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Institute for Hormone and Fertility Research (R.I., M.B., A.-N.S., R.D., N.H., E.K., A.K.M.), University of Hamburg, 22529 Hamburg, Germany; and Department of Human Anatomy (E.H., H.M.C.), University of Oxford, United Kingdom
Address all correspondence and requests for reprints to: R. Ivell, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany.
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Introduction |
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To characterize the expression and regulation of the RLF gene in more detail, we have extended our previous study in the mouse (3), examining both ovary and testis, and have raised specific antibodies against a recombinant mouse RLF protein. Additionally, we have examined RLF gene expression in differently stimulated mouse Leydig cell cultures and in the testes of both wild-type and hypogonadal (hpg) mice through different stages of development. These studies confirm the pattern of expression of RLF at the peptide level, and they support a constitutive regulation for the gene, at least in the adult Leydig cell, and its suitability as a differentiation marker.
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Materials and Methods |
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8
weeks) were obtained either from Charles River (NBLT wild-type strain),
from Jackson Laboratories (w/wv azoospermic mutant mice and
wild-type littermates), or from the breeding colony at the Department
of Human Anatomy, Oxford University (homozygous hypogonadal
hpg mice and wild-type littermates, of different postnatal
ages, as indicated). Homozygosity of the latter strain was checked by
PCR of genomic DNA from tail clips, as previously described (11). To
induce pubertal differentiation in the hpg mice, these were
injected sc with 2 IU human CG (hCG) (Sigma, Deisenhofen, Germany)
twice daily, until death by cervical dislocation, at the times
indicated.
RNA was extracted from the frozen tissues, pooled from at least three
different animals for each stage (except liver: one sample only), with
a modification of the single-step procedure of Chomczynski and Sacchi
(12) using the RNA-Clean reagent (AGS, Heidelberg, Germany). Northern
hybridization was performed on 1.2% agarose gels using the
formaldehyde/morpholinoproprionic sulfate procedure (13), blotting the
electrophoresed RNA to nylon membranes (Nytran, Schleicher and
Schüll, Dassel, Germany) by overnight capillary transfer.
Hybridization probes specific for mouse RLF, mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or ß-actin
transcripts (specific activity >5 x 108 cpm/µg)
were radiolabeled by random primer extension in the presence of
[
32P]deoxycytidine triphosphate (Amersham-Buchler,
Braunschweig, Germany), as previously described (3). Where indicated,
Northern blots were quantified by phosphorimager (Storm 840; Molecular
Dynamics, Sunnyvale, CA). In situ transcript hybridization
was performed exactly as described elsewhere (14) using complementary
RNA probes derived by in vitro transcription in the presence
of digoxigenin-labeled uridine 5'-triphosphate (Boehringer-Mannheim,
Mannheim, Germany).
RT-PCR
Single-stranded cDNA was prepared by RT from total RNA, exactly
as described previously (6). Five microliters of this cDNA were used to
prime the PCR reaction, which, after denaturation for 3 min at 95 C,
used standard conditions of denaturing for 0.5 min at 95 C, annealing
for 1 min at 65 C, and extension for 1 min at 72 C, for a total of 30
cycles. Five microliters of the resulting reactions were
electrophoresed on 2% agarose gels, and the PCR products were
visualized by UV irradiation of the ethidium bromide-stained products.
For detection of RLF-specific transcripts, two oligonucleotide primers
were prepared from the mouse RLF cDNA sequence (3) (forward:
5'-CGCGCCGCTGCTACTGATGC; reverse: 5'-GGGCCTGTGGTCCTTGCTTACTGC) to yield
a 495-bp DNA fragment spanning the intron 1 splice junction.
Additionally, the RLF-specific PCR products were transferred to
nylon membranes and were hybridized to an internal antisense
41-mer (5'-GACCCAGCGCTAGACCCGCAGCTTCCTCGGCAGGCTTCTCA), end-labeled
using [
32P]ATP and T4 polynucleotidyl
kinase. Radioactive products were visualized by exposure to
autoradiography film (Kodak X-Omat R, Eastman-Kodak, Rochester, NY) .
As a control for the integrity of the extracted RNA, primers were also
prepared from the mouse GAPDH sequence (15) (forward:
5'-GGGGTGAGGCCGGTGCTGAGTA; reverse: 5'-TTGGGGGCCGAGTTGGGATAGG) to yield
an 818-bp PCR fragment. PCR parameters were exactly as above. The
described experiments were repeated twice, with similar results.
