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Endocrinology Vol. 140, No. 8 3416-3421
Copyright © 1999 by The Endocrine Society

ARTICLES

Glial Cell Line-Derived Neurotropic Factor Stimulates Sertoli Cell Proliferation in the Early Postnatal Period of Rat Testis Development

Jianguo Hu, Hiroki Shima and Hachiro Nakagawa

Research and Development Center, BML, Inc. (J.H., H.N.), Matoba 1361–1, Kawagoe-shi, Saitama 350-1101; and the Department of Urology, Hyogo Medical College (H.S.), Nishinomiya, Hyogo 663, Japan

Address all correspondence and requests for reprints to: Dr. Jianguo Hu, Research and Development Center, BML, Inc., Matoba 1361–1, Kawagoe-shi, Saitama 350-1101, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glial cell line-derived neurotropic factor (GDNF) was first characterized by its actions on central nervous system neurons. GDNF messenger RNA was expressed in many peripheral tissues in addition to brains. We demonstrated that GDNF had a marked stimulatory effect on the proliferation of Sertoli cells during the early postnatal period of rat testis development. The action of GDNF was examined in in vitro organ culture using testicular fragments from 6-day-old rats. [3H]Thymidine incorporation into testicular fragments cultured for 3 days was significantly stimulated by GDNF in the presence of FSH. Neither LH nor testosterone had such synergism with GDNF. The stimulation was GDNF dose and [3H]thymidine exposure time dependent and was specifically inhibited by both anti-GDNF and anti-GDNF receptor (RET) antibodies. Immunohistological section labeled with 5'-bromo-2'-deoxyuridine at the end of in vitro culture demonstrated a dramatic increase in both total and labeled Sertoli cells after combined treatment with FSH and GDNF. These findings suggest that GDNF might play an important role in regulation of Sertoli cell number as a local factor in early postnatal period of rat testis development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLIAL CELL line-derived neurotropic factor (GDNF), a distantly related member of the transforming growth factor-ß superfamily, was originally isolated based on its ability to promote the survival and differentiation of dopaminergic neurons in primary culture (1, 2). It was also demonstrated that GDNF was an extremely potent survival factor for motoneurons in vitro (3, 4, 5, 6, 7). The study of GDNF-deficient mice indicated that the mutant mice appeared normal during the development of dopaminergic and spinal motoneurons, but failed to develop kidneys and to innervate the gastrointestinal tract (8, 9, 10). GDNF, as a neurotropic factor, was detected in almost every brain region of all ages, ranging from embryonic day 11.5 to adulthood, as far as its expression was examined by RT-PCR (11). It was also reported, however, that GDNF messenger RNA (mRNA) was intensively expressed in the noncentral nervous system (non-CNS) organs and tissues, such as kidney, intestine, stomach, muscle, cartilage, lung, and blood. These findings suggested that GDNF might play some physiological role in regions other than CNS (11, 12, 13, 14).

Just as glial cells provide neurons with tropic factors in CNS, Sertoli cells nurture germ cells in testis. Sertoli cells extend to the interior of lumen of the tubule all the way from the basement membrane of the seminiferous epithelium and constitute the blood-testis barrier to provide an environment essential for germ cell differentiation. Sertoli cells are also known as an endocrine organ from which several hormones, such as Müllerian inhibitory factor, estradiol, and inhibin, are secreted, and play a special role in nurturing and controlling the spermatogenesis process. The total number of Sertoli cells populating the adult rat is established within about 2 weeks after birth, with their proliferation being very active during the late fetal and early postnatal period and their mitotic divisions stopping by day 16 (15, 16). At that very important stage, GDNF is found to be highly expressed in testis. Ribonuclease protection assay revealed that GDNF expression in the testis increased during development and peaked on postnatal day 7, subsequently decreased during the second and third postnatal weeks, and was lowest in the adult. Expression of GDNF mRNA in the Sertoli cell line TM4 suggested that in the testis, GDNF might be derived from Sertoli cells (12).

