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From the * Lab of Molecular Endocrinology and
Signal Transduction, Institute of Biology and Experimental Medicine-CONICET,
Buenos Aires, Argentina; the Department of
Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary;
and the Department of Biological Chemistry,
School of Sciences, University of Buenos Aires, Buenos Aires, Argentina.
| Correspondence to: Carolina Mondillo, PhD, Laboratory of Molecular Endocrinology and Signal Transduction, Institute of Biology and Experimental Medicine (IByME-CONICET), Vuelta de Obligado 2490, CP 1428, Buenos Aires, Argentina (e-mail: mondillo{at}dna.uba.ar). |
| Reprint requests to: Erna Pap, PhD, Department of Genetics, Cell- and Immunobiology, Semmelweis University, Nagyvárad tér 4, Budapest, Hungary (e-mail: nyierna{at}dgci.sote.hu). |
| Received for publication March 22, 2006; accepted for publication August 21, 2006. |
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Abstract |
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Key words: Steroidogenesis, human choriogonadotropin
The presence of HA in animal gonads is indisputable (Nistal et al, 1984; Van Thiel et al, 1987; Gaytan et al, 1992; Almeida et al, 1996; Hibi et al, 2001). Very recent results proved the existence of a histaminergic system in the human testis as well (Albrecht et al, 2005). Most importantly, HA has been previously suggested to affect processes related to male fertility, such as gonadal development, spermatogenesis, and sexual behavior. However, the precise role of the amine in the testis is still poorly understood.
In particular, histaminergic regulation of Leydig cell physiology has not been fully investigated yet, and the limited evidence available so far seems contradictory. Mayerhofer et al (1989) demonstrated that HA can stimulate in vitro testosterone synthesis in the golden hamster; meanwhile, HA-deprived histidine decarboxylase gene (Hdc) knockout (HDC KO) mice showed increased in vivo testicular steroid production (Pap et al, 2002). To complicate the picture, HA was recently reported to exert a dual concentration-dependent effect on steroidogenesis in rat Leydig cells, as well as in the MA-10 mouse Leydig tumor cell line. H1 and H2 receptor subtypes were detected in such cells using radioligand binding assays, but no molecular evidence was shown to support these observations (Mondillo et al, 2005).
To further evaluate a direct role for testicular HA in modulation of Leydig cell physiology, we performed experiments on highly purified Leydig cells in culture, isolated from testes of HDC KO and wild type (WT) mice.
We measured testosterone levels in the incubation media and evaluated Cyp11a1 expression in the cells using quantitative real-time polymerase chain reaction (PCR) assays. Cyp11a1 gene (herein denoted CYP11A) codes for cholesterol side chain cleavage cytochrome P450 (P450scc), the main quantitative regulatory enzyme of steroid synthesis. We also performed quantitative real-time PCR assays to study expression levels of H1 and H2 receptor genes (Hrh1 and Hrh2, herein denoted H1 and H2) in both experimental models, providing molecular evidence that Leydig cells are direct targets of HA action.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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2.4-kb fragment extending from the SpeI site in intron 5 to the
PstI site in exon 9 with a PGK-neor cassette. The
cloned fragments were linearized and introduced into the ES cell line R1
selected by G418 and ganciclovir. Out of 6 long and accurate polymerase chain
reaction (LA-PCR) positive clones aggregated with CD1 morulae, 3 were
confirmed to be homologous recombinants by Southern blotting. WT and HDC KO
mice were bred in our transgenic facility. Normal rodent diet (HA content >50 µmol/g) was provided ad libitum (Charles River, Hungary). Adult male mice from multiple litters (3 to 5 months old) were used in this study. Mice were killed with ether anesthesia. Their testes were removed and kept in 4°C phosphate buffered saline buffer (PBS) until culture preparation was started. All procedures were carried out under sterile conditions.
Preparation of Leydig Cell Culture
For each experiment, 12 to 14 testes of WT and HDC KO mice were used to
obtain interstitial cells, and these pooled cells were used for subsequent
Leydig cell isolation.