Antibody preparation
The amino acid sequence of mouse RLF is only 51% homologous to
that from pig or human. Therefore, the complete mouse RLF coding region
was subcloned into the expression vector pGEX-6T as part of a
glutathione-S-transferase (GST) fusion protein construct (14).
Expression of the resultant fusion protein in Escherichia
coli and purification of the protein by means of Ni-Sepharose
chelation chromatography, taking advantage of the hexahistidine
cassette also encoded by this expression vector, were exactly as
described previously for the endozepine-like peptide (14). The purified
GST-RLF fusion protein was then used to raise polyclonal antisera in
six rats, following a conventional immunization protocol (16). The
resulting antisera were tested by Western blotting against the
immunizing antigen. The serum with the highest apparent titer (no. R63)
was used for all subsequent immunological analyses.
Immunohistochemistry
Immunohistochemistry for RLF, using the rat polyclonal antiserum
no. R63, diluted 1:1000, followed a conventional double PAP-ABC
combination protocol, as described elsewhere (14). As negative
controls, the primary antiserum was replaced by the preimmune rat serum
from the same animal that yielded antibody no. R63. Because the
antibody was raised against a GST-RLF fusion protein, the immune
reaction was also performed in the presence of excess GST protein
(Sigma). Furthermore, an antibody raised against a similar
bacteriogenic GST protein, made using the same expression vector but
without a fusion insert (courtesy of Dr. Wolfgang Northemann, Freiburg,
Germany), was tested in the same protocol and shown not to react in the
mouse testis and ovary. Where indicated, sections were counterstained
with hemalaun to emphasize cell nuclei and were mounted in Faramount
aqueous mounting medium (Dako Diagnostika, Hamburg, Germany). To
compare the expression pattern of mouse RLF in the ovary with that of
the related hormone relaxin (RLX), immunohistochemistry was also
performed on successive sections, using a polyclonal rabbit antirat
relaxin antibody (no. 267, courtesy of Dr. O. David Sherwood, Urbana,
IL), which has been used specifically to identify relaxin in the mouse
ovary (17).
Cell culture
Primary cultures of adult mouse Leydig cells were prepared, as
described elsewhere (18), by mechanical disintegration of the testes,
followed by Percoll gradient centrifugation. Cells were plated out in
DMEM (Gibco, Eggenstein, Germany) containing 1%
penicillin/streptomycin (ICN Biomedicals, Eschwege, Germany), 1%
L-glutamine, 20 mM HEPES, and 10% FCS (Gibco).
After 12–16 h, this medium was replaced by similar medium, but with
the FCS substituted by 0.1% BSA, and supplemented with either 2 or 5
ng/ml hCG (Boehringer-Mannheim), 0.2 µM rat
atrionatriuretic peptide (ANP; Saxon Biochemicals, Bissendorf,
Germany), 0.2 or 0.4 µg/ml testosterone (Sigma), or 1 mM
8Br-cAMP (Boehringer-Mannheim), as indicated in the figure legends; and
incubation continued for an additional 24 h, unless otherwise
indicated. At the end of this time, medium was removed and cells were
lysed directly in RNA-Clean for RNA extraction and subsequent Northern
hybridization. Similar experiments were also performed using the mouse
Leydig tumor cell-line MA10 and, initially, Waymouth MB 752/1 medium
(ICN Biomedicals) additionally containing 1% penicillin/streptomycin,
1% L-glutamine, 15 mM HEPES, 1.12 g/liter
NaHCO3, and 10% horse serum (Gibco). After 24 h
incubation, the culture medium was replaced by a similar medium, but
lacking the horse serum and containing supplementary stimulatory
agents, as above.
The extracted RNA was analyzed by Northern hybridization for RLF- and GAPDH-specific transcripts, as described above. For RLF transcripts in primary Leydig cells, autoradiographic exposure was for 30 min using Biomax film (Eastman-Kodak); for the the RLF transcripts in MA10 cells, exposure was for 24 h using the same film. GAPDH transcripts required exposures of 48 h. All experiments were repeated at least twice, with similar results.
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). Whereas the testis, as
expected, shows the strongest signal for RLF mRNA, weak specific
signals also are detectable in most of the ovarian samples, with
further weak signals evident in the epididymis and prostate. The liver
proved to be consistently negative.
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, B and C), compared with
the control, using a sense probe (Fig. 2A). In the interstitial cells
of the testes of adult hpg mice (Fig. 2D), a specific
hybridization signal is not detectable under the conditions chosen here
to compare RLF gene expression in different mouse strains, but the
signal becomes weakly positive upon longer times of chromogen
development (not shown).
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) and
w/wv (not shown), but not in the hpg mouse
testes (Fig. 3C). Control sections of wild-type testes incubated with
preimmune serum (Fig. 3A) or with anti-GST antibodies (not shown) were
negative.
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), pregnancy
(Fig. 4, B–D), and postpregnancy periods (not shown). Not all corpora
lutea stain with equal intensity; particularly in the cycle, the same
ovary will have both weakly and highly expressing corpora lutea (Fig. 4A). There seems also to be positive staining for RLF in some stromal
cells (Fig. 4B). At no stage could RLF-immunoreactivity be detected in
follicles. Positive chromogen was also absent in the control, using
preimmune serum (Fig. 4E).
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, F–I) to determine whether the two related
peptides might be regulated by similar mechanisms. Although RLX shows
similar immunoreactivity only in corpora lutea, there is a clear
difference in the temporal pattern of expression. RLX positive cells
are absent in corpora lutea from the cycle and early pregnancy (Fig. 4, F and G), only appearing in corpora lutea of day 11.5 of pregnancy and
later (Fig. 4, H and I). There was no immunostaining in the control
using preimune serum (Fig. 4J).
RLF expression through development in the testes of wild-type and
hypogonadal (hpg) mice
Northern hybridization indicated that in wild-type mice the RLF
gene becomes up-regulated in the testis during puberty (Fig. 5A, lanes 3 and 4). Indeed,
phosphorimager analysis of similar Northern hybridizations indicates a
4-fold increase in RLF mRNA between days 20 and 30 (not shown).
However, there is nevertheless a hybridization signal already in
testicular RNA from prepubertal and neonatal mice (Fig. 5A, lanes 1 and
2). When a similar analysis is performed for the hpg mouse,
whose testes are maintained in a prepubertal status because of the lack
of gonadotropin stimulation, as expected, adult mice show only a very
weak RLF mRNA signal (Fig. 5A, lane 8). However, when looking at the
change through development, there seems to be a stronger signal in the
neonatal period (day 5) than later on (Fig. 5A, lane 5). A more
detailed Northern analysis of this immediate postnatal period in
wild-type testes (Fig. 6) shows a
significant decrease in RLF mRNA on day 3, followed by a recovery on
days 4 and 5, compatible with the differentiation switch associated
with the replacement of the fetal population of Leydig cells by a new
prepubertal population.
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). Concomitant
with the up-regulation of the RLF mRNA at puberty seen in Northern
hybridization, there are obvious signals indicating RLF gene
transcripts in the interstitial cells of wild-type mice, and in no
other testicular compartment (Fig. 7). At this time, the hpg
mouse testes are negative. In the younger hpg mice, the day
1 and day 5 testes show a pattern of specific in situ
hybridization for RLF mRNA that is quantitatively and qualitatively
indistinguishable from that in wild-type mice at this time, again only
in the interstitial cells.
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). Unlike
the distribution of the gene transcripts visualized by in
situ hybridization, the pattern of RLF protein expression is less
clear. In the perinatal period (days 1 and 5) for both wild-type and
hpg mice, there is apparently specific staining in all
compartments of the testis, including the seminiferous tubules (Fig. 8). Only sporadically is specific staining seen more strongly in the
cytoplasm of interstitial (presumably Leydig) cells. By day 10, this
pervasive staining seems maximal, at a time when the specific mRNA is
greatly reduced. There seems to be only very limited staining within
interstitial cells. However, by day 15, the pervasive staining is
virtually absent from the seminiferous tubules and only sporadic in the
interstitial compartment. By contrast, strong punctate staining for RLF
is seen in the interstitial cells only. This trend increases at day 20.
Here, there is now no pervasive staining at all, and all interstitial
cells exhibit a single dense spot of specific RLF staining. By day 30,
the punctate staining pattern gives way to a more evenly distributed
cytoplasmic distribution within the interstitial cells, very similar to
that seen in the adult mouse testis.