Sympathetic neurons are known to require continuous support of neurotropic factors; GDNF was thus found to be expressed in some sympathetic-innervatal organ, such as kidney, stomach, lung, and salivary gland. Testis also receives sympathetic innervation from the inferior mesenteric ganglion, although it is not directly innervated from sympathetic trunk. In rat testis, postmeiotic cells appear at the age of about 25 days, whereas somatic Sertoli cells constitute the bulk of the seminiferous epithelia during early postnatal development (17). Temporal expression of GDNF mRNA in testis is correlated with the expansion of the Sertoli cell population, although it decreases with an increase in germ cells at later stages (12).

The physiological response to GDNF requires the presence of a glycosyl-phosphatidylinositol-linked protein (GDNFR-{alpha}) that is expressed on GDNF-responsive cells and binds GDNF with high affinity. At the same time, GDNF promotes the formation of a physical complex between GDNFR-{alpha} and the orphan tyrosine kinase receptor Ret (GDNFR-ß), thereby inducing its tyrosine phosphorylation (18). We confirmed the expression of GDNFR-{alpha}1 and -ß (Ret) in rat testis using RT-PCR, but did not identify in which cell type they were expressed (unpublished data). Cao and et al. reported that Ret protein might function in the regulation of cell growth and differentiation during mouse embryogenesis and sperm differentiation, because Ret expression was ubiquitous in day 10.5–13.5 mouse embryos, but was restricted in adult mice, with the highest level of expression in spermatocytes (19). High level expression of both GDNF and GDNFR in the testis strongly implicates the potential role of GDNF in testis development. Therefore, we used in vitro organ culture of testicular fragments from 6-day-old rats to investigate its action on Sertoli cells in early postnatal period.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression of GDNF in Escherichia coli
Using rat embryonic 18 (E18) brain complementary DNA as a template, complementary DNA containing GDNF mature protein region was amplified by PCR with an order primer (5'-GACGGATCCAGCCCAGAGAATTCCAGAGGG) and a reverse primer (5'-GCGCCCGGGTCAGATACATCCACACCGTTTAG). The PCR product was digested with BamHI and SmaI and directionally inserted into prokaryotic expression vector pQE. Escherichia coli JM109 was transformed with expression plasmid and induced by isopropyl ß-D-thiogalactopyranoside.

Preparation of recombinant GDNF
Recombinant GDNF was isolated from supernatant of sonicated E. coli lysate by Ni2+-NTA-agarose. Western blot using anti-GDNF antibody showed a high specificity between rGDNF and anti-GDNF antibody. Recombinant GDNF existed as a 15-kDa band.

Preparation of anti-GDNF antibody
Recombinant GDNF (1 mg/ml PBS) was emulsified with 1 ml complete Freund’s adjuvant and sc injected into a New Zealand White rabbit. The rabbit was given two booster injections at biweekly intervals by injecting half of the original amount of antigen emulsified with an equal volume of incomplete Freund’s adjuvant. The IgG fraction was isolated from the antiserum by precipitation with caprylic acid followed by ammonium sulfate fractionation (20). The antibody fraction was further purified by an immunoaffinity column conjugated with recombinant GDNF. The purity of the antibody was checked by SDS-PAGE.

Immunoblot analysis
GDNF isolated from GDNF-expressed E. coli were mixed with an equal volume of sample buffer (10% glycine, 1% 2-mercaptoethanol, 2% SDS, and 0.015% bromophenol blue in 80 mM Tris-HCl) and boiled for 3 min. The proteins were electrophoresed on a 5–20% gradient SDS-polyacrylamide minigel and transferred onto a nitrocellulose membrane. After blocked with Block Ace (Dainippon Pharmaceutical, Osaka, Japan), the membrane was immunostained by anti-GDNF antibody (0.5 µg/ml) for 3 h at room temperature and washed with PBS containing 0.05% Tween-20. The membrane was incubated with antirabbit IgG-horseradish peroxidase solution (0.5 µg/ml) for another 3 h at room temperature. After extensive washing, the bands were detected using DuPont Western blot chemiluminescence reagent as described by the manufacturer (DuPont Pharma Radiopharmaceuticals, Wilmington, DE).

