis will provide information on the cellular morphology of the entire length of the coiled epididymal duct and of the relative sperm and cell content of the ductular lumen. In the dog and primate, the longitudinal section of the epididymis may also include some of the efferent ducts. The transverse section through the rodent prostate should provide comparative morphology of the dorsolateral lobes and ventral lobes of the tissue and may also include cross-sections of the vas deferens and of the associated ampullary gland of the vas. The secretory products of the various lobes and of the ampullary gland have different affinities for eosin. The differential staining pattern can be used to identify various regions (Lee and Holland, 1987; Yuan et al., 1987). In the dog and the primate, the prostatic acini should appear more or less homogeneous with no regional variation. Using Basic Protocol 2, perfusion fixation should provide tissue with a minimum of artifacts. All interstitial capillaries should be open and empty of erythrocytes. There should be no shrinkage of tubules away from the interstitial tissue and no shrinkage of germ cells away from Sertoli cells. Vacuoles between adjacent Sertoli cells should be minimal or nonexistent and there should be no sloughing of germ cells into the tubular lumen. When viewed by electron microscopy, the morphology of the smooth endoplasmic reticulum and mitochondria in the basal Sertoli cell cytoplasm are good indicators of fixation quality. Toluidine bluestained resin sections can be used to distinguish the spermatid acrosome in all species including the dog, although the thinness of the section results in many acrosomes being incompletely sectioned

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or out of the plane of section. Glycol methacrylateembedded, immersion-fixed tissue generally results in less tubular and cellular shrinkage than paraffin-embedded tissue and shows improved resolution of nuclear and cytoplasmic features. It too suffers from reduced numbers of complete acrosomes in the plane of section.

method, multiple perfusion stations are required, and animals must be staggered through the various procedures. With three perfusion stations and two people carrying out the perfusions, it is feasible to complete 20 animals in a normal working day. (Sprando, 1990).

