BIOLOGY OF REPRODUCTION 48, 40-45 (1993)
Localization of Transforming Growth Factor 1 and [12 during Testicular
Development in the Rat'
KATJA J. TEERDS 2'3 and JENNIFER H. DORRINGTON 4
Department of Cell Biology and Histology,3 Faculty of Veterinary Medicine, University of Utrecht
Utrecht, The Netherlands
Banting & Best Department of Medical Research,4 University of Toronto, Toronto, Ontario, Canada
ABSTRACT
The transforming growth factor s (TGFBs) affect the metabolic activities of the somatic cells of the testis. Sertoli cells,
peritubular/myoid cells, and germ cells contain mRNA for TGFO, and/or TGF~2,. We have used immunohistochemical techniques
to determine, in vivo, when TGFO, and TGFO2 are present in the rat testis during development and have identified the precise
localization of these growth factors.
The most pronounced changes in TGFP immunoreactivity occurred during spermatogenesis. TGF[, predominated in spermatocytes and early round spermatids, but as the spermatids elongated around stages VIII-IX of the cycle, the TGF,I levels
declined. TGF2, was undetectable in spermatocytes and early round spermatids, but as spermiogenesis progressed, around stages
V-VI, the spermatids rapidly acquired TGFP,. The intense staining for TGF,32 was maintained as the spermatids elongated. TGF, 1
immunoreactivity was detected in Sertoli cells throughout testicular development. TGF[3, was found in fetal Sertoli cells, but
became undetectable rapidly after birth. In fetal animals the Leydig cells contained TGFO, and TGF2,; after birth TGFO, persisted
whereas TGFO, became undetectable in the Leydig cells. Prior to puberty, TGF[, and TGF2, were absent in a portion of the
Leydig cells; when the adult stage was reached, TGFO, was no longer detectable and TGF[, staining was faint to absent.
In conclusion, our novel findings show that TGF, and TGF{ 2 are present in vivo in testicular cells at clearly defined stages
of their differentiation. From the changes in the pattern of localization of these peptides, we conclude that they are strictly
regulated in the somatic cells and the germ cells. An important outcome of the precise localization studies in vivo is that they
have pointed to the stages of spermatogenesis at which TGFP is likely to play a physiological role in growth and/or differentiation.
INTRODUCTION
The gonadotropins LH and FSH are essential endocrine
regulators of the normal physiological functions of the testis [1]. However, because the coordination of events that
establishes the repeating cycle of spermatogenesis proceeds in the presence of relatively constant levels of circulating gonadotropins, it has been suggested that factors
other than gonadotropins are also involved. The precise
regulation of Sertoli cell and Leydig cell proliferation and
development in the fetal and prepubertal testis, and the onset and maintenance of germ cell proliferation in the prepubertal and adult animal, have implicated growth-promoting factors synthesized in the testis as mediators or
modulators of gonadotropin action. A number of locally
produced peptide factors that affect the growth and metabolic activities of testicular cell types have been identified
in the testis. These factors include insulin-like growth factor
I (IGF I) [2] transforming growth factor ot (TGFct) [3-5],
transforming growth factor 13(TGF3) [6-8], interleukin 1
[9], and seminiferous growth factor [10].
The TGF[3s are polypeptide growth factors that are multifunctional regulators of both growth and development in
many different tissues. To date, five different forms of TGF[3
have been identified, and three have been reported to be
present in the testis [11-13]. In the mouse testis [14] and
in the rat testis [15], TGFP,3 and TGF3 mRNAs have been
shown to be expressed in Sertoli cells and peritubular/myoid
cells throughout testicular development. TGFPf2 mRNA appears to be expressed predominantly in the prepubertal
testis [6, 15]. In the adult mouse testis, TGFP,3 mRNA expression has also been found in germ cells [14]. Furthermore,
in vitro studies have shown that both Sertoli cells and peritubular/myoid cells secrete a TGFf3-like factor as assessed
by binding to TGF3 receptors and from its bioactivity [6].
