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Endocrinology Vol. 140, No. 3 1492-1498
Copyright © 1999 by The Endocrine Society

ARTICLES

Function of Stem Cell Factor as a Survival Factor of Spermatogonia and Localization of Messenger Ribonucleic Acid in the Rat Seminiferous Epithelium1

Harri Hakovirta2, Wei Yan2, Marko Kaleva, Fuping Zhang, Katariina Vänttinen, Patricia L. Morris, Molle Söder, Martti Parvinen and Jorma Toppari

Departments of Anatomy (H.H., M.P.), Physiology (W.Y., M.K., F.Z., K.V., J.T.), and Pediatrics (J.T.), University of Turku, Turku, Finland; The Population Council (H.H., P.L.M.), New York, New York; and Pediatric Endocrinology Unit (O.S.), Karolinska Institute, Stockholm, Sweden

Address all correspondence and requests for reprints to: Dr. Jorma Toppari, Department of Physiology, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: jorma.toppari{at}utu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To address the possibility that stem cell factor (SCF) is a paracrine regulator of germ cell development in the adult rat testis, stage-specific distribution of SCF messenger RNA (mRNA) was investigated with Northern blot and in situ hybridization analyses. The highest levels of SCF mRNA were found in stages II–VI of the rat seminiferous epithelial cycle, whereas the lowest levels were in stages VII–VIII. Intermediate levels of SCF mRNA were detected in stages IX–XIV–I of the cycle. The expression of the SCF gene was found to be developmentally regulated, and the expression pattern followed the process of Sertoli cell proliferation and differentiation during postnatal life. The effect of mouse recombinant SCF on spermatogonial DNA synthesis was studied using an in vitro tissue culture system for stage-defined seminiferous tubules. A significant increase in DNA synthesis in spermatogonia could be detected when tubule segments from stage XII were cultured in the presence of 100 ng/ml SCF for 48 h (P < 0.05) and 72 h (P < 0.01). This observation was further confirmed with autoradiographic analyses; almost a 100-fold increase in thymidine incorporation in the SCF-treated (100 ng/ml) tubule segments was observed compared with that in untreated samples. The results of the present study suggest that SCF is a Sertoli cell-produced paracrine regulator and acts as a survival factor for spermatogonia in the adult rat seminiferous epithelium in a stage-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IMPORTANCE of stem cell factor (SCF) in germ cell development was first shown by the observations that mutations at the mouse gene loci White spotting(W) or Steel (Sl) result in defective germ cell development in both the testis and ovary (1, 2, 3, 4). Later, a protooncogene, tyrosine kinase receptor, c-Kit, and its ligand SCF were determined to be encoded at the W (c-Kit) and Sl (SCF) loci, respectively (5, 6, 7, 8, 9, 10). The gene encoded in the Sl locus is a transmembrane protein expressed as two alternatively spliced isoforms, either 220 amino acids lacking the exon coding for the peptidase cleavage site or 248 amino acids containing the peptidase cleavage site (11, 12). This factor has been named SCF, steel factor, mast cell growth factor, and c-Kit ligand (9, 13, 14, 15, 16, 17).

Immunohistochemical studies in the adult mouse testis suggested that the SCF receptors are localized in the proliferating germ cells, spermatogonia, and preleptotene spermatocytes (18). These results were further supported by the observation that highly purified type A spermatogonia isolated from the immature rat testes were the site of c-Kit messenger RNA (mRNA) and protein synthesis (19). Pachytene spermatocytes, round spermatids, or Sertoli cells show no detectable c-Kit mRNA expression (19) or immunoreactivity (18).

Both forms of SCF mRNA have been reported to be abundant in mouse testes at all ages (20). The major site of SCF synthesis in the testis appears to be Sertoli cells (21).

To address the possibility that SCF is a paracrine regulator of germ cell development in the adult rat testis, stage-specific distribution of SCF mRNA was investigated using both in situ and Northern hybridization analyses. The effects of mouse recombinant SCF on spermatogonial DNA synthesis were measured using a tissue culture method for staged seminiferous tubules that we previously developed for study of the effects of growth factors on stage-specific DNA synthesis (22).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague-Dawley rats, aged 1 day, 5 days, 10 days, 20 days, 30 days, 40 days, and 2–3 months, were used as experimental animals. They were housed two per cage in a controlled environment at 21 C with a 14-h light, 10-h dark cycle with free access to water and food.

