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DOI: 10.1369/jhc.4A6249.2004
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Journal of Histochemistry and Cytochemistry
Volume 52 (6): 813-820, 2004
Copyright ©The Histochemical Society, Inc.

Quantification of In Situ Hybridization Signals in Rat Testes

Touji Kimura, Jun Kosaka, Takako Nomura, Teruo Yamada, Yukari Miki, Koji Takagi, Takashi Kogami and Junzo Sasaki

Department of Cytology and Histology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Correspondence to: Junzo Sasaki, MD, PhD, Dept. of Cytology and Histology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikatacho, Okayama 700-8558, Japan. E-mail:sasakij{at}md.okayama-u.ac.jp


   Summary
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We performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouin's fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through "posterization" of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections.

(J Histochem Cytochem 52:813–820, 2004)

Key Words: quantification • posterization • in situ hybridization • rat • testis • ribosomal RNA


   Introduction
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 Introduction
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 Literature Cited
 
DNA ARRAY ANALYSIS can reveal the underlying genes and proteins involved in pathological and physiological changes. However, it cannot detect the cells in which such molecules are up- and/or downregulated, information needed to understand the role of the constituent cells of tissues that react to such changes. To analyze the data from cDNA array analyses in full, it is essential to localize gene products in tissue sections by quantitative in situ hybridization (ISH).

We have studied the gene expression of reactive oxygen species (ROS)-related enzymes in the reproductive organs using ISH histochemistry (Sasaki et al. 1994Go; Nomura et al. 1996Go; Mori et al. 2001Go). In female rats, the expression of manganese superoxide dismutase was up- and downregulated cyclically in the theca interna, granulosa, and lutein cells in ovulated follicles and in the epithelial cells of the uterus and vagina during the estrous cycle and ovulatory process. In male rats, phospholipid hydroperoxide glutathione peroxidase was expressed stage-specifically during spermatogenesis in adulthood. From these results, we concluded that hydroperoxide has important roles in the functioning of male and female reproductive organs (Nomura et al. 1996Go; Mori et al. 2001Go). We hypothesized that the activities of ROS in the reproductive organs were regulated during the estrous cycle and spermatogenesis at a transcriptional level. We therefore tried to estimate the amount of each transcript in certain cell types. However, such estimates tend to be subjective.

Radioactive ISH quantitations have been used extensively in the field of neuroscience (Baskin and Stahl 1993Go). The use of 35S-labeled probes seemed suited to ISH of the CNS because neurons are separated from one another by glial tissues. However, there could be severe background problems in tissues such as the epithelium. This weak point in the histochemical method can affect the results of cDNA array analyses.

In this study we tried to quantify ISH signals using a posterization of images of the rat testis. This strategy revealed differences in the amounts of rRNA during spermatogenesis.


   Materials and Methods
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 Materials and Methods
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 Literature Cited
 
All animal experiments were performed according to the Guidelines for Animal Experiments of the Okayama University Graduate School of Medicine and Dentistry. All reagents were of the highest grade commercially available.

Tissue Preparation
Fifteen minutes before the initiation of perfusion fixation, adult male Wistar rats (CLEA Japan; Osaka, Japan) were injected IP with 130 IU/kg of heparin (Sprando 1990Go) and then anesthetized with pentobarbital (50 mg/kg). They were perfused via the left ventricle with 4% paraformaldehyde (PA) or Bouin's fixative (375 ml saturated aqueous picric acid, 125 ml 37% w/w stock formaldehyde, and 25 ml glacial acetic acid). The testes were carefully isolated and immersed in the same fixative for more than 6 hr. The specimens were dehydrated with ethanol, substituted with benzene, and embedded in paraffin. Paraffin sections (5 µm thick) were mounted on silane-coated slides.

