Morphology of the Fetal Rat Testis Preserved in Different Fixatives

Abstract

Histopathological examination of the testes of exposed fetuses and neonates is important in assessing the developmental effects of environmental toxins, including sex hormone modulators. Modified Davidson’s fluid (mDF) has been suggested as a superior substitute for Bouin’s fluid for fixation of adult animal testes. We compared the morphology of fetal rat testes stained with hematoxylin and eosin (H&E) or immunochemically after fixation in 10% neutral buffered formalin (NBF), Bouin’s fluid, or mDF. Fixation in mDF resulted in more sharply defined nuclear detail and better preservation of cellular cytoplasm on H&E-stained sections of rat testes on gestation day 19. Use of Bouin’s fluid did not allow satisfactory detection of apoptotic cells by fluorescent terminal deoxynucleotide transferase-mediated deoxy-UTP nick labeling. Staining with the immunoperoxidase system and the conventional chromogen diaminobenzidine tetrahydrochloride to visualize 5-bromo-2-deoxyuridine-positive cells demonstrated that the number of positive nuclei and intensity of staining were similar with all 3 fixatives. Immunostaining for cytoskeletal protein vimentin was more intense and provided better details of the Sertoli cell cytoplasm with formalin fixation than with mDF. Our study demonstrates that fixation in mDF provided better morphologic detail in the fetal rat testis compared with 10% NBF and Bouin’s fluid and illustrates the importance of establishing the correct fixation conditions for each immunostaining protocol.

Introduction

Histopathological examination of the testes of exposed fetuses and neonates is important in assessing the developmental effects of environmental toxicants, including sex hormone modulators (Mylchreest et al., 1999; Gray et al., 2001; OECD, 2001; Barlow and Foster, 2003; Sharpe et al., 2003). The rat is the preferred species for developmental studies involving environmental toxicants, and data from rat bioassays are commonly used in risk assessment. Modified Davidson’s fluid (mDF) has been proposed as superior to formalin, Bouin’s, Zenker’s, and Helly’s fixatives for adult animal testes because it provides excellent histology, is safer than Bouin’s, and is easy to make and use (OECD, 2001; Latendresse et al., 2002). Adult rat testes fixed in mDF have less tissue shrinkage artifact and better morphology than testes fixed in Bouin’s fluid or 10% neutral buffered formalin (NBF). On hematoxylin and eosin (H&E) stained sections nuclear detail is also better preserved in mdF-fixed adult testes (Latendresse et al., 2002). Staining of the rat acrosome with periodic acid Schiff reagent was more apparent after fixation in mDF compared with conventional Davidson’s fluid (Latendresse et al., 2002). However, certain fixative components such as acetic acid or methanol can disrupt cytoskeleton proteins during fixation and therefore compromise immunodetection of these proteins. Fixation in 10% NBF resulted in stronger vimentin staining in the human tonsil than Bouin’s fixative (Williams et al., 1997). Fixation of the rat eye in Bouin’s fluid gave poor results when immunofluorescence was used, although this fixative was satisfactory for the human eye (Mirshahi et al., 1999). We compared morphology of the gestation day (gd) 19 fetal rat testis fixed in 10% NBF, mDF, or Bouin’s fluid using H&E stained sections. Since apoptosis and cell proliferation play a key role in the development of the testis and are likely targets for developmental toxicants (Creasy, 2001; Sharpe et al., 2003; Boekelheide et al., 2004), we assessed sensitivity and specificity of detecting apoptotic cells using terminal deoxynucleotide transferase-mediated deoxy-UTP nick labeling (TUNEL) technique and immunostaining for the cell proliferation marker 5-bromo-2-deoxyuridine (BrdU) in this tissue fixed in mDF, Bouin’s fluid, or 10% NBF. We also evaluated immunostaining for cytoskeletal protein vimentin, a commonly used marker for Sertoli cell cytoplasm (Pelliniemi et al., 1993).