Organ culture
Testicular tissue obtained from a P6 male Wistar rat was cut into approximately 1-mm3 fragments and arranged on a 13-mm polyvinylidene difluoride membrane filter (Millipore, MA) kept on a steel grids in a 35-mm culture dish (Nunc, Copenhagen, Denmark). Medium was added into the dish until the lower surface of the grid became wet. For culture, Eagle’s MEM with Eagle’s salts supplemented with 2 mM glutamine, 15 mM HEPES, nonessential amino acids (single strength), 100 IU/ml penicillin, and 100 mg/ml streptomycin was used.

Rat FSH (UCB-Bioproducts S.A., Braine-l’Alleud, Belgium), recombinant GDNF, bovine LH (Biogenesis, New Fields, UK), testosterone enanthate (Wako, Osaka, Japan), and anti-GDNF and anti-RET antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to the culture medium either alone or in combination at the concentrations indicated in the figure legends. Tissue fragments were cultured for 3 days at 32 C in a humidified atmosphere of 5% CO2 in air.

During the last 5 h of culture, testicular fragments were labeled with either 3.5 µCi/ml [methyl-3H]thymidine (Amersham, Aylesbury, UK) or bromodeoxyuridine (BrdU) and 5-fluoro-2'-deoxyuridine (cell proliferation kit, Amersham). At the end of labeling, fragments were washed twice with PBS and processed for further analysis.

Thymidine incorporation into DNA
Testicular fragments labeled with [3H]thymidine were incubated overnight with 0.5 mg/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) in 50 mM Tris, 100 mM EDTA, 100 mM NaCl, and 1% SDS at 55 C. DNA was then extracted with phenol-chloroform-isoamyl alcohol and resuspended in TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). The radioactivity of each specimen was measured by liquid scintillation spectrometry.

The DNA content of the same sample was determined by a fluorometric assay using Hoechst 33258 (Sigma Chemical Co.) as a fluorescent dye. Each 2-µl sample was mixed with 1 ml dye solution (0.1 µg/ml Hoechst in 0.1 M NaCl, 10 mM Tris-HCl, and 1 mM EDTA, pH 7.4). Fluorescence was immediately measured using a Hitachi F-3010 fluorescence spectrophotometer (Hitachi Scientific Instruments, Inc., Hialeah, FL) at 365/460-nm (excitation/emission) wavelengths, with salmon sperm DNA as a standard. The radioactive content was expressed in counts per min/µg DNA.

BrdU incorporation
Testicular fragments labeled with BrdU were fixed in Bouin’s fluid and dehydrated. After embedded in paraffin, 3-µm thick serial neighboring sections were prepared, and BrdU-incorporated cells were detected by anti-BrdU monoclonal antibody and antimouse IgG antibody conjugated with peroxidase (cell proliferation kit, Amersham), followed by counterstain of hematoxylin and eosin Y.

The numbers of total Sertoli cell gonocytes, and BrdU-labeled Sertoli cell gonocytes were determined by analyzing 30 coronal sections of seminiferous tubules, selected at random, and expressed as the mean ± SE/seminiferous tubule.

c-Kit antibody immunostain
The neighboring sections prepared above were incubated by affinity-purified polyclonal anti-c-Kit antibody (Santa Cruz Biotechnology, Inc.) and visualized by Vectastain ABC Kit (Vector Laboratories, Inc., Burlingame, CA).

Statistics
Statistical analysis of differences between control and treated testicular fragments was performed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of GDNF on [3H]*thymidine incorporation into P6 testicular fragments
Testicular fragments from 6-day-old rats were cultured for 3 days in the presence or absence of GDNF and with or without FSH (Fig. 1Go). Recombinant GDNF alone induced a slight increase in the incorporation of [3H]thymidine (117%). FSH significantly stimulated incorporation compared with the effect of plain medium (199%; P < 0.01). The combination of GDNF and FSH resulted in a marked increase in the incorporation of [3H]thymidine compared with the effect of FSH treatment only or medium only (410%; P < 0.01 and P < 0.001, respectively).



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  		Figure 1. Effect of GDNF and FSH on [3H]thymidine incorporation in testicular fragments from 6-day-old rats. Fragments were cultured for 3 days in the presence or absence of FSH (200 ng/ml) and recombinant GDNF (100 ng/ml) and then labeled with [3H]thymidine for 5 h. Incorporation of [3H]thymidine into DNA was measured as described in Materials and Methods. Each bar is represented as counts per min/µg DNA (mean ± SEM; n = 4). Statistical difference was analyzed by Student’s t test. *, P < 0.05; ***, P < 0.001.