The cells of the testis respond to TGF3 in a number of
ways: Leydig cells respond by increased DNA synthesis [16]
and changes in steroidogenesis [7,17-19], FSH-induced
aromatase activity in rat Sertoli cells is inhibited [20], and
interactions between Sertoli cells and peritubular/myoid
cells are promoted in culture [6].
The responses of testicular cells depend upon the local
concentrations of TGF3 [18]. To identify the cell populations in the testis that contain significant levels of TGF,31
and TGF,32 during testicular development in vivo, we have
localized these growth factors in fetal, neonatal, prepubertal, and adult rat testis using specific antibodies.
Accepted August 30, 1992.
Received June 18, 1992.
'The research of KJ.T. has been made possible by a fellowship of the Royal
Netherlands Academy of Arts and Sciences.
2
Correspondence: Katja J. Teerds, Ph.D., Department of Cell Biology and Histology, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80.157, 3508 TD
Utrecht, The Netherlands. FAX: 31-30-516853.
MATERIALS AND METHODS
Antibodies
The anti-TGFP3 polyclonal antibody-anti-LC-(1-30)-was
raised against a synthetic peptide corresponding to the first
40
LOCALIZATION OF TGF {3DURING TESTICULAR DEVELOPMENT
30 amino acids of mature TGF31 and was purified by Flanders et al. [21]. The TGFP,3 antibody principally recognizes
intracellular TGFP,1 at the site of synthesis and does not
show any cross-reactivity with TGF3 2 [21, 22]. The specificity of the immunohistochemical staining for TGF,3' using
this antibody has been demonstrated in organs of mice [22]
and in immature and adult rat ovaries [23, 24]. The TGF32
polyclonal antibody was raised against a synthetic peptide
corresponding to the first 29 N-terminal amino acid residues of TGFi32 by Van den Eijnden-Van Raaij et al. [25]. This
anti-TGFP 2 peptide antiserum did not show any cross-reactivity with TGF3I and completely neutralizes the growthinhibitory effect of TGF3 2 on mink lung carcinoma
(ML-CC164) cells. It is specific for TGF,32 in several immunological assays, including ELISA, and in immunoblotting and immunofluorescent experiments. The specificity of
the immunohistochemical staining for TGF32 has been
demonstrated in P19 embryonic carcinoma cells, in the
TGFi 2-producing African green monkey epthelial cell line
BSC-1 [25], and in the rat ovary [24]. Biotinylated horse antirabbit immunoglobulin G, obtained from Vector Laboratories (ABC-peroxidase staining kit elite, Vector Labs, Burlingame, CA), was used as a second antibody.
Treatment of Animals
Adult (12 wk old), (pre)pubertal (21-35 days old), neonatal (7 days old), and fetal (Days 19-21 postcoitus) male
Wistar rats were used. Rats were killed by cervical dislocation or by CO 2 asphyxiation without pretreatment.
Immunohistochemical Staining
Testis tissue was either immersion-fixed (fetal testes and
7-day-old testes) or perfusion-fixed (21-day-old testes and
adult testes) in 4% paraformaldehyde, followed by post-fixation in Bouin's solution consisting of 0.9% picric acid, 9%
formaldehyde, and 5% acetic acid. The tissue was dehydrated and embedded in paraffin. The immunostaining
technique was performed on 5-Rm sections according to
Teerds and Dorrington [24]. Briefly, sections were first deparaffinized, and endogenous peroxidase was blocked with
1% H2 02 in methanol; sections were then rinsed with 0.01
M Tris-buffered-saline (TBS, pH 7.4) and incubated with 0.1
M glycine in TBS. Subsequently the slides were incubated
with hyaluronidase (1 mg/ml; Sigma Chemical Co., Poole,
Dorset, UK) in sodium acetate buffer. After this step, the
slides were washed with TBS, blocked with 5% normal horse
serum, and incubated overnight at 4C with the polyclonal
antibody against TGFI (gift from Dr. K.C. Flanders) or the
polyclonal antibody against TGF,32 (gift from Dr. AJ.M. Van
den Eijnden-Van Raaij). After this incubation, the slides were
rinsed with TBS and treated with a biotinylated horse antirabbit polyclonal antibody (Vector Labs). Slides were again
washed in TBS and subsequently incubated with the components A and B of the ABC staining kit (Vector Labs). Bound
41
antibody was visualized by incubating the slides with a 0.6mg/ml solution of 3,3'-diaminobenzidine tetrachloride
(Sigma) and 0.03% H20 2 in TBS for 2-5 min. The slides
were counterstained with hematoxylin.