Microdissection of the seminiferous tubule segments, tissue culture, and assessment of DNA synthesis
Stages I, V, VIIa, VIII–IX, and XII of the seminiferous epithelial cycle were selected, because they contain cells at representative phases of mitotic and meiotic DNA synthesis (23). During culture times of 24, 48, and 72 h, these stages differentiate through all stages of the cycle. Two-millimeter seminiferous tubule segments were isolated under a transilluminating stereomicroscope in a laminar flow hood for in vitro analyses of DNA synthesis. Stages were identified as described previously (24). Twenty 2-mm long tubule segments were transferred onto 96-well culture plates in 10 µl PBS and incubated at 34 C for 24, 48, and 72 h in 100 µl Ham’s F-12-DMEM (Life Technologies, Paisley, UK) supplemented with 0.1% BSA (Sigma Chemical Co., St. Louis, MO), G-penicillin (60 mg/liter; Sigma Chemical Co.), and streptomycin (500 mg/liter; Sigma Chemical Co.) in a humidified atmosphere containing 5% CO2 in air. Recombinant mouse SCF (Genzyme Transgenics Corp., Cambridge, MA) was added at concentrations of 0, 20, and 100 ng/ml. Tubules were pulse labeled during the last 4 h of the culture by adding 20 kBq [methyl-3H]thymidine (185 gigabecquerels/mmol; TRA 120, Amersham, Aylesbury, UK).

The cultures were harvested on filter discs (934-AH, Whatman, Clifton, NJ) with a continuous flow of distilled water for 1 min. A scintillation wax (MeltiLex A 1450–441, Wallac, Turku, Finland) was melted on the filters, and the radioactivity was measured by a flat bed liquid scintillation counter equipped with two parallel detectors (1450 Microbeta, Wallac, Turku, Finland). Four separate experiments were performed, each with three replicate samples. For autoradiography, the labeled seminiferous tubules were carefully squashed between microscope slides and coverslips and frozen in liquid nitrogen (25). The coverslips were removed by flipping with a scalpel, and the frozen squash preparations were fixed in ethanol-glacial acetic acid (3:1, vol/vol) for 30 min and air-dried. The slides were dipped in Kodak NTB-3 nuclear track emulsion (Eastman Kodak Co., Rochester, NY), exposed for 2 days, developed, and stained with hematoxylin. Positively labeled cells were counted in 2-mm segments of seminiferous tubules under a miocroscope.

Preparation of riboprobes
The rat SCF complementary DNA cloned into pGEM3Z was a gift from Amgen, Inc. (Thousand Oaks, CA). The cloned plasmid containing a 560-bp insert was linearized with HindIII or EcoRI restriction enzymes for preparation of antisense and sense probes, respectively, in the presence of [35S]UTP (for in situ hybridization) or [32P]UTP (for Northern hybridization; Amersham). In vitro transcription reactions were performed essentially as recommended by the manufacturer of the polymerases (Promega Corp., Madison, WI). For in situ hybridization, sense and antisense RNA probes were adjusted to the same radioactivity.

In situ hybridization
Testes were fixed in 10% buffered formalin at room temperature for 24 h, dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Sections (5 µm) were cut, and in situ hybridization was performed as described previously (26). The results of the in situ hybridization were quantified by counting the number of positively labeled Sertoli cells identified in the darkfield per 100 Sertoli cell nuclei identified in the brightfield. Cells were counted from randomly selected cross-sections from stages II–VI, VII–VIII, IX–XII, and XIII–XIV–I of the cycle. The number of positively labeled Sertoli cells from the sense reaction was subtracted from that of the antisense result. Two separate countings gave similar results.

RNA extraction and Northern blot hybridization
Northern hybridization analyses were applied to study stage-specific distribution of SCF mRNA synthesis in the seminiferous epithelium. Segments of seminiferous tubules at stages II–VI, VII–VIII, IX–XII, and XIII–I were microdissected for mRNA analysis. Total RNA was extracted by a single step method (27). The quality and quantity of RNA were determined by measuring optical densities at 260 and 280 nm by UV spectrophotometry (Beckman Coulter, Inc., Fullerton, CA). The OD ratios of absorbance at 260 nm to that at 280 nm were between 1.7–1.9. RNA samples (10 µg) were fractioned on 1% agarose gel in the presence of formaldehyde. The gel was stained with ethidium bromide to verify the even loading of RNA. RNA was then transferred onto nylon membranes (Hybond-N, Amersham). Hybridizations were performed according to the instructions of the membrane manufacturer. After baking for 2 h at 80 C, filters were prehybridized in 50% formamide, 3 x SSC (standard saline citrate), 5 x Denhardt’s solution (1 mg/ml Ficoll, 1 mg/ml polyvinylpyrrolide, and 1 mg/ml BSA), 1% SDS, and 10% dextran sulfate containing 100 µg/ml heat-denatured calf thymus DNA and 100 µg/ml yeast transfer RNA at 65 C for 6–16 h. Hybridization was performed at the same temperature for 16–24 h by adding 32P-labeled probe. The filters were washed twice for 15 min each time with 2 x SSC at room temperature, followed by two washes of 45 min in 0.2 x SSC-0.1% SDS at 65 C and two washes of 30 min in 0.1 x SSC at room temperature. Filters were exposed to Kodak XAR-5 film at -80 C between intensifying screens. Northern blotting analyses on the stage-specific expression of SCF gene in the rat seminiferous epithelium were repeated 10 times independently using 10 rats, aged 60–70 days.