Testes were decapsulated and dispersed by shaking (10 minutes, 80 cycles/min, 34°C) in a tissue culture flask containing 0.025% collagenase, in Medium 199 (M199) supplemented with 0.1% BSA (1 mL/testis). The crude cell suspension was filtered twice through Nitex monofilament. Interstitial cells were pelleted by centrifugation and washed twice. The cells were then resuspended in 5 mL of a 1.7 mmol Tris, 140 mmol NH4Cl solution, pH 7.2, and incubated for 10 minutes at 37°C. This procedure eliminates red blood cells and does not affect the cell response to gonadotropin stimulation. The unfractionated interstitial cell preparation was resuspended in 10 mL M199-0.1% BSA. Testicular macrophages were separated by differential attachment to plastic culture plates (34°C, 15 minutes). Unattached cells were centrifuged, and the cell pellet was then purified by fractionation on a 5-layer Percoll density gradient (21%, 26%, 34%, 40%, and 60%). The purity of Leydig cells obtained was assessed by histochemical staining for 3-ß-hydroxysteroid dehydrogenase activity (Payne et al, 1980; Del Punta et al, 1996; Mondillo et al, 2005). Isolated Leydig cells were plated in 24-well plates and maintained under a water-saturated atmosphere of 5% CO2 and 95% air at 34°C in a 1:1 mixture of Ham's F-12: Dulbecco modified Eagle medium, supplemented with 15 mmol NaHCO3, 20 mmol Hepes, pH 7.4, 0.1% Gentamicin, 10 µg/mL transferrin, 5 µg/mL vitamin E, and 5% fetal bovine serum (unless otherwise stated, all materials were purchased from Sigma-Aldrich Ltd, St Louis, Mo).
Steroid Concentration Measurements
Testosterone was measured from media with ELISA kit (R and D Systems, Inc,
Minneapolis, Minn) after 5 hours of incubation in the absence or presence of
human choriogonadotropin (hCG) (Human Co, Gödöllö, Hungary) at
concentrations ranging from 0.45 to 9 mIU/mL, as indicated in each figure
legend). Results are expressed as the mean ± SEM. Statistical analysis
was performed by one-way ANOVA followed by Tukey's multiple comparisons test.
P less than .05 was considered significant.
Expression of Enzymes and Receptors
Total RNA was collected from the cells after a 35-minute incubation in the
absence or presence of 9 mIU/mL hCG.
Total Cytoplasmic RNA Preparation
Briefly, samples were homogenized using TRI reagent (Sigma-Aldrich Ltd).
This was followed by extraction with chloroform, precipitation with
isopropanol, and final wash with alcohol. For H1 and H2 receptor studies, RNA
was treated by RNase-free DNase (Promega Corp, Madison, Wis).
Reverse Transcription Polymerase Chain Reaction
Reverse transcription (RT) of an aliquot of total cytoplasmic RNA (1 µg)
to cDNA was catalyzed by multireverse transcriptase (Perkin–Elmer) in
the presence of 1 mM of each dNTPs, RNase inhibitor, random hexamers, 10PCR
buffer, and MgCl2 (Promega) to a total volume of 40 µL. The RT
mix was incubated at 42°C for 10 minutes for reverse transcription,
followed by 95°C for 10 minutes to inactivate the reverse transcriptase.
The cDNAs were stored at –20°C until use.
Quantitative Real-time PCR
The assays were performed based on Assays-on-Demand Gene Expression
Products Protocol (Applied Biosystems). Briefly, target cDNA samples
synthesized from the RNA sample were amplified by AmpliTaq Gold DNA polymerase
in the TaqMan Universal PCR Master Mix, using sequence-specific primers and
TaqMan MGB probe. QRT-PCR was carried out using an ABI Prism 7000 Sequence
Detection System (Applied Biosystems, Foster City, Calif), and the associated
ABI 2.0 software was used to analyze the data and determine the threshold
count regarding relative quantification of gene expression experiments
referring to Sequence Detection System User Bulletin #2 (P/N 4303859). The
primers and probes were designed using the Assays-on-Demand Gene Expression
Product list of Applied Biosystems.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Relative Expression of CYP11A Gene
WT and HDC KO Leydig cells were incubated with or without 9 mIU/mL hCG, and
total RNA was extracted from the cells. The RNAs were reverse-transcribed, and
real-time amplification was performed as described in "Materials and
Methods." hCG showed no significant inducing effect on CYP11A relative
expression levels in either WT or HDC KO Leydig cells. However, results
indicate a small tendency for basal CYP11A relative expression rate to be
lower in HDC KO Leydig cells (Figure
3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Our observations appear to conflict with previous in vivo results obtained in HDC KO mice, which indicated elevated testicular steroid levels compared to WT mice (Pap et al, 2002). However, experiments carried out using testicular homogenate may not be directly comparable to those using isolated Leydig cells, because of the presence of cell types other than Leydig cells in the in vivo case, which might exert some influence on the steroidogenic process via paracrine secretions. Considering those recent reports that indicate the presence of HA receptors in peritubular and germinal cells (Albrecht et al, 2005), such cells might also be affected by the lack of HA; more dramatically, testes of HDC KO mice lack endogenous HA from the very beginning of their embryonic development. Thus, the experiments carried out in this study—that is, using purified Leydig cells—enable a more direct determination of the effects of HA deficiency on Leydig cell function.
Our present observations also indicate that HDC KO Leydig cells cannot respond to LH/hCG stimulus as effectively as WT cells do. In this regard, even after a 5-hour induction in the presence of maximally stimulating hCG, testosterone levels synthesized by HDC KO Leydig cells reached only the levels of untreated WT cells. Given that occupation of as little as 10% of the total LH/hCG receptors in Leydig cells suffices to induce maximal steroid production (Catt et al, 1974; Barañao and Dufau, 1984), it seems quite improbable that the decreased testosterone accumulation in KO Leydig cells can be attributable to a reduction in the number of LH/hCG receptors. P450scc plays a central role in the steroidogenic pathway leading to testosterone synthesis in Leydig cells (Nolan and Payne, 1990). The interplay between intracellular HA and different cytochrome P450 enzymes has been described in several studies (Brandes et al, 1998; LaBella et al, 2000). Therefore, we evaluated the relative expression rate of CYP11A gene in WT and HDC KO Leydig cells, under basal conditions and after hCG treatment. We found no statistically significant differences, but a tendency for CYP11A relative expression rate to be lower in HDC KO Leydig cells. Also, we cannot discard the possibility of appearing differences at post-transcriptional level or alteration of other key regulatory step(s) in the steroidogenic pathway leading to androgen synthesis, potentially before the action of P450scc enzyme.
Interestingly, Leydig cell ultrastructure is seriously altered in adult HDC KO mice, and testicular weight is significantly reduced already in 7-day-old litters, at a time when the testes have not yet descended from the abdomen. Importantly, lack of HDC does not interfere with the general morphology and distribution of GnRH neurons in these mice, which also show normal hypothalamic Gnrh1 mRNA expression (Pap et al, 2002). Thus, although LH is the primary factor influencing morphology and function of Leydig cells (Lejeune et al, 1996), both important to maintain their steroidogenic potential, the absence of endogenously produced HA in HDC KO mice may probably result in embryonic modifications of testicular ontogenesis, leading to subnormal adult Leydig cell function. This hypothesis is reinforced by previous research indicating that testicular HA concentration is normally higher in the immature gonad than in the adult one (Zieher et al, 1971), suggesting the occurrence of important HA-dependent events during testicular development.
Very recently, we reported the existence of H1 and H2 receptors on the membrane of rat Leydig cells and MA-10 mouse Leydig tumor cells, based on binding studies (Mondillo et al, 2005). The work represents the only evidence available so far regarding the presence of HA receptors in purified Leydig cells. However, it has been argued that HA receptor subtypes cannot be distinguished solely on the basis of pharmacological studies. Also, HA-deprived HDC KO mice were previously shown to have altered expression levels of H1 and H2 receptor genes in certain tissues (Fitzsimons et al, 2001). Thus, we evaluated the expression rates of H1 and H2 receptor genes in WT and HDC KO Leydig cells, under basal conditions and after hCG treatment. In agreement with previous biochemical studies, we detected basal expression of H1 and H2 receptor genes in WT Leydig cells. Moreover, hCG showed a significant stimulatory effect. These results provide the first molecular evidence for the expression of H1 and H2 receptor subtypes in isolated Leydig cells and reveal an inducing effect of hCG on the gene expression levels of such receptor subtypes. In addition, these findings complement our recently described results, which showed that H2 receptor activation can potentiate the steroidogenic effect of hCG in rat Leydig cells and MA-10 mouse Leydig tumor cells. Also, H1 receptor activation was shown to exert a minor but significant contribution to the potentiating effect of HA (Mondillo et al, 2005).