Methods

Animals

Timed-mated Sprague–Dawley rats were obtained from Charles River Laboratories, Inc. (Raleigh, NC) on gd 0, defined as the day that sperm were identified in the vagina. The animals were housed according to federal guidelines for the care and use of laboratory animals. The dams were maintained on a standard 12-hour light-dark cycle, and water and food (NIH-07 rodent chow, Zeigler Brothers, Gardener, PA) were provided ad libitum. On gd 19, dams were euthanized by CO₂, and fetuses were removed by Caesarian section and decapitated. The fetuses were kept in ice-cold, phosphate-buffered saline until dissection. Under a dissecting microscope, the fetuses were sexed, the head, the chest, legs and the tail of the male fetuses were trimmed, the abdominal cavity opened, and the liver and intestines removed. The left testis with epididymis was removed for other assays, while the right was left in situ.

Tissue Processing and Staining

The trimmed fetal carcasses were immersed in a fixative for 24 hours. The fixatives used were 10% NBF (Fisher, Pittsburg, PA), Bouin’s fluid (Sigma, St. Louis, MO), or mDF. mDF was prepared as described previously (Latendresse et al., 2002) and used within 1 month. The recipe for mDF is 30% of a 37–40% formaldehyde, 15% ethanol, 5% glacial acid, and 50% distilled water. After fixation, the carcasses were transferred to 70% ethanol and kept there until processed (except for those fixed in Bouin’s fluid, which were transferred to 50% ethanol for 48 hours and then to 70% ethanol). Testes with epididymides were dissected from the abdomen, processed through graded alcohols, cleared in xylene, and embedded in paraffin. Sections of 5 μm were cut and stained (1) with H&E for overall morphological evaluation; (2) with the terminal deoxynucleotide transferase-mediated deoxy-UTP nick labeling (TUNEL) technique for detection of apoptotic cells or (3) immunologically for BrdU or vimentin. Testes from at least 2 pups were fixed in each of the 3 fixatives, and at least 5 sections from each testis were stained by each of the methods.

Harris hematoxylin was purchased from Fisher, and alcoholic eosin was made from powder (Fisher). H&E staining using our routine method for rat testes fixed in Bouin’s or 10% NBF (Mylchreest et al., 1999; Shultz et al., 2001; Barlow and Foster, 2003) was pale in mDF-fixed testes. Therefore, the procedure for staining of mDF-fixed testes was modified by increasing the time for staining with hematoxylin from 5 to 15 minutes and with eosin from 16 to 40 seconds, and by decreasing the time for acid alcohol decolorization after hematoxylin staining from 5 to 4 seconds.

BrdU was given to the dams by intraperitoneal injection (50 mg/kg) 1 hour before euthanasia to label cells in S phase. Immunohistochemistry for BrdU and vimentin was performed using the Dako EnVision+ horse radish peroxidase with diaminobenzidine tetrahydrochloride (DAB) chromogen system (Dako, Carpinteria, CA). Antigen retrieval was performed using Dako target retrieval solution in a decloaking chamber (Biocare Medical, Walnut Creek, CA) for 20 minutes at 95°C. Slides were allowed to cool to 85°C in the chamber. After a rinse in distilled water, slides were placed in Tris-buffered saline containing 0.05% Tween 20 (TBST) and loaded on the Dako Autostainer for the remainder of the procedure. Endogenouse peroxidases were blocked for 5 minutes using 3% hydrogen peroxide (Sigma). Dako protein block buffer was applied for 5 minutes to block positive-charged sites on the sections. Antivimentin antibody (Dako) was diluted at 1:200, and anti-Brdu antibody (Caltag Labs, Burlingame, CA) was diluted at 1:12,000 in Dako antibody diluent. Primary antibodies were applied for 30 minutes. After rinsing with TBST, labeled polymer (Dako) was applied for 15 minutes. Following rinses in TBST, DAB was applied for 5 minutes. Slides were rinsed in distilled water and stained with hematoxylin (Dako) for 1 minute. Slides were then rinsed with TBST, removed from the Autostainer, dehydrated through ascending grades of alcohol, and cleared in xylene. Slides were cover-slipped using Permount mounting medium (Fisher).

TUNEL was performed using an Apop Tag Red kit (Intergen, Purchase, NY) according to the manufacturer’s protocol for indirect detection using a fluorescent-labeled antibody. Vectashield mounting media with DAPI (Vector Laboratories, Burlingame, CA) was used for counterstaining and slide mounting. Rhodamine and DAPI staining was analyzed on a BX61 Olympus epifluorescence microscope.