Literature Cited Time Considerations

Immersion fixation and embedding tissue in paraffin wax is the most economical methodology in terms of time and cost. Because all the procedures are more or less standard routine for a histology laboratory, the processing, embedding, and staining is largely automated. Overall time depends on the number of tissues sampled and weighed from each animal and the number of animals processed. For a routine study size (e.g., two to five treatment groups, each with five to ten animals), allowing 48 hr fixation time plus 1 day processing and embedding into paraffin and 1 day sectioning and staining, sections could be ready for evaluation within a week. Processing for glycol methacrylate can also be automated using routine, paraffin-embedding processors up to the point of resin infiltration. Processing for epoxy resin can be automated through to the point of embedding, but this requires a purpose-designed machine to handle the small tissue size and the viscuous resin mixtures. The alternative is to carry out the procedure in individual bottles, carefully draining and filling each with new solution. Embedding in resin requires special molds and special polymerization conditions and can take up to 48 hr for adequate polymerization. Sectioning of resin blocks requires the use of a special, automated microtome that uses prepared glass or tungsten carbide knives and is a technically demanding procedure requiring at least 1 to 2 days training and several days practice to become proficient. With glycol methacrylate, consistent polymerization of the resin and control of the relative hydration of the resin during sectioning can also add to the difficulty and the time taken to produce acceptable sections. Overall, the size of the tissue block and the proficiency and experience of the technician will determine the time taken to prepare adequate resin sections, but, as a guide, it probably takes at least twice to three times longer to prepare a 2- to 3-m resin section than it does to prepare a paraffin section. The duration of the perfusion means that this is also a time- and labor-intensive technique. If large numbers of animals are to be fixed by this
Bancroft, 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 glycol methacrylate-embedded testes. Toxicol. Pathol. 12:221-227. Environmental Protection Agency (EPA). 1998 Health Effects Test Guidelines, Report No. OPPTS 870.3800, Reproduction and Fertility Effects. pp. 1-12. EPA 712-C-98-208. Office of Prevention, Pesticides and Toxic Substances (OPPTS), U.S. EPA, Washington, D.C. Evans, H.E. 2000. Guide to the Dissection of the Dog, 5th ed. W.B. Saunders, Philadelphia. Feldman, D.B. and Seeley, J.C. 1988. Necropsy Guide: Rodents and the Rabbit. CRC Press, Boca Raton, Fla. Frederick, P.M. and Doorn, L.G. 1973. A technique for perfusion of rat testis in situ through internal spermatic arteries. J. Reprod. Fertil. 35:117-121. Harleman, J.H. and Nolte, T. 1997. Testicular toxicity: Regulatory guidelinesthe end of formalin fixation? Toxicol. Pathol. 25:414-417. Hayat, M.A. 1989. Principles and techniques of electron microscopy: Biological applications, 3rd ed. CRC Press, Boca Raton, Fla. Hess, R.A. 1998. Effects of environmental toxicants on the efferent ducts, epididymis and fertility. J. Reprod. Fertil. Suppl. 53:247-259. Hess, R.A. and Moore, B.J. 1993. Histological methods for evaluation of the testes. In Methods in Toxicology, Vol. 3, Part A. Male Reproductive Toxicology (R.E. Chapin and J.J. Heindel, eds.) pp. 86-94. Academic Press, San Diego. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). 1994. Tripartite Harmonized ICH Guideline S5A: Reproductive Toxicology: Detection of toxicity to r epr oduction f o r medicinal products/CPMP/ICH/386/95. Fed. Regist. 59:4874648752. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). 1996. Tripartite Harmonized ICH Guideline S5B: Reproductive Toxicology: Male fertility studies/CPMP/ICH/136/95. Fed. Regist. 61:15360. Lee, C. and Holland, J.M. 1987. Anatomy, histology and ultrastructure, prostate, rat. In Monographs on Pathology of Laboratory Animals: Genital System (T.C. Jones, U. Mohr, and R.D. Hint, eds.) pp. 239-251. Springer-Verlag, New York.

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Organization for Economic Cooperation and Development (OECD). 1995. Reproduction/developmental toxicity screening test. In Guideline for Testing of Chemicals, No. 421 (adopted July 27, 1995) pp. 1-10. OECD, Paris. Organization for Economic Cooperation and Development (OECD). 2001. Proposal for updating Guideline 416: Two generation reproduction toxicity study. In Guideline for Testing of Chemicals, No. 416 (adopted January 22, 2001) pp. 1-13. OECD, Paris Russell, L. and Burguet, S. 1977. Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture. Tissue Cell 9:751-766. Russell, L.D., Ettlin, R., Sinha Hikim, A.P., and Clegg, E.D. 1990. Tissue Preparation for Evaluation of the Testis. In Histological and Histopathological 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 testis through the heart using heparin. In Histological and Histopathological Evaluation of the Testis (L.D. Russell, R. Ettlin, A.P. Sinha Hikim, and E.D. Clegg, eds.) pp. 277-280. Cache River Press, 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 background lesions 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 ductus deferens (ampullary gland), rat. In Monographs on Pathology of Laboratory Animals: Genital System. (T.C. Jones, U. Mohr, and R.D. Hint, eds.) pp. 229-234. Springer-Verlag, New York.

Key References
Hess, R.A. and Moore, B.J. 1993. See above. Provides a detailed comparison of the results achieved using different combinations of fixatives and embedding procedures to examine the testes. Also provides technical details for perfusion fixation and subsequent processing and embedding in resin for light and electron microscopy. Russell et al., 1990. See above. Provides various protocols for fixation, tissue processing, and staining of the testis and discusses the advantages and disadvantages of various methods. Sprando, R.L. 1990. See above. Provides detailed technical guidance on the art of perfusion fixation for the testes.

Contributed by Dianne M. Creasy Huntingdon Life Sciences East Millstone, New Jersey

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