Probe Preparation
A well-preserved segment (accession number #V01270; 6035–6068, 5'-gccgccgcaggtgcagatcttggtggtagtagca-3', total 34-mer) of rat 28S rRNA was used for the detection of rRNA previously reported by Yoshii et al. (1995)Go. An oligonucleotide probe (34-mer) labeled with digoxygenin at the 5' end complementary to the segment of 28S rRNA was purchased from Takara Bio Inc., Japan. It is referred to here as the DIG probe.

For the control experiment, unlabeled probes or DIG-labeled 34-mer oligonucleotides of random sequences were prepared.

An alternative DIG-labeled probe was prepared using a multilabeling method (Sasaki et al. 1998Go; Mori et al. 2001Go) as follows. Annealed double stranded 5'-phosphorylated oligonucleotides containing sense (5'p-aattc-gccgccgcaggtgcagatcttggtggtagtagca-a-3') and antisense (5'p-agctt-tgctactaccaccaagatctgcacctgcggcggc-g-3') sequences (34-mer) with EcoR I or Hind III restriction sites as protruding cohesive ends (total 40-mer) were purchased as lyophilized products. They were then subcloned into pBluescript I KS(–). The resultant plasmid DNA was linearized with EcoR I or Hind III and used as a template for the synthesis of labeled sense or antisense riboprobes. DIG-labeled riboprobes were synthesized by in vitro transcription using T3/T7 RNA polymerase in the presence of DIG-UTP according to the instructions provided by the manufacturer of the RNA labeling kit used (Roche Diagnostics; Mannheim, Germany). After DNase treatment, the product was ethanol-precipitated and the pellet was resuspended in the hybridization mixture. Limited hydrolysis after DNase treatment was omitted because the probes were less than 100-mer long. These probes are referred to as ML probes.

Hybridization
Sections were dewaxed with toluene and ethanol, then treated sequentially with 0.2 N HCl, 1 µg/ml proteinase K in 2 mM CaCl2 and 20 mM Tris-HCl buffer (pH 7.5), and 2 mg/ml of glycine in Dulbecco's PBS and were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) to prevent nonspecific binding of the probes. The slides were washed with 2 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate) and then prehybridized in a solution containing 50% formamide and 2 x SSC at 45C for more than 1 hr. Hybridization was performed on slides using 20 µl of a hybridization mixture containing 50% formamide, 2 x SSC, 1 mg/ml tRNA, 0.5 mg/ml sonicated salmon sperm DNA, 2 mg/ml bovine serum albumin, 10% dextran sulfate, and the riboprobe (~2 µg /ml) at 45C for 16 hr in a moist chamber. The slides were then rinsed three times with 2 x SSC and 50% formamide at 45C for 20 min each. After further rinsing of the slides with 0.1 x SSC, anti-DIG alkaline phosphatase conjugate (1:2500) was placed on the sections at room temperature for 2 hr. After more rinsing of the slides, the enzyme-catalyzed color reaction was continued overnight with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt according to the instructions provided with the kit. The sections were counterstained with methyl green and observed with a light microscope.

For the classification of spermatogenesis in rat testes, the serial sections were stained with periodic acid–Schiff (PAS)–hematoxylin (Hess 1990Go; Russell et al. 1990Go).

Quantification of Signals
The stained sections were observed under an Axioplan Zeiss microscope using a x20 Plan Neofluar lens. Photos obtained with an AxioCam CCD camera were saved in the tagged-image files format (TIFF) at a size of 1300 x 1030 pixels (~3.8 MB). Images were separated into five tones from dark to light with posterization tools from Adobe Photoshop 5.0 (Adobe Systems; Tucson, AZ). These five tones are expressed as numerals from grade 5 to grade 1: grade 5 black, grade 4 dark gray, grade 3 gray, grade 2 light gray, and grade 1 white. First, figures were analyzed at low magnification and early primary spermatocytes were identified as grade 5 black. Then we saved pictures that included more than four tubules in which cells showing all five tones were present at one time. The mean value in each cell population was obtained from five or six tubules.