Densitometric analysis of Northern hybridization results
The x-ray films of Northern blotting results were first scanned by a UMAX scanner (Super Vista S-20, Binuscan, Inc., NY) and a Binuscan Photoperfect software package (Binuscan, Inc.). The images were saved as TIFF-type files (1.tif, Microsoft Corp. and Aldus Co., NY) and then quantified by TINA 2.0 densitometric analytical system (Raytest Isotopenmesgerate GmbH, Straubenhardt, Germany) according to the manufacturer’s instruction.

Statistical analysis
In all of the Northern hybridization analyses, the densitometric values of the signals of SCF mRNA were first normalized to 28S ribosomal RNA signals, and the highest densitometric value was designated 100%. Other values were expressed as the percentages of the highest one. The values from all experiments were pooled for calculation of the means and their SEs and for one-way ANOVA and Student-Newman-Keuls multiple comparison test to determine the significant differences between different experimental groups using SAS 6.12 (SAS Institute, Inc., Cary, NC). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of SCF mRNA
Specific hybridization signals were observed in basal compartment of the seminiferous tubule (Fig. 1AGo: a, A). The most intense signals were confined to the cytoplasm of Sertoli cells, mainly around Sertoli cell nuclei (Fig. 1AGo: b, B). Specific staining was not found in Leydig cells. Due to the resolution limit of the method used, it could not be ruled out that some signals might came from spermatogonia and/or spermatocytes as well. Quantification by counting the number of positively labeled Sertoli cells per 100 Sertoli cell nuclei identified in the brightfield revealed that the highest expression levels of SCF mRNA were in stages II–VI and XIII–I. In stages VII–VIII, very few Sertoli cells were positive, indicating the lower expression levels of SCF mRNA (Fig. 1BGo).



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  		Figure 1. In situ localization and quantification of SCF mRNA expression in adult rat testis. A, In situ hybridization of SCF mRNA in adult rat testis. The brightfield (A–C) and corresponding darkfield (a–c) photomicrographs of hematoxylin-stained sections from an adult rat testis are shown. In low magnification (a, A), stages VI and I show different intensities of hybridization signals. In high magnification (b, B), signals are mainly found to be confined to the cytoplasm of Sertoli cells. Background level hybridization signals are shown in the sense control (c, C). Arrows point to the Sertoli cell nuclei. Note that bundles of elongated spermatids give strong autofluorescent signals that do not represent any labeling. B, Quantification of SCF expression levels in four pooled stages (II–VI, VII–VIII, IX–XII, and XIII–I) by counting the number of positively labeled Sertoli cells per 100 identified Sertoli cell nuclei. Bar, 50 µm.

 
Stage-specific distribution of SCF mRNA in the seminiferous epithelium
SCF mRNA was most abundant in stages II–VI of the seminiferous epithelial cycle. Intermediate levels of SCF mRNA were detected at stages IX–XII and XIII–I of the cycle, whereas the lowest level was found in stages VII–VIII of the cycle (Fig. 2AGo). The differences among the four pooled stages of the rat seminiferous tubules were significant (Fig. 2BGo; n = 10; P < 0.05).



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  		Figure 2. Stage-specific expression of SCF gene in the rat seminiferous epithelium. A, Northern hybridization analysis (upper panel) of SCF gene expression in different stages of the rat seminiferous epithelial cycle. Total RNA was isolated from stages II–VI, VII–VIII, IX–XII, or XIII–I of the rat seminiferous epithelial cycle. Sixteen micrograms of total RNA were loaded in each lane and hybridized with 32P-labeled SCF antisense riboprobe. The exposure time was 7 days. The locations of the 28S and 18S ribosomal RNAs are marked on the right, and ethidium bromide (EtBr) staining of the 28S RNAs of the samples in the blot of the upper panel after blotting onto the nylon membrane is shown in the bottom panel for evaluation of even loading and transfer of the RNA. B, Quantitative analysis of stage-specific expression pattern of the SCF gene in the rat seminiferous epithelium. ADU, Arbitrary densitometric unit (defined as the percent expression level in stages II–VI). Each bar represents the mean ± SEM of independent experiments repeated 10 times. Stages with different letters are significantly different (n = 10; P < 0.05)

