Toxicologic Pathology

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Immersion Fixation Methods for Glycol Methacrylate-embedded Testes Robert E. Chapin, Monica D. Ross and James C. Lamb Toxicol Pathol 1984 12: 221 DOI: 10.1177/019262338401200303 The online version of this article can be found at: http://tpx.sagepub.com/content/12/3/221

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Toxrco~ocrcPATHOLOGYlSSN:0132-6233 Copyright 0 1984 by the Society of Toxicologic Pathologists

Vol. 12, No. 3, 1984 Printed in U.S.A.

Immersion Fixation Methods for Glycol methacry late-embedded Testes* Robert E. Chapin, Monica D. Ross, and James C. Lamb National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709 ABSTRACT Routine processing of testicular tissue through 10% neutral buffered formalin (NUF) into paraffin produces severe cellular shrinkage which obscures most of the morphologic detail. The following studies were performed to compare different combinations o f immersion fixatives and embedding media for optimal cellular detail in the final histologic sections. We examined sections of testes from rats, mice, and rabbits fixed in either NBF, Bouin’s, Zenker’s, or Helly’s fixatives, and embedded in either standard paraffin or glycol methacrylate. The results were similar in all 3 species. In paraffin, Bouin’s or Helly’s fixatives produced the fewest artifacts, while in glycol methacrylate, NBF-fixed tissue showed the greatest intracellular detail and preservation. The merits and limitations of each method are discussed for the rat, with exceptions for the other species noted where appropriate. The use of glycol methacrylate as the support medium for sectioning makes high-quality tissue sections available from formalin-fixed testes.

INTRODUCTION The safety of new drugs and industrial chemicals is routinely assessed in studies using laboratory animals. Virtually all of these studies incIude the histopathologic study of tissues. Commonly, the fixative.used in these studies is 10% neutral buffered formalin (NBF). The tissue blocks are then embedded in paraffin and generally cut at 5-10 pm. This provides reasonable fixation and resolution for most tissues, but ceIluIar shrinkage is a problem in the testis, and much of the cellular detail is obscured. This study was performed to evaluate the resolution and microscopic detail provided by fixation of testes from rats, mice, and rabbits in either NBF, Bouin’s, Helly’s, or Zenker’s after embedment in either paraffin or glycol methacrylate (GMA). METHODS

Bouin’s, NBF, Helly’s, and Zenker’s fixatives were formulated to standard composiAddress all correspondence l o Dr. R. E. Chapin at the above address.

tion (8).The animals used were adu t Fisc ier 344 rats (300-350g, n = 20),CD-1 mice (3842 g, n = lo), and New Zealand White rabbits (2-3 kg, n = 10)obtained from Charles River Laboratories (Kingston, NY, rats and rabbits) or Jackson Laboratories (Bar Harbor, ME, mice). Housing conditions were as follows: lights on from 8 a.m. to 8 p.m., temperature 72 zk 2”F, and 50 f 10% relative humidity. Rats and mice were allowed ad libitum access to NIH 31 diet, and rabbits received Feed A ad libitum; both diets were purchased from Ziegler Brothers (Gardners, PA). All animals had unrestricted access to filtered tap water. All animals were euthanized by asphyxiation with carbon dioxide. Testes were removed, and the tunica was punctured in 4 places with a 16-gauge needle befor‘e the testis was placed in the fixative. The testes from each animal were placed in different fixatives. Testes were fixed for 24 hr in NBF without agitation, and a transverse section through each testis was removed, dehydrated through graded alcohols and xylcne, and embedded in paraffin (Paraplast, obtained through Fisher Scientific Products, Pittsburgh, PA).


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lular shrinkage, would show no chromatin aggregation in round spermatids or Sertoli cells, would be free of cytoplasmic granularity or shrinkage, and would allow easy identification of the proacrosomal granule or acrosomal cap. This situation is generally pbserved in animals which have been fixed by vascular perfusion (1, 5). The effects of the different fixatives and embedding media were comparable for all three species. A summary of findings for rat is presented in Table I. For the following discussion, “grain” will refer to a granulated appearance of the area in question, either cytoplasmic or nuclear. This granularity may be the result of protein aggregation during the fixation process. Formalin-fixed, paraffin-embedded tissue will be described to demonstrate the criteria by which the slides were scored. In formalin-fixed, paraffinembedded testis (Fig. I), there were no spaces between the Leydig cells and the seminiferous tubules, showing that there was minimal tubular shrinkage. The interstitial Leydig cells appeared to have mild nuclear chromatin aggregation, and in any given group of Leydig cells, there were judged a moderate number of vacuoles. Whether these were inter- or intracellular was undetermined. For the germinal epithelium, the most severe artifacts were wide spac,es, which appeared to be extracellular due to the lack of cellular membranes between adjacent nuclei. The cytoplasmic area was also markedly reduced. Thus, the presence and severity of “grain” in the cytoplasm could not be determined. This shrinkage artifact also obscured the acrosome

An adjacent cross-section was processed through graded alcohols to 95% ethanol and embedded in GMA (Sorvall Division, DuPont, Wilmington, DE). Tissues in Bouin’s solution were fixed for 24 hr, sections were rinsed in 50% ethanol until clear, and embedded in paraffin after complete dehydration or GMA after processing through 95% ethanol. Tissues in Helly’s and Zenker’s fluids were fixed for 24 hr, and sections were washed in running water for 24 hr before complete dehydration and embedment in paraffin or processed through 95% ethanol before embedment in GMA. All blocks were cut at 5 pm (paraffin)or 3 pm (GMA). Slides from Helly’sand Zenker’s-fixed tissue were rinsed in 0.5% iodine solution for 10 min and cleared in 5% sodium thiosulfate solution for 5 min before staining. Sections were stained with periodic acid-Schiffs to highlight the acrosomal system of developing spermatids; nuclei were stained with hematoxylin (8). The GMAembedded tissue was stained according to instructions from the Sorvall Manual. The stage of spermatogenesis in the seminiferous tubules was classified according to published criteria (6). Sections were compared to those fixed for other studies by vascular perfusion (I, 5). Sections from immersion-fixed tissues were scored by an observer unaware of which method was used for preparation. Artifacts were scored as minimal or not present (-), mild (+), moderate (++), or severe (+++). RESULTS

An ideal section of testis for morphologic examination should be free of tubular or celTABLE I-Summary

TOXICOLOGIC PATHOLOGY

of Effects Seen with Different Fixatives in Paraffin on GMA

Preparative Met hod”

Effect

Paraffin NBF

Tubular Contraction Leydig Cells Chromatin Aggregation Cytoplasmic Shrinkage Germ Cells Chromatin Aggregation Acrosornal Obfuscation Cytoplasmic Grain Cytoplasmic Shrinkage Sertoli Cells Chromatin Aggregation Cytoplasmic Shrinkage

-

+ ++ ++ ++ ND +++ ++ +++

B

+ + + ++ ++ ++ + ++ +

GMA H

++ +

+ + + + + + +

Z

NBF

++ +

-

-

+

+++ ++ +++ + ++ +

-

+ ++ + +

B

+ +

-

++ + ++

-

++ -

H

++ + + + + + + -

2

++

+ + +++ ++ ++ + ++

-

NBF, 10%neutral buffered formalin; 8, Bouin’s fluid; H, Helly’s fluid; Z, Zenker’s fluid; GMA, glycol methacrylate. Grading criteria as follows: key: ND, not determined; -, absent-to-minimal; mild; ++, moderate; +++, severe. r,

+,


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FIG. 1-A, seminiferous tubules from rat, fixed in NBF, embedded in paraffin; tubules a r e in stages VII (top) and VI. Note marked shrinkage artifact inside tubules and difficulty in distinguishing acrosomes (arrowheads). x 450. B, seminiferous tubules from rat, fixed in NBF, embedded in CMA; tubules a r e in stages V I (top) and 111. Note reduced cellular shrinkage relative t o paraffin-embedded, NBF-fixed testis, and improved acrosomal resolution (arrowheads). x 450.

and proacrosomal granule, and it was difficult to determine the stage of spermatogenesis by standard criteria (6). Chromatin appeared clustered and dense. Because Sertoli cell cytoplasm interdigitates among the germ cells, the large intercellular spaces were deemed a result of Sertoli cell cytoplasmic contraction as well as a germ cell shrinkage.

The other fixatives were scored as indicated in Table I. Helly’s fixative (Fig. 2A) caused moderate seminiferous tubular shrinkage, and mild artifacts in the other categories observed. Of the four different fixatives used before paraffin embedment, Helly’s yielded the fewest artifacts in the acrosome and nucleus, both important in describ-


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FIG. 2-A, seminiferous tubules from rat, fixed in Helly’s, paraffin-embedded; tubules are in stages VII (left) and II. The section demonstrates good acrosomal resolution (arrowheads) and slight nuclear and cytoplasmic granularity. x 450. B, seminiferous tubule from rat, fixed in Helly’s, CMA-embedded; tubules are in stage Ill. These sections are characterized by lack of “grain,” little cellular shrinkage, and excellent arosomal resolution (arrowheads). x 450.

ing post-treatment toxicity (below). Bouin’s (Fig. 3A) consistently produced slightly more artifactual changes in the acrosome and nucleus than did Helly’s, while inducing slightly less tubular shrinkage overall. Zenker’s (Fig. 4A), although producing less tubular shrinkage than Bouin’s or Helly’s fixatives, produced significant “grain” and chromatin aggregation, both of which interfered with nuclear and acrosomal resolution. Generally, the same effects noted in rat tissue were present in mouse and rabbit tissue. The murine germ cells appeared slightly less prone to shrinkage and “grain” compared to rat tissue. Rabbit tissue evidenced more chromatin aggregation with all fixatives than was shown by the other species. In GMA, formalin-fixed tissue showed less cellular shrinkage than that seen after embedment into paraffin. Staining was less intense in these tissues than in those fixed in other fixatives. However, formalin-fixed testes also showed little cytoplasmic “grain,”

and mild to moderate nuclear “grain.” In addition, acrosomal resolution was excellent. When embedded in GMA, Bouin’s-fixed tissue from all three species showed less cellular shrinkage than when embedded in paraffin, although Bouin’s still produced more nuclear and cytoplasmic “grain” than Helly’s. Fixation in Zenker’s fluid consistently resulted in the most severe chromatin aggregation and cytoplasmic “grain,” which impeded acrosomal resolution. Cellular detail was poorest in formalin-paraffin tissue, whereas it was best in formalin-GMA processed tissue.

DiscussioN Fixation of testis in 10% neutral buffered formalin, followed by embedment in paraffin, is common practice in many routine histopathologic studies. This method results in chromatin aggregation and severe shrinkage of the germinal and Sertoli cells. This makes it difficult, if not impossible, to identify the


Vol. 12, No. 3,1984


225

FIG. 3-A, seminiferous tubules from rat, fixed in Bouin’s, paraffin-embedded; tubules are in stage I I (bottom) and VII. Note moderate cytoplasmic and nuclear granularity and acrosomal resolution (arrowheads). x 450. B, seminiferous tubules from rat, fixed in Bouin’s, CMA-embedded; tubules are in stage VI (left) and VII. Moderate acrosomal resolution and nuclear chromatin clumping are present with minimal shrinkage artifact. x 450.

proacrosomal granule in many round spermatids. While fixation in Zenker’s reduced cell shrinkage, aggregation of chromatin became the limiting factor in morphologic interpretation of testes fixed in Zenker’s. Most of these artifacts were alleviated somewhat by fixation in Bouin’s. The extent of nuclear and cytoplasmic “grain” was less severe in Helly’s-fixed tissue, and the acrosomal system was more easily discernable after Helly’s fixation than after immersion in the other fixatives. Embedding formalin-fixed testes in GMA resulted in a dramatic reduction of the cellular shrinkage artifact seen in paraffinembedded tissue. This resulted in sections which showed slight cellular shrinkage, but little or no “grain” or aggregation of chrornatin. One might speculate that these differences between sections fixed in the same solution but embedded in different media could result from the difference in dehydration required for embedment. While com-

plete dehydration is required prior to embedding in paraffin, GMA contains about 5% water, and complete dehydration is unnecessary. Thus, the relative lack of artifact in the GMA-embedded sections might be related to less dehydration-induced strain imposed on the tissue. Also of note is that, of the eight different processing methods tested above, the spermatid acrosome was most easily seen in tissue that was fixed in NBF followed by embedment in GMA. Proper choice of fixative and histologic technique is essential for accurate interpretation of chemically-induced morphologic changes in the testis. The morphologic pattern of lesion development in the testis can be related to the mechanism of action of the compound in question. For example, compounds that interfere with microtubular function produce different lesions than compounds that disrupt the hormonal milieu or blood flow to the testis (9-11,respectively]. Preservation of nuclear detail free of artifact


226



FIG. 4-A, seminiferous tubules from rat, fixed in Zenker's, paraffin-embedded; tubules are in stages VI (top) and VII. Note severe nuclear chromatin aggregation and moderate acrosomal resolution (arrowheads). x 450. B, seminiferous tubules from rat, fixed in Zenker's, GMA-embedded; tubules are in stage VII (top) and VI. Cytoplasmic granularity is decreased compared to paraffin, but chromatin aggregation is severe. x 450.

is important because changes in nuclear staining are frequently the first indication of cell death (3, 13). Early lesion detection is enhanced by using methods which interfere least in observing possible nuclear changes. Preservation of the acrosomal structure allows the investigator to determine if the lesion preferentially occurs in one or more

tubular stages of spermatogenesis, as has been shown for several toxicants 11, 2 , 4 , ? 1 13). As more compounds are examined and found to exhibit stage specificity in the testis, new insights may be gained on both the mechanism of action of those compounds, and also on the way the testis responds to toxicants.


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Although not fully detailed above, the relative ranking of the fixatives was the same for all three species examined. This suggests that these techniques may be applied to a broader range of species. Each fixative has it’s own limitations with regards to histologic artifact, and some histochemical stains may be precluded by using a particular fixative (see Reference 7 for review). Additionally, while tissue handling for paraffin has been automated to a large extent, this is not yet true for GMA. Thus, the choice of fixative and embedding medium is often a compromise between several conflicting situations, and can only be made by each investigator dependent on need. It is noteworthy that formalin-fixed testes can yield remarkably good sections when embedded in GMA. If paraffin embedding is preferred, Helly’s- or Bouin’s-fixed tissues are superior to sections from formalin-fixed tissues. ACKNOWLEDGMENTS The authors are grateful for the excellent technical assistance of Phyllis Merritt, Nell Peedin, Page MacLain, and Tina Jones; for the continued support and encouragement of Dr. Bern Schwetz; and to Drs. Robert Maronpot, Hank Soleveld, Charles Montgomery, and Scott Eustis for their comments on the manuscript. REFERENCES 1. Chapin RE, Morgan KT, and Bus JS (1983). The

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