Control experiments were carried out for all the age
groups, and representative data are shown in Figure 1, A
and B. In these control experiments the polyclonal TGF3
antibodies were omitted from the procedure, or preimmune rabbit serum was employed instead of the first antibodies. In these experiments, no immunostaining was found
in the interstitial and tubular tissue (Fig. 1A). Moreover, incubation with an immunoglobulin of a related IgG class did
not result in any detectable immunostaining (Fig. 1B).
RESULTS
Localization of TGFP, and TGFP2 Immunoreactivity
during Testicular Development
In the fetal testis, the Sertoli cells in the primitive seminiferous tubules stained positively for both TGFP, and for
TGF3 2, but staining was not detected in gonocytes (Fig. 2,
A and B). In the interstitial compartment, the fetal Leydig
cells stained positively for both TGFP, and TGF 2 (Fig. 2,
A and B). The Leydig cells were identified by their large
spherical nuclei with characteristic distribution of heterochromatin.
In 7-day-old neonatal testes, the gonocytes have differentiated into spermatogonia, which are located between the
Sertoli cells close to the basal lamina of the seminiferous
tubule. The Sertoli cells contained detectable amounts of
TGF,13 (Fig. 2C), whereas TGF,32 immunostaining was faint
to undetectable (Fig. 2D). Also in the Leydig cells at this
stage, TGFP, predominated over TGF,32 (Fig. 2, C and D).
In sections of testis of 21-day-old rats, Sertoli cells continued to contain immunoreactive TGFP,. Because meiosis
is initiated during the prepubertal period, the germ cells,
spermatocytes, and round spermatids acquired immunoreactive TGFP, (Fig. 2E). Immunoreactivity for TGF,32 was
not detected within the seminiferous tubules of these animals (Fig. 2F). Immunoreactivity for TGF, and TGF32 was
found in the Leydig cells; however, the proportion of positive cells was variable. Leydig cells that were located in
clusters usually stained positively, whereas isolated Leydig
cells were more often negative (Fig. 2, E and F). During
further testicular development, the number of TGFfP, and
TGF,3 2-positive Leydig cells decreased, and they became
negligible at the age of 35 days.
In the adult testis, immunoreactivity for TGFP, persisted
in the Sertoli cells. Whereas spermatocytes and round spermatids contained detectable immunoreactivity for TGF3,,
this diminished and became undetectable as the spermatids
became elongated around stages VIII-IX of the cycle of the
seminiferous epithelium (Fig. 1, C and D). The most striking changes in TGF,32 immunoreactivity were displayed
42
TEERDS AND DORRINGTON
FIG. 1. Immunohistochemical localization of TGFP, and TGF3 2 in the adult rat testis. A) Control section incubated with preimmune serum instead of
primary antibody. B) Control section incubated with an antibody of a related TgG class. C and D) Sections incubated with TGF31 antibody. E and F) Sections
incubated with TGFP2 antibody. Sertoli cells are indicated by arrowheads, Leydig cells by arrows, spermatocytes by small asterisks, spermatids by large
asterisks. The stages of the cycle of the seminiferous epithelium are indicated by Roman numerals. A-D: x 475 (bar = 21 lam); E and F: x 360 (bar =
27.8 im).