 
SCF gene expression during rat testicular development
Northern hybridization analyses revealed that the SCF mRNA was at low levels from 1–5 days after birth, dramatically increased at 10 days of age, and peaked around 20 days of age. Thereafter, it decreased and remained at low levels, which were, however, higher than those at 1 and 5 days of age (Fig. 3Go).



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  		Figure 3. SCF gene expression during testicular development in the rat. A, Northern hybridization analysis of SCF gene expression during testicular development of the rat. Total RNA was isolated from testes of 1-day-old (1d), 5-day-old (5d), 10-day-lod (10d), 20-day-old (20d), 30-day-old (30d), 40-day-old (40d), or 60-day-old (60d) rats. Each lane contains 20 µg total RNA. The exposure time was 4 days. The locations of the 28S and 18S ribosomal RNAs are marked on the right. The bottom panel shows the ethidium bromide (EtBr) staining of the 28S RNAs of the samples in the blot of the upper panel after blotting onto the nylon membrane to evaluate even loading and transfer of the RNA. The expression of SCF gene peaks at 20 days of postnatal life. B, Quantification of SCF mRNA levels at different ages of rat testes. Each bar represents the mean ± SEM of independent experiments repeated four times.

 
Effects of SCF on stage-specific DNA synthesis in the adult rat seminiferous epithelium
A significant (n = 12; P < 0.05) stimulation of thymidine incorporation was observed when stage VIII–IX tubule segments were cultured in the presence of 100 ng/ml SCF for 24 h. Stage XII tubules cultured in the presence 100 ng/ml SCF for 24 h showed a tendency to increased thymidine incorporation (Fig. 4AGo). With the same culture conditions, thymidine incorporation at stage XII was significantly stimulated at 48 h (P < 0.05) and 72 h (P < 0.01; Fig. 4Go, B and C). By using autoradiography, we detected the cellular origin of the increased DNA synthesis. Stimulated cells from cultured stage XII tubules were found to be type A and intermediate type spermatogonia as judged by autoradiography (Fig. 5Go, A and B). According to autoradiographic quantification, SCF-treated tubule segments showed an approximately 100-fold increase in thymidine incorporation compared with the control value (Fig. 5CGo).



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  		Figure 4. The [3H]thymidine incorporation (counts per min; mean ± SEM; n = 12; normalized to 2 mm of tubule) at stages I, V, VIIa, VIII–IX, and XII of seminiferous epithelium cultured for 24 (A), 48 (B), and 72 h (C) with 0, 20, and 100 ng/ml mouse recombinant SCF. No significant effects on stage-specific DNA synthesis could be detected at any time point studied at stages V, VIIa, and I of the cycle. SCF stimulated the thymidine incorporation of stages VIII–IX of the cycle cultured for 24 h. At stage XII of the cycle, the thymidine incorporation of spermatogonia was stimulated significantly after 48 and 72 h in culture with 100 ng/ml SCF. The progression of the stages during 0, 24, 48, and 72 h of culture is as follows: stage V->V->VI->VII, stage VIIa->VIIb->VIIc->VIII, stage VIII-IX->IX->XI->XII, stage I->I->III-IV->V, and stage XII->XIV->I->I, respectively. (n = 12). **, P < 0.01; *, P < 0.05 (compared with controls).

 


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  		Figure 5. Autoradiography of the seminiferous tubules from stage XII of the seminiferous epithelial cycle cultured for 72 h in the absence (A) and presence (B) of recombinant human SCF (100 ng/ml). The tubule segments from stage XII of the cycle proceed to stage I of the cycle during the culture. Spermatogonia in the control tubules show minimal grain accumulation (A), whereas spermatogonia in the tubule segments cultured in the presence of 100 ng/ml SCF show abundant grain accumulation. C, Quantification of the labeled cells in the 2-mm tubule segment from stage XII cultured for 72 h in the absence and presence of SCF (100 ng/ml). The control tubule segment shows 100 times fewer labeled germ cells compared with the SCF-stimulated tubule segment. Bar, 25 µm; n = 4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Earlier studies have suggested that SCF plays an important role during germ cell development (1, 2, 3, 4, 19). These studies were based on the Sertoli cell-germ cell coculture method lacking the microenvironment provided by more advanced germ cells. Also, the spermatogonia in these studies were isolated from the testis of immature rodents. In the present study, the effects of SCF on stage-specific DNA synthesis were analyzed using a tissue culture system for adult rat seminiferous tubules isolated from specific developmental stages of the epithelium that provides closer physiological microenvironment for the proliferating germ cells.

A study by Dym et al. (19) has shown that SCF stimulated DNA synthesis in vitro in 7- to 8-day-old mouse testes enriched at that age with type A and B spermatogonia. Therefore, the role of SCF action on germ cell development has been suggested to be the regulation of cell proliferation during testicular development. The present observations demonstrate that in the adult rat seminiferous epithelium, SCF also stimulated DNA synthesis at defined stages (XII–XIV and I–III) of the epithelial cycle. As type A4 and intermediate spermatogonia either proliferate or degenerate, and a cell cycle arrest in these cells has never been shown, the increase in DNA synthesis should result from an increased number of surviving spermatogonia rather than from stimulation of new spermatogonia into the cell cycle. The significant SCF-induced stimulation of thymidine incorporation at stages VIII–IX of the cycle suggests that SCF might also function as a survival factor for preleptotene spermatocytes.

Immunohistochemical observation has shown that the SCF-like immunoreactivity in Sertoli cells is located basally at stages X–II of the murine seminiferous epithelium around the type A2, A3, and A4 spermatogonia. No immunoreactivity was detected in the maturing germ cells (20). In the present study SCF mRNA levels were observed to be most abundant at stages II–VI, suggesting that the increasing number of proliferating germ cells might regulate the production of SCF in the Sertoli cells. The slight discrepancy between the present observation for SCF mRNA and earlier immunohistochemical observations by Manova et al. (20) may be due to the posttranscriptional regulation of SCF gene expression. For example, high mRNA levels may precede the high protein levels during the seminiferous epithelial cycle.

In the present study, the stage-specific distribution of SCF mRNA followed the pattern of stage-specific FSH-stimulated cAMP production along the cycle of the seminiferous epithelium (28), suggesting that the FSH stimulation of SCF expression in the seminiferous epithelium is mediated by cAMP, although other regulatory pathways cannot be excluded. Our study of the stage-specific regulation of SCF gene expression has confirmed that the up-regulation of SCF gene expression by FSH is mediated through the cAMP/PKA pathway (29).

During rat testicular development, SCF gene expression was highly regulated. The developmental expression pattern is consistent with the process of Sertoli cell proliferation and maturation during postnatal life. The decrease in SCF mRNA levels after 20 days of age might result from the dilution of Sertoli cell mRNA by germ cell mRNA, as germ cell number increases markedly from this age onward. Based on the unchanging number of Sertoli cells per testis after day 20 and the localization of SCF to the Sertoli cell, it is likely that there may be no change in terms of SCF expression level per Sertoli cell. This is supported by a recent report by Blanchard et al. (30) showing that the expression level of membrane SCF is relatively low on days 5 and 7, but becomes and stays high after day 11 of postnatal life, whereas the soluble SCF mRNA levels at different ages remain relatively constant. However, our findings on the developmental expression pattern of SCF gene differ from those by Munsie et al. (31) showing that SCF mRNA peaks on day 5 and decreases thereafter, and we cannot offer a clear explanation for the discrepancy, although the time points studied are slightly different in the two studies. According to the Northern blotting results in the previous study, the researchers appear to have analyzed two blots from two different sets of experiments for of 1–5 and 6–35 days of age. If so, that may have influenced their interpretation of the results, as samples on two separate blots from different times of experiments may not be comparable due to the variation in experimental condition. We repeated our analyses four times independently with identical results.

The present results suggest that SCF is a Sertoli cell-produced paracrine regulator and acts as a survival factor for spermatogonia in the adult rat seminiferous epithelium in a stage-specific manner. The molecular mechanisms by which SCF mediates the survival of spermatogonia remain an interesting subject for further studies.


   Footnotes
 
1 This work was supported by grants from EU DGXII Biomed 2 Program BMH4-CT96–0314, EU Biotechnology Program BIO4-CT96–0183, the Academy of Finland, and Turku University Central Hospital (to J.T.); the Children’s Cancer Fund, the Magnus Bexqvall Foundation, and Swedish Medical Research Council Project 8282–11412 (to O.S.); NIH Grants HD-29428 and 13541 (to P.L.M.); and the Turku University Foundation (to W.Y.). Back

2 These authors contributed equally to this work and should both be considered first authors. Back

Received May 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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