   Results
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 Materials and Methods
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Figure 1 shows the detection of 28S rRNA with DIG probes (Figures 1a and 1c) and ML probes (Figures 1b and 1d) in 4% PA-fixed (Figures 1a and 1b) and Bouin's-fixed (Figures 1c and 1d) paraffin sections. Compared with the brownish color depth, which was a final enzymatic product resulting from the conjugation of alkaline phosphatase with anti-DIG antibodies, round spermatids at stage VII in 4% PA-fixed sections hybridized with ML probes (Figure 1b) showed the most intense positive signals among the four cases under the routine experimental conditions described in Materials and Methods. For control experiments, DIG-labeled 34-mer oligonucleotides of random sequences and sense ML probes for rRNA were used for the experiments using DIG-labeled probes (Figures 1a and 1c) and ML probes (Figures 1b and 1d), respectively (data not shown).



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  		Figure 1

Detection of 28S rRNA with DIG probes (a,c) and ML probes (b,d) in 4% paraformaldehyde-fixed (a,b) and Bouin-fixed (c,d) paraffin sections. Bar = 0.5 mm.

 
However, in the 4% PA-fixed sections, the fixation caused shrinkage of the seminiferous tubules, resulting in a widening of the lymphatic space among the tubules (Figures 1a and 1b, arrows). For this reason, the quantitative analysis was performed using the Bouin's-fixed sections shown in Figure 1d. Figure 2a shows rat testis hybridized with antisense ML probes for rRNA. The brownish color shows the presence of 28S rRNAs. Figure 2b shows rat testis hybridized with sense ML probes for rRNA, in which specific signals were not observed.



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  		Figure 2

Detection of 28S rRNA with ISH using ML probes in rat testis. Bars: A = 1.0 mm; B = 0.2 mm.

 
To quantify RNA signals, it was essential that the signals were not distributed unevenly among seminiferous tubules. It was postulated that the germ cells at the same stage of development in neighboring tubules express the same level of rRNA. We obtained reproducible results in several tubules. The top left tubules in Figures 3a and 3c show stage V, and both the center tubule in Figure 3a and the bottom right tubule in Figure 3c show stage IX. These sets of tubules show equal levels of rRNA. The top right tubule in Figure 3a, center right tubule in Figure 3c, bottom left tubule in Figure 3e, and top center tubule in Figure 3g show stage XIV, as determined by the presence of mitotic figures and/or secondary spermatocytes. Dividing cells (Me1 and Me2) in these tubules showed equal levels of rRNA. The reaction products in secondary spermatocytes were weaker than those in diplotene spermatocytes (D) (Figures 3a, 3c, 3e,and 3g), and early step 1 spermatids contained very small amounts of rRNA (Figure 3g).




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  		Figure 3

Detection of 28S rRNA with ISH using ML probes in rat seminiferous tubules (a,c,e,g). Each image is separated into five tones (b,d,f,h). The left two tubules in g are the same as the right two tubules in a. Arabic numerals indicate steps of spermatid differentiation. Bar = 0.1 mm.

 
From these findings (Figures 3a–3g), we considered the results to be reproducible. Next, we analyzed the data quantitatively. The analysis of rRNA amounts in spermatogonia was not performed by posterization because the cell numbers were too small and the effect of overlapping cells was not negligible.

The specimens fixed with Bouin's fixative and hybridized with ML probes could easily be analyzed quantitatively using a posterization of the images (Figures 3b, 3d, 3f, and 3h). Images were separated into five tones from dark to light, expressed as numerals from grade 5 to grade 1 as described in Materials and Methods. The mean value of each cell population was obtained from five or six tubules, and the results are summarized in Figure 4 . The amount of rRNA hybridizable with the probe was grade 5 black in early primary spermatocytes, grade 4 dark gray in primary spermatocytes, grade 3 gray in diplotene spermatocytes, and grade 2 light gray or grade 1 white in spermatids.