during spermiogenesis. TGF3 2 was not detectable in Sertoli
cells or in germ cells prior to meiosis, nor in early round
spermatids. As the round spermatids reached stages V-VI of
the cycle, they rapidly acquired immunoreactive TGF3 2 so
that during the process of elongation the staining was very
intense (Fig. 1, E and F). Staining for TGFP, and TGFP2 was
faint or absent in Leydig cells and other interstitial cells in
the adult testis (Fig. 1, C-F).
LOCALIZATION OF TGF
DURING TESTICULAR DEVELOPMENT
43
FIG. 2. Immunohistochemical localization of TGFp, and TGF,3 2 in the developing rat testis. A, C, and E) Sections incubated with TGF,3 antibody. B,
D, and Fl Sections incubated with TGF02 antibody. A and B show serial sections of fetal testis tissue in which gonocytes are indicated by asterisks. Those
gonocytes that are completely in focus clearly show the absence of cytoplasmic immunostaining around their nucleus. C and D show sections of neonatal
(7-day-old) testis, and E and F of prepubertal (21-day-old) testis tissue. In A-F, Sertoli cells are indicated by arrowheads and Leydig cells by arrows, while
spermatocytes and spermatids in E and F are indicated by asterisks. x 950 (Bar = 10.5 pLm).
DISCUSSION
Since most cells produce TGFP when they are maintained in culture [13], the extrapolation of in vitro findings
and the significance for the in vivo situation is tenuous. Sertoli cells, peritubular/myoid cells, and germ cells contain
mRNA for TGFP, and the somatic cells secrete TGFI3-like
bioactivity [6, 14]. These in vitro studies show the potential
of the cells in a chemically defined medium, but give little
information about their expression in vivo in an environment influenced by cell-cell interactions and endocrine,
44
TEERDS AND DORRINGTON
paracrine, and autocrine factors. In the present study, we
have used immunohistochemical techniques to determine
when TGFP,3 and TGF32 are present in the testis during
development and have identified the precise localization of
these growth factors. These studies have revealed clearly
defined patterns of localization of TGF3 in somatic cells
and in germ cells as they progress through spermatogenesis.
The most pronounced changes in the levels of TGF{ immunoreactivity occurred during spermatogenesis. A clear
demarcation in the localization of TGFP,3 and TGF32 was
apparent during germ cell development. TGF[3 predominated in spermatocytes and early round spermatids, but as
the spermatids differentiated and elongated, around stages
VIII-IX of the cycle of the seminiferous epithelium, TGF[I3
immunostaining decreased and became undetectable. Conversely, TGF32 was undetectable in spermatocytes during
the prophase of meiosis and after the formation of early
round spermatids; but as spermiogenesis progressed, around
stages V-VI of the cycle, the cells rapidly acquired very high
levels of TGFP,2 . The intense staining for TGF 2 was maintained as the spermatids elongated. Previously, Skinner and
Moses [6] showed that freshly isolated germ cells released
from seminiferous tubules of adult rat testes during a short
incubation do not contain detectable levels of mRNA for
TGF{I. Since the population of germ cells retrieved by this
method would contain predominantly the more differentiated spermatids, our observations on the localization of
TGFP, are consistent with the mRNA detection analysis.
Watrin et al. [14] have also shown that spermatocytes and
round spermatids from the adult mouse testis express TGFP,
mRNA.
TGF{3 and TGFI 2 are different gene products with similar functional activities; nevertheless, in some cells differential effects of the growth factors have been observed
[11, 13]. The marked transition from TGFP, to TGF3 2 during spermiogenesis suggests that TGFP, and TGFP, exert
different effects during germ cell development. Since growth
factors in general diffuse short-range to exert their effects
on neighboring cells, two physiological roles of TGF in
germ cells can be envisaged. First, the TGFI3 in germ cells
may be secreted and subsequently activated to act in an
autocrine fashion to promote the differentiation of the germ
cells themselves. Second, the TGFPi released from the germ
cells at different stages of development may provide a means
by which they interact and communicate with neighboring
regions of Sertoli cells with which they are in intimate contact. It has been suggested that TGF3 is a chemotactic agent
in the interaction between Sertoli cells and peritubular/
myoid cells [6]. Similarly, TGFP released by germ cells may
provide a chemotactic agent to attract and maintain the Sertoli cell cytoplasmic extensions that surround them
throughout their development.
Sertoli cells from prepubertal rat [6] and pig [8, 20, 2628] testes express TGF{3 mRNA. Our immunohistochemical
technique showed that TGFt was not restricted to Sertoli
cells during the prepubertal stages but was detected in Sertoli cells throughout development in the rat testis. Both
TGFP, and TGF,32 were present in Sertoli cells from fetal
animals, but subsequently TGF32 immunostaining decreased rapidly after birth and was no longer present in
Sertoli cells of immature animals, whereas TGFPI3 predominated throughout adult life. The observation that TGF32
immunoreactivity declined in Sertoli cells expands recent
findings by Mullaney et al. [15], who reported in a preliminary study that, with the initiation of active germ cell growth,
the mRNA expression of TGF{2 in Sertoli cells rapidly decreases to undetectable levels. It is known that TGF] inhibits the actions of FSH on Sertoli cells, such as aromatase
activity, by attenuating cAMP levels [20, 26]. Sertoli cell functions may, therefore, be modulated by the TGF3 synthesized by both Sertoli cells and by the germ cells that are
associated with Sertoli cells at different stages of the cycle
of the seminiferous epithelium.
In the interstitial compartment of fetal animals, the Leydig cells contained TGF, 1 and TGF,32. After birth, TGF{I3
persisted whereas the levels of TGF{2 immunoreactivity declined. Prior to puberty TGFP,f and TGF 2 were absent in
a portion of the Leydig cells, and when the adult stage was
reached, TGFP, was no longer detectable and TGF3 2 levels
were low. TGF[3 immunoreactivity declined in Leydig cells
at the time at which TGFot immunostaining becomes intense [5]. The inverse relationship between TGF, and TGFot
in the Leydig cells and the switch from TGF3 to TGFoa before puberty may be in response to the steroidogenic requirements of the seminiferous epithelium; TGF{ inhibits
androgen production by Leydig cells [17, 19] and may play
a physiological role in this regard prior to puberty. TGFta,
on the other hand, promotes steroidogenesis in Leydig cells
[29], and it furthermore interacts with IGF-I and LH to promote Leydig cell proliferation, which occurs prior to puberty [16, 30]. The stimulatory effects of TGFo, IGF-I and
LH on Leydig cell proliferation can be partially inhibited by
TGF3 (Khan and Dorrington, unpublished observations).
Therefore, the decrease in the levels of TGF3 from the Leydig cells may allow these cells to increase their secretion
of steroids and to stimulate their rate of proliferation.
In summary, our novel findings show that TGF{I and
TGF,32 are present in vivo in testicular cells at clearly defined stages of their differentiation. Furthermore, from the
pattern of localization of TGFI3 and TGF32 immunoreactivity, it can be concluded that the levels of these peptides
are strictly regulated in the somatic cells and the germ cells.
An important outcome of these precise localization studies
in vivo is that they have pointed to the stages of spermatogenesis at which TGF[ is likely to play a physiological
role in growth and/or differentiation.
ACKNOWLEDGMENTS
The authors thank Dr. K.C. Flanders (Laboratory of Chemoprevention, National
Cancer Institute, National Institutes of Health, Bethesda, MD) and Dr. A.J.M. Van den
LOCALIZATION OF TGF 1i DURING TESTICULAR DEVELOPMENT
Eijnden-Van Raaij (Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands) for the TGFP,l and TGFP,2 polyclonal antibodies. We
are grateful for the skillful technical assistance of Mrs. M. de Boer-Brouwer, Mr. H.
van Oudheusden, Mr. A. Tieleman; and to Mr. AN. Van Rijn and Mr R. Scriwanek
(Department of Cell Biology, Medical School, University of Utrecht, The Netherlands)
for preparing the photomicrographs.
REFERENCES
1. Steinberger E. Hormonal control of mammalian spermatogenesis. Physiol Rev
1971; 51:1-36.
2. Handelsman DJ, Spaliviero JA, Scott CD, Baxter RC. Identification of insulin-like
growth factor-I and its receptors in the rat testis. Acta Endocrinol 1985; 109:543549.
3. Holmes SD, Spotts G, Smith RG. Rat Sertoli cells secrete a growth factor that
blocks epidermal growth factor binding to its receptor. J Biol Chem 1986; 261:40764080.
4. Skinner MK, Takacs K, Coffey RJ.Transforming growth factor-a gene expression
and action in the seminiferous tubule: peritubular cell Sertoli cell interactions.
Endocrinology 1989; 124:845-854.
5. Teerds KJ, Rommerts FFG, Dorrington JH. Immunohistochemical detection of
transforming growth factor-a in Leydig cells during the development of the rat
testis. Mol Cell Endocrinol 1990; 69:R1-R6.
6. Skinner MK, Moses HL. Transforming growth factory gene expression and action
in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Mol Endocrinol 1989; 3:625-634.
7. Avallet O, Perrard-Sapori MH, Vigier M, Skalli M, Saez JM. Comparison of the
effects of transforming growth factor-I and porcine Sertoli cell conditioned medium on porcine Leydig cell function. In: Cooke BA, Sharpe RM (eds.), Molecular
and Cellular Endocrinology of the Testis, vol. 50. New York: Raven Press; 1988:
319-324.
8. Avallet O, Vigier M, Albaladejo V, de Cesaris P, Saez JM. Transforming growth
factor p gene expression in cultured porcine Sertoli and Levdig cells: effects of
hormone and growth factors. In: Proceedings of the 6th European Workshop on
Molecular and Cellular Endocrinology of the Testis; 1990; Abstract D13.
9. Khan SA, Schmidt K,Hallin P, Di Pauli R, De Geyter C, Nieschlag E. Human testis
cytosol and ovarian follicular fluid contain high amounts of interleukin-l-like
factor(s). Mol Cell Endocrinol 1988; 58:221-230.
10. Bellvd AR, Zheng W. Growth factors as autocrine and paracrine modulators of
male gonadal functions. J Reprod Fertil 1989; 85:771-793.
11. Sporn MB, Roberts AB, Wakefield LM, De Crombrugghe B. Some recent advances
in the chemistry and biology of transforming growth factor-3. J Cell Biol 1987;
105:1039-1045.
12. Roberts AB, Sporn MB. Transforming growth factor-Il. Adv Cancer Res 1988; 51:107145.
13. Roberts AB, Sporn MB. The transforming growth factor-is. In: Sporn MB, Roberts
AB (eds.), Peptide Growth Factors and Their Receptors I. Berlin, New York, Heidelberg, London, Paris, Tokyo, Hong Kong: Springer Verlag; 1990: 419-472.
14. Watrin T, Scotto L,Assoian RK, Wolgemuth DJ. Cell lineage specificity of expression of the murine transforming growth factor-13 and transforming growth factor-1, genes. Cell Growth &Differ 1991; 2:77-83.
45
15. Mullaney BP, Glenn B, Skinner MK Cell-cell interactions in the testis: the role
of transforming growth factors. Biol Reprod 1991; 44(suppl. 1): Abstract 520.