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)
showed that, already by day 3 of treatment, substantial amounts of RLF
mRNA are detectable, attaining wild-type levels (estimated by
phosphorimager) by day 12. Immunohistochemical analysis, however,
indicated that RLF protein translation is markedly delayed by
comparison (Fig. 3D). Only at day 6 can specific immunostaining be
seen, and then sporadically in a few cells only per interstitial
cluster. Even at 12 days, when the RLF mRNA level is high, still less
than half the interstitial cells stain positively for RLF. It is
interesting also to note that the staining is not homogenous within the
cells, as in adult wild-type Leydig cells (Fig. 3E), but seems to be
concentrated in a perinuclear, possibly Golgi-related spot within each
positive cell (Fig. 3F), even after 16 days of treatment. This punctate
pattern of RLF protein expression is very similar to that seen during
pubertal development in wild-type mouse testes (Fig. 8).
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). However,
treatment with a variety of effectors known to influence other Leydig
cell parameters under similar conditions has no effect whatever on the
level of RLF mRNA expression. Because MA10 cells themselves produce
progesterone, rather than testosterone, the latter was also applied to
these cells, to look for any possible autocrine influence of this
steroid, but without effect (Fig. 10).
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The w/wv azoospermic mutant mouse has a defective c-kit gene, the receptor for the Sertoli cell product Steel or stem cell factor (23). The lack of expression of this receptor on premeiotic spermatogonia leads to a complete arrest in spermatogenesis. The gonadal axis in these mice is intact, with the result that the Leydig cells seem to show a normal, wild-type phenotype and produce normal circulating levels of testosterone (24). Consequently, also RLF expression in Leydig cells conforms to the wild-type phenotype, with in situ hybridization showing intense signals for RLF mRNA and an adult-type immunohistochemical staining pattern (not shown). In contrast, the hpg mouse testes are azoospermic because of a defective gonadal axis caused by a deletion in the hypothalamically expressed gene for GnRH, with consequent gonadotropin deficiency (19, 25, 26). Testosterone levels are very low, and the lack of testicular RLF expression indicates that the Leydig cells in this mutant seem to be arrested in a prepubertal state of differentiation.
Analysis of the change in RLF mRNA levels through postnatal testicular development indicates, as shown previously (3), an increase through puberty concomitant with the establishment of the pituitary-gonadal axis. However, in both wild-type and hpg mice, there is a significant and equivalent expression of RLF mRNA in the immediate postnatal testis. Morphological analysis of testes at this time shows that the RLF mRNA is quite strongly expressed in Leydig cells, although one should be cautious in overinterpreting nonradioactive in situ hybridization signals with what is effectively not a highly quantitative methodology. Phosphorimager analysis of Northern hybridizations of total testis RNA shows that, in this immediate postnatal period (days 1–5), RLF mRNA represents about one tenth of that in the adult testis, where the Leydig cells represent a smaller proportion of the testis tissue. In the immediate postnatal testis, there would seem to be significant immunospecific staining for RLF within both the seminiferous tubules and the interstitial space. This pervasive, intratubular staining disappears in the wild-type testis by about day 15 of postnatal age. This is the time in early puberty when another kind of RLF immunoreactivity is detected, namely a punctate, highly intense staining within the interstitial cells only. This staining increases through puberty and leads finally to the more diffuse cytoplasmic staining exclusively in the Leydig cells of the adult wild-type mouse. Because it has not been possible to obtain Sertoli cells completely free of contaminating Leydig cells, it cannot yet be determined whether the RLF-like epitopes in the postnatal seminiferous tubules are caused by diffusion from the interstitial compartment or are the product of low-level local gene expression not detectable with the in situ hybridization protocol used. The marked presence of RLF protein in the perinatal period would suggest that RLF might have a function related to pre- or perinatal physiology, as well as in the adult testis.
Because it is the pituitary-gonadal axis, but not the testis per se, that is defective in the hpg mouse, application of a gonadotropin, such as LH or hCG, can lead to a recovery of Leydig cell function and, hence, testosterone production (19). hCG not only stimulates Leydig cell steroidogenesis but probably also induces differentiation. This is reflected by the hCG-dependent up-regulation of the RLF transcripts in RNA extracted from hpg testes, and the slow induction of immunopositive cells. Two points are of interest here. First, the appearance of RLF epitopes is very slow, by comparison with the up-regulation of the mRNA, suggesting an arrest in translation of the RLF mRNA in early Leydig cell differentiation. This view is supported by the punctate appearance of the immunostaining in these hCG-treated hpg mice, by comparison with adult wild-type mice, suggestive of a restriction of RLF epitopes to the Golgi complex, which is concentrated close to the nucleus in Leydig cells. Second, the appearance of RLF immunoreactivity occurs in a few cells only, scattered randomly throughout a Leydig cell cluster, numbers of positive cells increasing very slowly and randomly. This suggests that hCG-treatment is inducing a differentiation of preexisting Leydig cells stochastically, rather than encouraging proliferation of a particular phenotype, which would lead to an aggregated, clonal appearance of the RLF-positive cells. Nevertheless, the pattern of RLF expression induced by hCG in this model is very similar to that seen in wild-type mice going through normal puberty.
Mouse Leydig cells, in culture, also express the RLF gene. Primary adult Leydig cells express already high levels of RLF mRNA, but this expression cannot be influenced by hCG, ANP, or 8Br-cAMP (factors all known, under similar culture conditions, to have a major impact on Leydig cell signal transduction and steroidogenesis) (reviewed in Ref.27). This result also supports the view that the hCG-treatment of the hpg mice influences RLF expression by inducing Leydig cell differentiation, rather than simply by stimulating signal transduction within the existing Leydig cell population. The mouse Leydig tumor cell line, MA10, also expresses RLF mRNA, though at a lower relative level (3). In these cells, hCG and ANP are similarly without effect. Unlike primary adult Leydig cells, MA10 cells cannot produce testosterone. Because this steroid has been implicated in an autocrine-positive feedback system acting on androgen receptors within Leydig cells (reviewed in Ref.28), MA10 cells were treated also with exogenous testosterone. However, this parameter was also without effect, making it unlikely that testosterone could be the factor responsible for the up-regulation of RLF mRNA in vivo at puberty and in the hCG-treated hpg mice.
Taken together, all the data on RLF expression in the mouse testis support the view that the RLF gene is expressed in a differentiation-dependent, but constitutive, fashion within adult-type Leydig cells, and also in the preceding fetal population of Leydig cells, but with a different mode of expression. In the neonatal testis, fetal Leydig cells produce a moderate quantity of RLF mRNA in a fashion independent of the hpg mutation and, hence, of an active pituitary-gonadal axis. This mRNA gives rise to a protein product that is immediately released into the surrounding interstitial space, also possibly entering the seminiferous tubules, where RLF-mRNA is not detected by in situ hybridization, though low-level gene expression would not be detected with this technique. Intratubular staining persists up to about day 15, the time in mice when the effective Sertoli cell barrier of tight junctions is established (29), thus possibly supporting an interstitial origin for the intratubular RLF immunoreactivity in the perinatal period. Between days 5 and 15, the fetal Leydig cell population is replaced by an adult Leydig cell population, which is initially RLF-negative. This is the persistent Leydig cell population in the adult hpg mouse. RLF mRNA is expressed relatively quickly in these cells, as also in hpg mice upon hCG stimulation. However, RLF protein only seems to accumulate slowly, initially with a punctate pattern of subcellular staining, possibly restricted to the Golgi complex. Only toward the end of puberty, at about day 30 and onward, does the more homogenous (but exclusively Ledyig) cell pattern of staining prevail. The expression of RLF in these adult-type Leydig cells seems to offer a good indicator for the differentiation status of these cells.
Within the ovary, RLF immunoreactivity is expressed in the majority of luteal cells, both in the cycle and throughout pregnancy. The specificity of this staining is supported by the positive, albeit weak, RT-PCR signals indicating the presence of low levels of specific mRNA throughout the cycle, pregnancy, and lactation. There is no apparent variation in RLF expression within the luteal cells, in spite of time-dependent changes in hormonal parameters during the cycle and pregnancy, reflecting changes in luteal signal transduction. Nor is there any correspondence to the expression pattern of the related hormone RLX. However, not all corpora lutea express RLF to the same level. During the cycle and pregnancy, some corpora lutea stain less intensively than others. Nevertheless, within any one corpus luteum, staining intensity is always homogeneous for all luteal cells. In the cow, RLF gene expression has been identified at a high level within the theca cells of large follicles (6). Furthermore, granulosa cells can express RLF mRNA in this species, albeit at a low and variable level, and presumably give rise to some of the RLF-positive luteal cells seen already in the early-mid cycle corpus luteum (6). In the mouse, all follicle cells seem to be negative, though during the course of pregnancy, stromal cells also seem to show immunoreactive staining for RLF. The conclusion from a study of the mouse ovary is that, as in the testis, RLF expression seems to be constitutive, reflecting a differentiation phenotype in the mesonephric mesenchymal lineage leading to Leydig or luteal cells, rather than a hormonally activated state within a cell.
Preliminary studies, looking at the promoter of the mouse RLF gene, indicate that only a few hundred nucleotides of the 5' upstream promoter are sufficient for expression in Leydig cells (30, 31). Within this region are three apparent binding sites for the transcription factor steroidogenic factor 1 (SF1) (30, 31). In the mouse, SF1 is a major differentiation factor for gonadal tissues, mice deleted in this gene showing a severe disturbance of gonadal development (32). It seems possible that expression of the RLF gene might simply reflect the pattern of expression of SF1. However, the absence of marked RLF gene expression in the adrenal gland, where SF1 is also expressed to a high level, implies that additional cell-specific factors also may be involved. Taken together, all the information on RLF suggests that this new member of the insulin/relaxin/insulin-like growth factor family may act as a constitutive, differentiation-specific factor, possibly playing some role in gonadal development. Although we have been unable to identify effectors influencing acute RLF gene expression, it is also possible that the peptide may be regulated at a later step, such as translation or secretion, or by the controlled availability of specific binding proteins, much like its relative, IGF I.
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Acknowledgments |
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Footnotes |
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Received November 21, 1997.
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K. Bay, K. L. Matthiesson, R. I. McLachlan, and A.-M. Andersson The Effects of Gonadotropin Suppression and Selective Replacement on Insulin-Like Factor 3 Secretion in Normal Adult Men J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 1108 - 1111. [Abstract] [Full Text] [PDF]
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N. M. Robert, L. J. Martin, and J. J. Tremblay The Orphan Nuclear Receptor NR4A1 Regulates Insulin-Like 3 Gene Transcription in Leydig Cells Biol Reprod, February 1, 2006; 74(2): 322 - 330. [Abstract] [Full Text] [PDF]
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C. McKinnell, R. M. Sharpe, K. Mahood, N. Hallmark, H. Scott, R. Ivell, C. Staub, B. Jegou, F. Haag, F. Koch-Nolte, et al. Expression of Insulin-Like Factor 3 Protein in the Rat Testis during Fetal and Postnatal Development and in Relation to Cryptorchidism Induced by in Utero Exposure to Di (n-Butyl) Phthalate Endocrinology, October 1, 2005; 146(10): 4536 - 4544. [Abstract] [Full Text] [PDF]
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K. Bay, S. Hartung, R. Ivell, M. Schumacher, D. Jurgensen, N. Jorgensen, M. Holm, N. E. Skakkebaek, and A.-M. Andersson Insulin-Like Factor 3 Serum Levels in 135 Normal Men and 85 Men with Testicular Disorders: Relationship to the Luteinizing Hormone-Testosterone Axis J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3410 - 3418. [Abstract] [Full Text] [PDF]
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H. Obermann, I. Raabe, M. Balvers, B. Brunswig, W. Schulze, and C. Kirchhoff Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation Mol. Hum. Reprod., January 1, 2005; 11(1): 53 - 64. [Abstract] [Full Text] [PDF]
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C. Foresta, A. Bettella, C. Vinanzi, P. Dabrilli, M. C. Meriggiola, A. Garolla, and A. Ferlin A Novel Circulating Hormone of Testis Origin in Humans J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 5952 - 5958. [Abstract] [Full Text] [PDF]
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S. Hombach-Klonisch, J. Schon, A. Kehlen, S. Blottner, and T. Klonisch Seasonal Expression of INSL3 and Lgr8/Insl3 Receptor Transcripts Indicates Variable Differentiation of Leydig Cells in the Roe Deer Testis Biol Reprod, October 1, 2004; 71(4): 1079 - 1087. [Abstract] [Full Text] [PDF]
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 K. Kawamura, J. Kumagai, S. Sudo, S.-Y. Chun, M. Pisarska, H. Morita, J. Toppari, P. Fu, J. D. Wade, R. A. D. Bathgate, et al. Paracrine regulation of mammalian oocyte maturation and male germ cell survival PNAS, May 11, 2004; 101(19): 7323 - 7328. [Abstract] [Full Text] [PDF]
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F.-p. Zhang, T. Pakarainen, F. Zhu, M. Poutanen, and I. Huhtaniemi Molecular Characterization of Postnatal Development of Testicular Steroidogenesis in Luteinizing Hormone Receptor Knockout Mice Endocrinology, March 1, 2004; 145(3): 1453 - 1463. [Abstract] [Full Text] [PDF]
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F. Gaytan, M. L. Barreiro, J. E. Caminos, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, R. Paniagua, M. Nistal, F. F. Casanueva, et al. Expression of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in Normal Human Testis and Testicular Tumors J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 400 - 409. [Abstract] [Full Text] [PDF]
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R. Ivell, M. Balvers, R. J. K. Anand, H.-J. Paust, C. McKinnell, and R. Sharpe Differentiation-Dependent Expression of 17{beta}-Hydroxysteroid Dehydrogenase, Type 10, in the Rodent Testis: Effect of Aging in Leydig Cells Endocrinology, July 1, 2003; 144(7): 3130 - 3137. [Abstract] [Full Text] [PDF]
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T. Klonisch, K. Steger, A. Kehlen, W. R. Allen, C. Froehlich, J. Kauffold, M. Bergmann, and S. Hombach-Klonisch INSL3 Ligand-Receptor System in the Equine Testis Biol Reprod, June 1, 2003; 68(6): 1975 - 1981. [Abstract] [Full Text] [PDF]
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R. J. K. Anand, H.-J. Paust, K. Altenpohl, and A. K. Mukhopadhyay Regulation of Vascular Endothelial Growth Factor Production by Leydig Cells In Vitro: The Role of Protein Kinase A and Mitogen-Activated Protein Kinase Cascade Biol Reprod, May 1, 2003; 68(5): 1663 - 1673. [Abstract] [Full Text] [PDF]
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R. Ivell and S. Hartung The molecular basis of cryptorchidism Mol. Hum. Reprod., April 1, 2003; 9(4): 175 - 181. [Abstract] [Full Text] [PDF]
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M. Caprio, E. Fabbrini, G. Ricci, S. Basciani, L. Gnessi, M. Arizzi, A. R. Carta, M. U. De Martino, A. M. Isidori, G. V. Frajese, et al. Ontogenesis of Leptin Receptor in Rat Leydig Cells Biol Reprod, April 1, 2003; 68(4): 1199 - 1207. [Abstract] [Full Text] [PDF]
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R. Telgmann, R. A.D. Bathgate, S. Jaeger, G. Tillmann, and R. Ivell Transcriptional Regulation of the Bovine Oxytocin Receptor Gene Biol Reprod, March 1, 2003; 68(3): 1015 - 1026. [Abstract] [Full Text] [PDF]
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F. Gaytan, M. L. Barreiro, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, F. F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Immunolocalization of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in the Cyclic Human Ovary J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 879 - 887. [Abstract] [Full Text] [PDF]
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M.L. Barreiro, F. Gaytan, J.E. Caminos, L. Pinilla, F.F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Cellular Location and Hormonal Regulation of Ghrelin Expression in Rat Testis Biol Reprod, December 1, 2002; 67(6): 1768 - 1776. [Abstract] [Full Text] [PDF]
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R. Ivell and R. A.D. Bathgate Reproductive Biology of the Relaxin-Like Factor (RLF/INSL3) Biol Reprod, September 1, 2002; 67(3): 699 - 705. [Abstract] [Full Text] [PDF]
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 J. Kumagai, S. Y. Hsu, H. Matsumi, J.-S. Roh, P. Fu, J. D. Wade, R. A. D. Bathgate, and A. J. W. Hsueh INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent J. Biol. Chem., August 23, 2002; 277(35): 31283 - 31286. [Abstract] [Full Text] [PDF]
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H. F. Irving-Rodgers, R. A.D. Bathgate, R. Ivell, R. Domagalski, and R. J. Rodgers Dynamic Changes in the Expression of Relaxin-Like Factor (Insl3), Cholesterol Side-Chain Cleavage Cytochrome P450, and 3{beta}-Hydroxysteroid Dehydrogenase in Bovine Ovarian Follicles During Growth and Atresia Biol Reprod, April 1, 2002; 66(4): 934 - 943. [Abstract] [Full Text] [PDF]
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P. Koskimies, J. Levallet, P. Sipila, I. Huhtaniemi, and M. Poutanen Murine Relaxin-Like Factor Promoter: Functional Characterization and Regulation by Transcription Factors Steroidogenic Factor 1 and DAX-1 Endocrinology, March 1, 2002; 143(3): 909 - 919. [Abstract] [Full Text] [PDF]
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I. M. Adham, G. Steding, T. Thamm, E. E. Bullesbach, C. Schwabe, I. Paprotta, and W. Engel The Overexpression of the Insl3 in Female Mice Causes Descent of the Ovaries Mol. Endocrinol., February 1, 2002; 16(2): 244 - 252. [Abstract] [Full Text] [PDF]
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 P. J. O'Shaughnessy, H. Johnston, L. Willerton, and P. J. Baker Failure of normal adult Leydig cell development in androgen-receptor-deficient mice J. Cell Sci., January 9, 2002; 115(17): 3491 - 3496. [Abstract] [Full Text] [PDF]
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S.M.L. Chamindrani Mendis-Handagama and H.B. Siril Ariyaratne Differentiation of the Adult Leydig Cell Population in the Postnatal Testis Biol Reprod, September 1, 2001; 65(3): 660 - 671. [Abstract] [Full Text] [PDF]
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G. de Rienzo, F. Aniello, M. Branno, and S. Minucci Isolation and Characterization of a Novel Member of the Relaxin/Insulin Family from the Testis of the Frog Rana esculenta Endocrinology, July 1, 2001; 142(7): 3231 - 3238. [Abstract] [Full Text] [PDF]
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S. Hombach-Klonisch, S. Seeger, G. Tscheudschilsuren, J. Buchmann, B. Huppertz, G. Seliger, B. Fischer, and T. Klonisch Cellular localization of human relaxin-like factor in the cyclic endometrium and placenta Mol. Hum. Reprod., April 1, 2001; 7(4): 349 - 356. [Abstract] [Full Text] [PDF]
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T. Klonisch, J. Kauffold, K. Steger, M. Bergmann, R. Leiser, B. Fischer, and S. Hombach-Klonisch Canine Relaxin-Like Factor: Unique Molecular Structure and Differential Expression Within Reproductive Tissues of the Dog Biol Reprod, February 1, 2001; 64(2): 442 - 450. [Abstract] [Full Text]
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S. Nef and L. F. Parada Hormones in male sexual development Genes & Dev., December 15, 2000; 14(24): 3075 - 3086. [Full Text]
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M. Tomboc, P. A. Lee, M. F. Mitwally, F. X. Schneck, M. Bellinger, and S. F. Witchel Insulin-like 3/Relaxin-Like Factor Gene Mutations Are Associated with Cryptorchidism J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4013 - 4018. [Abstract] [Full Text]
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H. K. Deol, R. Varghese, G. F. Wagner, and G. E. DiMattia Dynamic Regulation of Mouse Ovarian Stanniocalcin Expression during Gestation and Lactation Endocrinology, September 1, 2000; 141(9): 3412 - 3421. [Abstract] [Full Text] [PDF]
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W. Pusch, M. Balvers, G. F. Weinbauer, and R. Ivell The Rat Endozepine-Like Peptide Gene Is Highly Expressed in Late Haploid Stages of Male Germ Cell Development Biol Reprod, September 1, 2000; 63(3): 763 - 768. [Abstract] [Full Text]
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 L. Gnessi, S. Basciani, S. Mariani, M. Arizzi, G. Spera, C. Wang, C. Bondjers, L. Karlsson, and C. Betsholtz Leydig Cell Loss and Spermatogenic Arrest in Platelet-derived Growth Factor (PDGF)-A-deficient Mice J. Cell Biol., May 29, 2000; 149(5): 1019 - 1026. [Abstract] [Full Text] [PDF]
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S. Y. Hsu Cloning of Two Novel Mammalian Paralogs of Relaxin/Insulin Family Proteins and Their Expression in Testis and Kidney Mol. Endocrinol., December 1, 1999; 13(12): 2163 - 2174. [Abstract] [Full Text]
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R. Bathgate, N. Moniac, B. Bartlick, M. Schumacher, M. Fields, and R. Ivell Expression and Regulation of Relaxin-Like Factor Gene Transcripts in the Bovine Ovary: Differentiation-Dependent Expression in Theca Cell Cultures Biol Reprod, October 1, 1999; 61(4): 1090 - 1098. [Abstract] [Full Text]
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K. J. Teerds, M. de Boer-Brouwer, J. H. Dorrington, M. Balvers, and R. Ivell Identification of Markers for Precursor and Leydig Cell Differentiation in the Adult Rat Testis Following Ethane Dimethyl Sulphonate Administration Biol Reprod, June 1, 1999; 60(6): 1437 - 1445. [Abstract] [Full Text]
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M. R. Zarreh-Hoshyari-Khah, A. Einspanier, and R. Ivell Differential Splicing and Expression of the Relaxin-Like Factor Gene in Reproductive Tissues of the Marmoset Monkey (Callithrix jacchus) Biol Reprod, February 1, 1999; 60(2): 445 - 453. [Abstract] [Full Text] [PDF]
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