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  		Figure 4

Quantitative analysis of 28S rRNA detected in rat seminiferous tubules. The vertical columns numbered with Roman numerals show cell association in cross-sectioned tubules (stages). The developmental progression of a cell is followed horizontally from left to right, and continues at the left of the cycle map one row up. Pl (preleptotene), L (leptotene), Z (zygotene), P (pachytene), and D (diplotene) show the subdivision of the prophase of the first meiotic division. Arabic numerals represent steps of spermatid differentiation. Spermatogonia are not indicated in the cycle map. SS, secondary spermatocytes. Original cyclic map from Russell et al. (1990)Go, with permission.

 

   Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
Fixation Methods and Intensity of ISH
Preservation of the tissue structure was essential for the quantification of signals obtained with the histochemical protocol. The testis is a suitable organ for quantification because germ cells undergo synchronized development and often show stage-specific expression of their genes. However, it was not easy to preserve the structure of the organs sufficiently. We applied the fixation method of Sprando (1990)Go, who reported that heparin, administered 15 min before whole-body fixation (cardiac route), dramatically increased the success of testicular perfusion. Bouin's fixative and paraformaldehyde were tried, and we used sections fixed with Bouin's fixative, which was reported to be best for the testis if the tissue is embedded in paraffin (Russell et al. 1990Go). In fact, more intense signals were observed in the 4% PA-fixed sections, but the fixation caused shrinkage of the seminiferous tubules, resulting in a widening of the lymphatic space among the tubules as well as the formation of spaces among germ cells (Figure 1). The shrinkage of tubules might cause condensation of signals in each cell or tissue.

rRNA as a Positive Control for ISH
In this model experiment, we selected 28S rRNA as the hybridizable target RNA in paraffin sections, as previously reported by Yoshii et al. (1995)Go, and used DIG-labeled probes prepared by the multiple-labeling method. This probe detected 28S rRNA in sections fixed in Bouin's as well as in PA-fixed sections.

The probe sequence was complementary to a well-conserved segment of 28S rRNA among species, so it could be used in the preparation of tissues from a wide range of species. Because proteinase digestion influenced the detectable amount of mRNA in paraffin sections of formalin-fixed tissues, it influenced the ISH rRNA staining intensity. Therefore, this probe could be used for assessments of RNA integrity and hybridizability in tissue sections, especially when false-negative results might be a problem in retrospective studies using formalin-fixed, paraffin-embedded tissues.

Molecules other than rRNA, such as EF-1{alpha} and ß-actin mRNAs, which are highly conserved among species, were used as a positive control in a nucleic acid assay (Gruber and Levine 1997Go). Because rRNA is present in great abundance in most cell types, it was used as target RNA for the evaluation of proteinase treatment in electron microscopic RNA ISH (Macville et al. 1996Go).

rRNA and Synthetic Activity
Koulouri et al. (1999)Go utilized oligo d(T) and 28S rRNA probes to detect cells containing polyadenylated mRNA and 28S rRNA. They reported that the use of both these markers provides insight into the synthetic activity of cells. In the present study, the amount of rRNA detected by ISH was greatest in the early spermatocytes and decreased throughout spermatogenesis, except for a slight increase in the spermatids after meiotic division. However, it was not clear whether or not these expression patterns reflected the synthetic activity of the spermatogenetic cells. This is because, in the testis, previous studies in vivo (Stefanini et al. 1974Go; Soderstrom and Parvinen 1976Go) and in vitro (Monesi et al. 1978Go) using the autoradiographic method clearly indicated that the uptake of [3H]-uridine increased in the mid-pachytene spermatocytes and not in spermatogonia. In the present study, the amount of rRNA was smaller in early step 1 spermatids than in steps 2–14 spermatids, confirming that rRNA genes become reactivated during spermiogenesis as described with the Ag-AS technique by Schmid et al. (1977)Go. The amount of rRNA in the cells exhibiting mitotic figures (Me1 and Me2 in Figures 3a, 3c, and 3e) markedly decreased. These results coincided with the changes in the rate of RNA synthesis during the cell cycle described in detail by Salem et al. (1998)Go. For examining the proliferative activity of cells, the use of histone mRNA is recommended because it degrades rapidly after S-phase is completed (Slowinski et al. 2002Go).

Perspectives and Conclusions
Both nonradioactive and radioactive ISH quantitations have been reported (Baskin and Stahl 1993Go; Larsson 1997Go), the latter having been used extensively in neuroscience. The background labeling of hybridization was greater with the 35S-labeled probe than with the nonradioactive probe. Therefore, the RI-labeled probe does not seem to be well suited for tissues in which different cell types are closely associated, as in the testis.

Many problems have been overcome for the quantification of mRNA in tissue sections. The first problem was whether or not there was linearity between the probe concentration and the ISH signals. Larsson and Hougaard (1994)Go used glass slides precoated with aminoalkylsilane for immobilizing oligonucleotides. In their model system, the optical density varied linearly with the logarithm of the target oligonucleotide (sense) concentration. In the present study, there was a possibility that a difference of one grade reflected a definite increase or decrease in the amount of hybridized probe. The next problem was whether or not the number of probe molecules per unit area or per cell cross-section accurately reflected the cellular mRNA content. Larsson (1997)Go pointed out that this might not always be the case and so it would be dangerous to assume that the number of probe molecules per cell corresponds to the number of mRNA molecules per cell. The accessibility of the probes to mRNA was different among the mRNAs. The variance in the degree of fixation reflected the degree of accessibility of probes among different tissue sections (Guiot and Rahier 1995Go). Proteolytic permeabilization depended on the degree of fixation and was critical (Singer et al. 1986Go; Larsson and Hougaard 1990Go). Unfortunately, different cell types had different optima for permeabilization. Moreover, enzymatic labeling often resulted in heterogeneously labeled probe molecules. The "specific activity" of such probes represented the mean of a wide range of differently labeled probe molecules (Larsson and Hougaard 1990Go). The other problem was that exact measurement of changes in cell volume, the volume of the structure, or the number of labeled cells was necessary for assessment of the exact change in the gene expression of a given tissue (McCabe and Bolender 1993Go).

ISH signals usually reflect only relative changes in the copy number of a gene and a relative assessment of the amount of signal in the section. Evaluations using + or ++ may be possible, although this does not show the dual change in the amount of signal.

For a precise quantitative analysis of ISH signals, the use of fluorescence may be essential, and fluorescence-labeled probes are being developed. Fluorescence-positive signals quantified under a confocal microscope will enable precise analyses using ISH.


   Acknowledgments
 
Supported by Grants in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We are grateful to Mr Shigeto Kanda (Department of Cytology and Histology, Okayama University Graduate School of Medicine and Dentistry) for technical assistance.


   Footnotes
 
Received for publication January 2, 2004; accepted January 29, 2004


   Literature Cited
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Baskin DG, Stahl WL (1993) Fundamentals of quantitative autoradiography by computer densitometry for in situ hybridization, with emphasis on 33P. J Histochem Cytochem 41:1767–1776[Abstract]

Gruber AD, Levine RA (1997) In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha). Histochem Cell Biol 107:411–416[Medline]

Guiot Y, Rahier J (1995) The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study. Histochem J 27:60–68[CrossRef][Medline]

Hess RA (1990) Quantitative and qualitative characteristics of the stages and transitions in the cycle of the rat seminiferous epithelium: light microscopic observations of perfusion-fixed and plastic-embedded testes. Biol Reprod 43:525–542[Abstract]

Koulouri O, Lappin DF, Radvar M, Kinane DF (1999) Cell division, synthetic capacity and apoptosis in periodontal lesions analysed by in situ hybridisation and immunohistochemistry. J Clin Periodontol 26:552–559[CrossRef][Medline]

Larsson LI (1997) Quantitative in situ hybridization. Endocrin Pathol 8:3–9

Larsson LI, Hougaard DM (1990) Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions. Histochemistry 93:347–354[CrossRef][Medline]

Larsson LI, Hougaard DM (1994) Glass slide models for immunocytochemistry and in situ hybridization. Histochemistry 101:325–331[CrossRef][Medline]

Macville MV, Van Dorp AG, Dirks RW, Fransen JA, Raap AK (1996) Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells. Histochem Cell Biol 105:139–145[CrossRef][Medline]

McCabe JT, Bolender RP (1993) Estimation of tissue mRNAs by in situ hybridization. J Histochem Cytochem 41:1777–1783[Abstract]

Monesi V, Geremia R, D'Agostino A, Boitani C (1978) Biochemistry of male germ cell differentiation in mammals: RNA synthesis in meiotic and postmeiotic cells. Curr Top Dev Biol 12:11–36[Medline]

Mori H, Nomura T, Seno M, Miki Y, Kimura T, Kogami T, Sasaki J (2001) Expression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in rat testes. Acta Histochem Cytochem 31:275–279[CrossRef]

Nomura T, Sasaki J, Mori H, Sato EF, Watanabe S, Kanda S, Matsuura J, et al. (1996) Expression of manganese superoxide dismutase mRNA in reproductive organs during the ovulatory process and the estrous cycle of the rat. Histochem Cell Biol 105:1–6[CrossRef][Medline]

Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED (1990) Staging for laboratory species. In Histological and Histopathological Evaluation of the Testis. Clearwater, FL, Cache River Press, 62–194

Salem C, El-Alfy M, Leblond CP (1998) Changes in the rate of RNA synthesis during the cell cycle. Anat Rec 250:6–12[CrossRef][Medline]

Sasaki J, Sato EF, Nomura T, Mori H, Watanabe S, Kanda S, Watanabe H, et al. (1994) Detection of manganese superoxide dismutase mRNA in the theca interna cells of rat ovary during the ovulatory process by in situ hybridization. Histochemistry 102:173–176[CrossRef][Medline]

Sasaki J, Yamamoto H, Nomura T, Matsuura J, Seno M, Sato EF, Inoue M (1998) Multiple-labeling of oligonucleotide probes for in situ hybridization. Acta Histochem Cytochem 31:275–279

Schmid M, Hofgartner FJ, Zenzes MT, Engel W (1977) Evidence for postmeiotic expression of ribosomal RNA genes during male gametogenesis. Hum Genet 38:279–284[CrossRef][Medline]

Singer RH, Lawrence JB, Villnave C (1986) Optimization of in situ hybridization using isotopic and non-isotopic deletion methods. Biotechniques 4:230–250

Slowinski J, Mazurek U, Bierzynska-Macyszyn G (2002) Histone mRNA in situ hybridization in assessing proliferative activity of normal and malignant cells. Folia Histochem Cytobiol 40:335–339[Medline]

Soderstrom KO, Parvinen M (1976) RNA synthesis in different stages of rat seminiferous epithelial cycle. Mol Cell Endocrinol 5:181–199[CrossRef][Medline]

Sprando RL (1990) Perfusion of the rat testis through the heart using heparin. In Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED, eds. Histological and Histopathological Evaluation of the Testis. Clearwater, FL, Cache River Press, 277–280

Stefanini M, De Martino C, D'Agostino A, Agrestini A, Monesi V (1974) Nucleolar activity of rat primary spermatocytes. Exp Cell Res 86:166–170[CrossRef][Medline]

Yoshii A, Koji T, Ohsawa N, Nakane PK (1995) In situ localization of ribosomal RNAs is a reliable reference for hybridizable RNA in tissue sections. J Histochem Cytochem 43:321–327[Abstract]


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