16. Khan S, Teerds K, Dorrington J. Growth factor requirements for DNA synthesis
by Leydig cells from the immature rat. Biol Reprod 1992; 46:335-341.
17. Lin T, Blaisdell J, Haskell JF. Transforming growth factor-I inhibits Leydig cell
steroidogenesis in primary culture. Biochem Biophys Res Commun 1987; 146:387394.
18. Benahmed M, Sordoillet C, Chauvin MA, De Peretti E, Morera AM. On the mechanisms involved in the inhibitory and stimulatory actions of transforming growth
factor-I on porcine testicular steroidogenesis: an in vitro study. Mol Cell Endocrinol 1989; 67:155-164.
19. Bebakar WMW, Honour JW, Foster D, Liu YL, Jacobs HS. Regulation of testicular
function by insulin and transforming growth factor-I. Steroids 1990; 55:266-270.
20. Morera AM, Esposito G, Ghiglieri C, Chauvin MA, Hartmann DJ, Benahmed M.
Transforming growth factor , inhibits gonadotropin action in cultured procine
Sertoli cells. Endocrinology 1992; 130:831-836.
21. Flanders KC, Thompson NL, Cissel DS, Van Obberberghen-Schilling E, Baker CC,
Kass ME, Ellingsworth LR, Roberts AB, Sporn MB. Transforming growth-factorIl1: histochemical localization with antibodies to different epitopes. J Cell Biol
1989; 108:653-660.
22. Thompson NL, Flanders KC, Smith JM, Ellingsworth LR, Roberts AB, Sporn MB.
Expression of transforming growth factor-I, in specific cells and tissues of adult
and neonatal mice. J Cell Biol 1989; 108:661-669.
23. Hernandez ER, Hurwitz A, Payne DW, Dharmarajan AM, Purchio AF, Adashi EY.
Transforming growth factor- 1 , inhibits ovarian androgen production: gene
expression, cellular localization, mechanisms(s), and site(s) of action. Endocrinology 1990; 127:2804-2811.
24. Teerds KJ,Dorrington JH. Immunohistochemical localization of transforming growth
factor-I, and -P2 during follicular development in the adult rat ovary. Mol Cell
Endocrinol 1992; 84:R7-R13.
25. Van den Eijnden-Van Raaij AJM, Koomneef I, Slager HC, Mummery CL, Van Zoelen E. Characterization of polyclonal anti-peptide antibodies specific for transforming growth factor 12. J Immunol Methods 1990; 133:107-118.
26. Benahmed M, Cochet C, Keramides M, Chauvin MA, Morera AM. Evidence for a
FSH-dependent secretion of a receptor transforming growth factor-I-like material by immature Sertoli cells in primary culture. Biochem Biophys Res Commun
1988; 154:1222-1231.
27. Saez JM, Avallet O, Naville D, Perrard-Sapori MH, Chatelain PG. Sertoli-Leydig
cell communications. In: Ewing LL, Robaire B (eds.), Regulation of Testicular
Function. Signaling Molecules and Cell-Cell Communication. Ann NY Acad Sci
1989; 565:210-231.
28. Morera AM, Chauvin MA, Feige 3, Guillaumot P, Keramideas M, Mauduit C, Sordoillet C, Benahmed M. Interactions between systemic hormones and local growth
factors in the control of the testis function: the example of gonadotropins and
transforming growth factor 3. In: Isidori A, Fabbri A, Dufau ML. (eds.), Serono
Symposia Publ 70. New York: Raven Press; 1990; 191-2025.
29. Verhoeven G, Cailleau J. Stimulatory effects of epidermal growth factor on steroidogenesis in Leydig cells. Mol Cell Endocrinol 1986; 47:99-106.
30. Hardy MP, Zirkin BR, Ewing LL. Kinetic studies on the development of the adult
population of Leydig cells in the testis of pubertal rat. Endocrinology 1989; 124:762771.