 
Dose response of GDNF upon FSH-dependent incorporation of [3H]thymidine
To investigate whether the effect of GDNF is dose dependent, testicular fragments were cultured for 3 days in the presence of FSH at a fixed concentration of 200 ng/ml and GDNF at increasing concentrations from 3–100 ng/ml. As shown in Fig. 2AGo, GDNF stimulated the incorporation of tritiated thymidine in a dose-dependent manner.



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  		Figure 2. Effects of dose of GDNF (A) and time of labeling (B) on FSH-dependent DNA synthesis in testicular fragments from 6-day-old rats. A, Fragments were cultured in the presence of 200 ng/ml FSH and various doses of recombinant GDNF for 3 days, and then labeled with [3H]thymidine for 5 h. B, Fragments were cultured in the presence of 200 ng/ml FSH and 100 ng/ml GDNF for 3 days and then labeled with [3H]thymidine for different periods. The incorporation of [3H]thymidine into DNA was measured as described in Materials and Methods. Each bar is represented as counts per min/µg DNA (mean ± SEM; n = 6).

 
Effect of exposure time on FSH-dependent incorporation of [3H]thymidine
For this purpose, testicular fragments previously cultured for 3 days in the presence of 200 ng/ml FSH and 100 ng/ml GDNF were exposed to [3H]thymidine from 5–24 h. As shown in Fig. 2BGo, the levels of [3H]thymidine incorporation were increased almost parallel to the exposure time.

Morphological analysis of testicular fragments labeled with BrdU from 6-day-old rats
To determine which cell types proliferated in response to FSH-GDNF treatments, testicular fragments were labeled with BrdU at the end of a 3-day culture period in the presence or absence of GDNF and with or without FSH. Within the seminiferous epithelium of 6-day-old rats, Sertoli cells as well as a few gonocytes were aligned along the basement membrane of the tubules. After testicular fragments were cultured in plain medium in vitro, histological organization of the seminiferous cords appeared to be well preserved, and labeled Sertoli cells were occasionally seen (Fig. 3AGo). In the presence of FSH, the labeling ratio of the seminiferous cord was increased (Fig. 3BGo and Table 1Go). When fragments were incubated with FSH and GDNF in combination, the diameter of the seminiferous cord was increased compared with that with plain medium control, and both the total number and the labeling ratio of Sertoli cells were markedly increased (Fig. 3CGo and Table 1Go).



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  		Figure 3. Photomicrographs of histological sections of testicular fragments from 6-day-old rats (x1000) after 3 days of culture in the presence of plain medium (A), 200 ng/ml FSH (B), and 100 ng/ml GDNF plus 200 ng/ml FSH (C) and then was labeled with BrdU for 5 h. Sections were immunostained with anti-BrdU antibody and counterstained by hematoxylin and eosin Y as described inMaterials and Methods. Arrow, Labeled Sertoli cell; black arrowhead, labeled gonocyte; white arrowhead, unlabeled gonocyte.

 

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  		Table 1. Number and labeling index of Sertoli cells and gonocytes in cultured testicular fragments

 
The gonocytes were confirmed by both hematoxylin-eosin stain and c-Kit immunostain. As shown in Table 1Go, the number of BrdU-positive gonocytes also increased after GDNF and/or FSH treatment.

Inhibition of the stimulatory effects by anti-GDNF and anti-RET antibodies
To further characterize the stimulatory effect, antibodies to GDNF and RET (the latter was recently defined as the ß-chain of GDNF receptor) were added to culture medium (Fig. 4Go). Addition of anti-GDNF antibody to the medium exerted no apparent effect on incorporation of [3H]thymidine into testicular fragments cultured for 3 days. In contrast, in the medium containing 30 ng/ml GDNF and 200 ng/ml FSH, the incorporation was markedly inhibited by both 4 µg/ml anti-GDNF and anti-RET antibodies (decrease from 264% to 165% and 203%, respectively; P < 0.01).