Images of stained sections were taken using a MagnaFire digital camera connected to a BX61 Olympus microscope and prepared for publication using Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA).

Results

H&E Stained Sections

In 10% NBF-fixed testes (Figure 1), chromatin was not sharply defined in the nuclei of cells above the basal layer of the seminiferous epithelium (suprabasal cells), although it was better defined in cells in the basal layer and interstitial cells. In Bouin’s-fixed testes (Figure 2), the clarity of nuclear detail, especially in suprabasal cells, was far better than with 10% NBF. In mDF-fixed testes (Figure 3), nuclear detail in suprabasal cells was even clearer. In 10% NBF-fixed fetal testes, there was some shrinkage artifact between semeniferous epithelium cells, which is a more serious problem in adult testes after formalin immersion fixation and paraffin embedding (Chapin et al., 1984; OECD, 2001; Latendresse et al., 2002). Mild shrinkage between seminiferous tubules was present only in 10% NBF-fixed fetal testes. Preservation of cells lying below the tunica albuginea was much better in mDF-fixed testes compared with Bouin’s fluid and 10% NBF. These cells and the tunica albuginea had significantly condensed cytoplasm in 10% NBF-fixed testes.

BrdU Immunostaining

Clarity of nuclear counterstaining was better with Bouin’s or mDF fixation than with 10% NBF (Figures 4, 5, and 6). The number of BrdU-positive cells in the seminiferous epithelium and the intensity of staining were similar, regardless of which fixative was used. Immunostained fetal testes fixed in Bouin’s had brown background, probably due to insufficient alcohol rinses to remove picric acid.

Vimentin Immunostaining

The intensity of staining was stronger in 10% NBF and Bouin’s-fixed testes compared to that fixed in mDF (Figures 7, 8, and 9). For all fixatives, staining was present in the cytoplasm of fetal interstitial and Sertoli cells. However, characteristic Sertoli cell cytoplasmic processes that extend between fetal germ cells were readily stained only in the testes fixed in 10% NBF (Figure 7, insert). Stained Sertoli cell processes were observed infrequently in Bouin’s- and mDF-fixed testes.

TUNEL Staining

Nuclear detail was clearer with mDF fixation compared with 10% NBF and Bouin’s (Figures 10, 11, and 12). The number and clarity of TUNEL-stained nuclei were similar in mDF- and 10% NBF-fixed testes. Bouin’s fixation, however, gave far worse visualization of TUNEL-positive nuclei. Fetal testes fixed in Bouin’s fluid had high nonspecific staining of orange color when the manufacturer’s TUNEL protocol was used.

Discussion

Fixation of the testis and other organs that cannot be trimmed to provide optimal penetration of a fixative and complete stabilization of histological features is a challenging task. Several studies demonstrated that Bouin’s fluid and mDF preserve morphology of the adult animal testis better than formalin (OECD, 2001; Latendresse et al., 2002). The size of the rat fetal testis eliminates constrains for fixative penetration, and 10% NBF has been successfully used for fixation of this tissue (Mylchreest et al., 1999; Barlow and Foster, 2003; Sharpe et al., 2003). In our study, fetal rat testes preserved in 10% NBF had minor cytoplasmic shrinkage in seminiferous tubules; however, interstitial cells, especially cells lying below the tunica albuginea, had significantly condensed cytoplasm. Formalin fixation is well known to cause condensation of cytoplasm due to its hyperosmocity and probable effects of formalin on the proteins in the plasma membrane. In contrast to 10% NBF, only minimal condensation of cellular cytoplasm was present in interstitial cells in testes fixed in Bouin’s fluid and mDF. Similar to the adult rat testis fixed in mDF, fetal rat testes had excellent nuclear resolution and distinct contours of the individual cells in both tubular and interstitial compartments. The intensity of H&E staining of mDF-fixed fetal testes was not as strong as that of 10% NBF-fixed testes but provided a good contrast between H&E colors. Overall, fixation in mDF resulted in more sharply defined cellular detail on H&E-stained sections of the rat fetal testis compared with 10% NBF or Bouin’s fluid.

Although cytoplasmic shrinkage in fetal testes fixed in 10% NBF was not as severe as that in adult testes, shrinkage artifact may compromise detection of tubular pathology in developmental toxicology studies. For example, swelling of the Sertoli cell cytoplasm has been noted in rat testes treated with testicular toxicants (Creasy, 2001). In our studies of developmental testicular toxicity of di-(n-butyl)phthalate, fetal Sertoli cell swelling was much more apparent in mDF-fixed testes (E. Kleymenova, manuscript in preparation) compared with testes fixed in 10% NBF. Stereology often complements hislopathological evaluation when cellular responses to testicular toxicants are studied (Bendsen et al., 2003; Tarka-Leeds et al., 2003; Akingbemi et al., 2004), but manual counting of cells is a burdensome task. Excellent preservation of cellular cytoplasm by mDF allows better discrimination and counting of individual nuclei that makes it possible to conduct computerized high-throughput cell counts.

Anti-BrdU staining is commonly used in rodent bioassays for quantification of cell proliferation in normal and chemically exposed tissues. In gd 19 rat testis, we observed BrdU immunoreactivity in Sertoli, peritubular, and interstitial cells (Figures 4–6). In the fetal testes fixed in mDF, all proliferating cell populations had distinct BrdU-positive nuclei that can be easily recognized and counted by computer software. Computerized high-throughput counts will facilitate determination of labeling indexes specific to cell type and quantification of effects of developmental toxicants on proliferation of specific cell populations in the fetal testis. In addition to providing excellent morphology of the fetal rat testis, use of mDF eliminates use of picric acid, a component of Bouin’s fluid. Problems associated with picric acid in Bouin’s fluid include biohazard properties of this chemical, and labor- and time-consuming protocols (Latendresse et al., 2002). Increased brown background on BrdU-stained sections of the fetal testis fixed in Bouin’s in our study underscores the necessity of thorough removal of picrates from the fixed tissue prior to staining.

Although very few cells undergo apoptosis in gd 19 rat testis, we were able to detect apoptotic gonocytes on some sections using TUNEL assay (Figures 10–12). The present results confirm previous observations (Mirshahi et al., 1999) that Bouin’s fixation gives poor results when immunofluorescence is used to stain rat tissues. In the TUNEL method, one possible solution to decrease nonspecific fluorescent staining is to decrease the time of treatment or the concentration of proteinase K. Decreasing incubation time and concentration of terminal deoxynucleotidyl transferase significantly lowered nonspecific staining in OCT-embedded fetal rat testes (E. Kleymenova, unpublished observations).

An article by Latendresse and colleagues (2002) that described use of mDF for fixation of adult animal testes demonstrated the utility of this fixative for immunohistochemistry based on the results of immunodetection of proliferating cell nucler antigen, androgen receptor, and spermatogonia marker protein gene product 9.5. In our study, immunostaining for cytoskeleton protein vimentin was more intense and provided better visualization of cytoskeleton in 10% NBF-fixed fetal testes compared with mDF. This observation is consistent with the established fact that acetic acid in a fixative alters cytoplasmic distribution of cytoskeletal proteins. Cytoplasmic distribution of vimentin in Sertoli cells can also be altered by toxic insult (Richburg and Boekelheide, 1996). Thus, in the toxicological studies that use vimentin distribution as an end point, the impact of a fixative on immunostaining outcome must be first evaluated.

Our TUNEL and vimentin staining results illustrate the importance of establishing the correct fixation conditions for each immunostaining protocol. Previously, Chapin and colleagues demonstrated that immonolocalization of cell-cell adhesion and signaling proteins associated with spermiation depends significantly on whether rats testes were fixed in formalin, in Bouin’s fluid, or by freezing (Wine and Chapin, 1999; Chapin et al., 2001). Together, these data strongly support the point that there is no ideal fixative that perfectly preserves testicular morphology and retains immunogenicity of all molecules. Since each antibody has its own fixative requirements and tolerance, the study goals will drive the decisions about which methods of fixation and immunostaing to employ.