In contrast to WT Leydig cells, hCG had no inducing effect on the relative expression rates of H1 or H2 receptor genes in KO Leydig cells. Most importantly, the hormone exerted an inhibitory effect on H2 receptor gene expression levels. Although the physiological significance of these findings requires further investigation, it seems clear that the absence of endogenously produced HA in HDC KO mice alters the way Leydig cells respond to hCG-induced regulation of gene expression levels of H1 and H2 receptors. Based on solid experimental evidence, G-protein-linked signal transduction pathways may converge and diverge at multiple levels, enabling cells to coordinate responses to diverse environmental stimuli (Brady and Limbird, 2002; Bakker et al, 2004). Both hCG and HA act through G-protein-dependent pathways. Thus, it is plausible that in the absence of HA, altered cross-talks of the transducing systems modify the response of HDC KO Leydig cells.
On the basis of the results reported herein, we suggest that prolonged HA deficiency in HDC KO mice affects various aspects of Leydig cell physiology, providing definite evidence that HA plays an important role as direct modulator of Leydig cell function and steroid synthesis in the testis.
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Acknowledgments |
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Footnotes |
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References |
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|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Almeida RG, Massoco CO, Spinosa HS, Bernardi MM. Perinatal astemizole exposure in the rat throughout gestation: long-term behavioral and anatomic effects associated with reproduction. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1996; 114: 123 –127.[CrossRef][Medline]
Bakker AR, Casarosa P, Timmermann H, Smit MJ, Leurs R.
Constitutively active Gq/11-coupled receptors enable signaling by co-expressed
G(i/o)-coupled receptors. J Biol Chem. 2004; 279: 5152
–61.
Barañao JLS, Dufau ML. Gonadotropin-induced changes in the LH receptors in cultured Leydig cells: measurement of the immunoactive bound hormone. In: Catt KJ, Dufau ML, eds. Hormone Action and Testicular Function. Annals of the New York Academy of Sciences, Vol 438 . New York, NY: The New York Academy of Sciences; 1984 : 594–597.
Brady AE, Limbird LE. G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction. Cell Signal. 2002; 14: 297 –309.[CrossRef][Medline]
Brandes LJ, Queen GM, LaBella FS. Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei, and chromatin from rat hepatocytes. J Cell Biochem. 1998; 69: 233 –243.[CrossRef][Medline]
Catt KJ, Tsuruhara T, Mendelson C, Ketelslegers J-M, Dufau ML. Gonadotropin binding and activation of the interstitial cells of the testis. In: Dufau ML, Means AR, eds. Hormone Binding and Target Cell Activation in the Testis. New York and London: Plenum Press; 1974 : 1–30.
Del Punta K, Charreau EH, Pignataro OP. Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology. 1996; 137: 5337 –5343.[Abstract]
Fitzsimons CP, Lazar-Molnar E, Tomoskozi Z, Buzas E, Rivera ES, Falus A. Histamine deficiency induces tissue-specific down-regulation of histamine H2 receptor expression in histidine decarboxylase knockout mice. FEBS Lett. 2001; 508: 245 –248.[CrossRef][Medline]
Gaytan F, Aceitero J, Lucena C, Aguilar E, Pinilla L, Garnelo P,
Bellido C. Simultaneous proliferation and differentiation of mast cells and
Leydig cells in the rat testis. Are common regulatory factors involved?
J Androl. 1992;13: 387
–397.
Hibi H, Kato K, Mitsui K, Taki T, Yamada Y, Honda N, Fukatsu H, Yamamoto M. The treatment with tranilast, a mast cell blocker, for idiopathic oligoospermia. Arch Androl. 2001; 47: 107 –111.[Medline]
Hill SJ. Distribution, properties and functional characteristics of three classes of histamine receptor. Pharmacol Rev. 1990; 42: 45 –83.[Abstract]
LaBella FS, Queen GM, Brandes LJ. Interactive binding at cytochrome P-450 of cell growth regulatory bioamines, steroid hormones, antihormones, and drugs. J Cell Biochem. 2000; 76: 686 –694.[CrossRef][Medline]
Lejeune H, Chuzel F, Thomas T, Avallet O, Habert R, Durand P, Saez J. Paracrine regulation of Leydig cells. Ann Endocrinol. 1996;57: 55 –63.[Medline]
Liu C, Ma X, Jiang X, Wilson SJ, Hofstra CL, Blevitt J, Pyati J, Li
X, Chai W, Carruthers N, Lovenberg TW. Cloning and pharmacological
characterization of a fourth histamine receptor (H(4)) expressed in bone
marrow. Mol Pharmacol. 2001; 59: 420
–426.
Mayerhofer A, Bartke A, Amador AG, Began T. Histamine affects testicular steroid production in the golden hamster. Endocrinology. 1989; 125: 2212 –2214.[Abstract]
Mondillo C, Patrignani Z, Reche C, Rivera E, Pignataro O. Dual role
of histamine in modulation of Leydig cell steroidogenesis via HRH1 and HRH2
receptor subtypes. Biol Reprod. 2005; 73: 899
–907.
Nistal M, Santamaria L, Paniagua R. Mast cells in the human testis and epididymis from birth to adulthood. Acta Anat. 1984; 119: 155 –160.[Medline]
Nolan CJ, Payne AH. Genotype at the P450scc locus determines differences in the amount at P450scc protein and maximal testosterone production in mouse Leydig cells. Mol Endocrinol. 1990; 4: 1459 –1464.[CrossRef][Medline]
Ohtsu H, Tanaka S, Terui T, Hori Y, Makabe-Kobayashi Y, Pejler G, Tchougounova E, Hellman L, Gertsenstein M, Hirasawa N, Sakurai E, Buzas E, Kovacs P, Csaba G, Kittel A, Okada M, Hara M, Mar L, Numayama-Tsuruta K, Ishigaki-Suzuki S, Ohuchi K, Ichikawa A, Falus A, Watanabe T, Nagy A. Mice lacking histidine decarboxylase exhibit abnormal mast cells. FEBS Lett. 2001;502: 53 –56.[CrossRef][Medline]
Pap E, Falus A, Mihalyi D, Borck H, Diel F, Pallinger E. Histamine regulates placental cytokine expression—in vivo study on HDC knockout mice. Placenta. 2006 [Epub ahead of print].
Pap E, Rácz K, Kovács JK, Varga I, Buzás E, Madarász B, Foldes C, Szalai C, Watanabe T, Ohtsu H, Ichikawa A, Nagy A, Falus A. Histamine deficiency in histidine decarboxylase gene knockout mice elevates male sexual steroid production. J Endocrinol. 2002; 175: 193 –199.[Abstract]
Payne AH, Downing JR, Wong KL. Luteneizing hormone receptors and testosterone synthesis in two distinct populations of Leydig cells. Endocrinology. 1980; 106: 1424 –1429.[Abstract]
Van Thiel DH, Gavaler JS, Heyl A, Susen B. An evaluation of the anti-androgen effects associated with H2 antagonist therapy. Scand J Gastroenterol Suppl. 1987; 136: 24 –28.[Medline]
Yatsunami K, Ohtsu H, Tsuchikawa M, Higuchi T, Ishibashi K, Shida
A, Shima Y, Nakagawa S, Yamauchi K, Yamamoto M, Hayashi N, Watanabe T,
Ichikawa A. Structure of the L-histidine decarboxylase gene. J Biol
Chem. 1994;269: 1554
–1559.
Zieher LM, Debeljuk L, Iturriza F, Mancini RE. Biogenic amine concentration in testes of rats at different ages. Endocrinology. 1971; 88: 351 –354.[Medline]
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