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  		Figure 4. Inhibition effects of anti-GDNF and anti-RET antibodies (4 µg/ml) on FSH-dependent DNA synthesis in testicular fragments from 6-day-old rats. Fragments were cultured for 3 days in the presence or absence of FSH (200 ng/ml) and recombinant GDNF (30 ng/ml) and then labeled with [3H]thymidine for 5 h. Incorporation of [3H]thymidine into DNA was measured as described in Materials and Methods. Each bar is represented as a percentage of the control value (mean ± SEM; n = 4). Statistical differences were analyzed by Student’s t test. *, P < 0.05; ***, P < 0.001.

 
Effect of combined treatment of GDNF with LH or testosterone
To confirm the specificity of effect, LH (100 ng/ml) and testosterone (10-7 or 10-6 M) were added to the medium in the presence or absence of 30 ng/ml GDNF. No increase in the incorporation of [3H]thymidine was observed even in testicular fragments concomitantly treated with GDNF (Fig. 5Go, A and B).



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  		Figure 5. Effects of testosterone (T) and LH on DNA synthesis in testicular fragments from 6-day-old rats. Fragments were cultured with testosterone at concentrations of 10-7 and 10-6 M (A) or 100 ng/ml LH (B) for 3 days in the presence or absence of GDNF (30 ng/ml). Data are expressed as percentages of control values (mean ± SEM; n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sertoli cells are reported to be stimulated by FSH during fetal and perinatal life (21, 22, 23, 24), but participation of factors other than FSH in the proliferation cannot be excluded. In line with this, Boitani et al. (25) reported that activin stimulated Sertoli cell proliferation in the presence of FSH in early postnatal rat testis. In the present study we demonstrated for the first time that GDNF, which is a strong neurotropic factor and expressed in testis, played a role in the proliferation of Sertoli cells.

It was shown in the present study that GDNF alone had no effect, but when coadministered with FSH, it dramatically stimulated the proliferation of Sertoli cells. This is very similar to the effect of activin, which is another member of the transforming growth factor-ß superfamily (25). The stimulation effect was inhibited by either anti-GDNF or anti-RET antibody, indicating that the effect was specific. FSH, the glycoprotein hormone secreted from gonadotropes in the pituitary after stimulation of GnRH, is known to directly act on Sertoli cells by binding to their specific membrane receptors with cAMP as a signal transducer. However, the role of GDNF in cell proliferation synergistically with FSH remains to be clarified.

To distinguish Sertoli cell from gonocytes, we used anti-c-Kit antibody immunostain as well as hematoxylin-eosin stains. Rat gonocytes migrate to the basement membrane during the first postnatal week, a change in position that is crucial for their survival. Gonocytes express c-Kit in newborn rat and develop the ability to migrate in Sertoli cell-gonocyte coculture (26). We investigated the labeling ratio of gonocytes (percentage of c-Kit-BrdU-positive cells of the total gonocytes). Both the number and labeling ratio of gonocyte tended to increase after GDNF and/or FSH treatment. GDNFR-ß was reported to be highly expressed in gonocytes (19), and recently, the presence of FSH and FSH receptor in both Sertoli and germ cells was reported (27). These findings suggest that the increase in the labeling ratio of gonocytes may directly result from GDNF and FSH and indirectly result from newly proliferated Sertoli cells.

There are many hormones other than FSH that participate in the differentiation and development of the testis. Testosterone is secreted from Leydig cells located in the interstitial of the testis. LH is another glycoprotein hormone that is secreted from anterior pituitary gland and stimulates Leydig cells to secrete testosterone. The testis is also stimulated by CG from the placenta to produce moderate quantities of testosterone during fetal development and a few weeks after birth. However, neither testosterone nor LH was proven to have a synergistic effect with GDNF on the division of Sertoli cells in our present experiment.

Our data obtained from organ culture of 6-day-old rat testis strongly suggest that GDNF, which is maximally expressed during the period of most vigorous expansion of the Sertoli cell population, plays an important role in cooperation with FSH in Sertoli cell proliferation in vivo during early postnatal life.


   Acknowledgments
 
The authors thank Dr. Yoshitake Nishimune, professor at the Institute for Microbial Diseases, Osaka University, and Dr. Kiyoshi Imai, vice-director of Hatono Research Institute, for helpful discussion and advice.

Received September 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals