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placental lactogen gene-cluster in the testis
Molecular and Cellular Endocrinology 130 (1997) 53 – 60
Organ-specific expression pattern of the human growth hormone/placental lactogen gene-cluster in the testis G. Untergasser a, W. Kranewitter a, P. Schwa¨rzler a, S. Madersbacher c, S. Dirnhofer a,b, P. Berger a,d,* a
Institute for Biomedical Aging Research of the Austrian Academy of Sciences, A-6020 Innsbruck, Austria b Department of Pathology, Uni6ersity of Innsbruck, A-6020 Innsbruck, Austria c Department of Urology, Uni6ersity of Vienna, Vienna, Austria d Institute for General and Experimental Pathology, Uni6ersity of Innsbruck, A-6020 Innsbruck, Austria Received 14 December 1996; accepted 24 March 1997
Abstract In addition to testosterone, the essential paracrine factor for spermatogenesis, a number of potential auto/paracrine regulatory substances such as b-endorphins, enkephalins, chorionic gonadotropin b, growth hormone-releasing hormone (GHRH) and insulin-like growth factor I (IGF-I) have been identified in the testis of various mammalian species. The latter findings prompted us to investigate a possible eutopic production of GH, placental lactogen (PL) and PRL in human testes. Specific expression of testicular GH/PL mRNA (n=20) was shown by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of primers designed to non-selectively amplify any transcript of the five GH/PL genes (GH-N, GH-V, PL-A, PL-B, PL-L). In contrast to the classical sites of production, the pituitary (exclusively GH-N transcripts) and the placenta (PL-A/B \99%, GH-VB1%), radioactive semiquantitative restriction enzyme analysis of the PCR-products revealed, that the testis has its own organ-specific pattern of GH/PL gene expression: PL-A/B \ GH-V ]PL-L =GH-N. All three organs express the single PRL gene, and testis and placenta show the alternative splice variant GH-V2. Immunological analyses by immunofluorometric assays for hPL-A/B, hGH-N and hPRL, demonstrated significant amounts of protein hormones in all testicular cytosolic homogenates (means: hPL 1.0 ng/g, hGH 5.1 ng/g and hPRL 58.7 ng/g tissue wet weight). Most noteworthy, hPL serum levels in an elderly age-matched healthy subjects (n=18) were B 0.02 ng/ml. The concept of purely endocrine functions of placental and pituitary-derived GH/PL needs to be reassessed, since human testicular synthesis of these molecules suggest auto/paracrine functions in the male reproductive tract. © 1997 Elsevier Science Ireland Ltd. Keywords: Growth hormone; Placental lactogen; Prolactin; Human testis; Eutopic production
1. Introduction The genes coding for the human (h) protein hormones prolactin (hPRL) and for the growth hormone/ placental lactogen (hGH/hPL) gene-cluster segregated from a common ancestral gene approximately 400 million years ago (Wallis, 1992). The members of this protein hormone family share genetic and subsequent * Corresponding author. Tel.: +43 512 58391924; fax: + 43 512 5839198; e-mail: [email protected]
structural, immunological and biological features. Located on chromosome 17 q22–24, the GH/PL genecluster is comprised of five highly related genes (GH-N, PL-L, PL-A, GH-V and PL-B), which evolved through a series of duplication events approx. ten million years ago, and share more than 90% nucleotide sequence identity in their coding and flanking regions (Chen et al., 1989). Despite these similarities, they are expressed in an organ-specific manner at high levels either in the anterior pituitary (GH-N) or as the other four genes, in the syncytiotrophoblast of the human placenta.
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G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
These placental hormones and alternative splice products of the GH-V and PL-A genes, GH-V2 and PL-A2, containing intron D, are coordinately induced during fetal development, increasing between weeks 12 and 20 of gestation and then plateauing through term (MacLeod et al., 1992). Their function during pregnancy is still a matter of debate, but they may have an important influence on the carbohydrate- and lipidmetabolism of mother and fetus. In addition, PL stimulates development and cell division of mammary gland epithelial cells (Handwerger, 1991). With primary support from the pituitary-derived gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH), the three major somatic cell types of the human testis (Leydig, peritubular and Sertoli cells) provide the hormonal milieu and essential factors for germ cell development and intra-testicular cell-cell interactions. Supporting the actions of gonadotropins, pituitary-derived GH and PRL play a modulatory role in the maintenance of testicular function (Zipf et al., 1978; Balducci et al., 1993; Chatelain et al., 1991). GH increases the numbers of testicular luteinizing hormone receptors (LH-R), and choriogonadotropin (hCG)-stimulated testosterone production in men and mice (Balducci et al., 1993; Chatelain et al., 1991). Evidence for an important role of PRL is seen in patients with hyperprolactinemia, who can be infertile, due to down-regulation of the LH-R on Leydig cells via the PRL-receptor (Zipf et al., 1978; Meiters, 1988; Bartke, 1980). Recently, it has been shown that, in the human testis, GH-releasing hormone (GHRH) (Berry et al., 1992; Ciampani et al., 1992) and the glycoprotein hormone subunits hCGb and LHb are transcribed and present in low amounts (Berger et al., 1994). Increasing evidence that locally produced factors including b-endorphin, enkephalins, dynorphins, oxytocin, activin, LH-releasing hormone (LHRH), GHRH, insulin-like growth factor I (IGF-I) (Skinner, 1991) and CGb might act in auto/paracrine fashions prompted us to investigate a possible eutopic expression of protein hormones in the human testis. 2. Materials and methods
2.1. Samples Testicular tissue (n =20) from previously untreated prostatic cancer patients undergoing subcapsular orchiectomy (age range, 68 – 83 years), one post-mortem pituitary (female, age 72 years; reason of death, cardiac failure), Jar and Bewo choriocarcinoma cells (106 cells) and human term placentae (n =3) were snap frozen in liquid nitrogen and kept at − 70°C until used for RNA analysis in the reverse transcription polymerase-chainreaction (RT-PCR).
One gram of testicular tissue (n = 11) was homogenized on ice in 5 ml phosphate buffer saline (PBS) (Ultra-Turrax, Janke and Kunkel, Stauffen, Germany). After centrifugation (10 000 × g, 20 min, 4°C) the aqueous cytosolic extract was stored at −20°C until analyses for hGH/hPRL/hPL content by immunofluorometric assays (IFMA). Phenylmethane-sulfonyl fluoride (Merck, Darmstadt, Germany) was added as protease inhibitor (1 mM).
2.2. Immunofluorometric assays for hPL-A/B, hGH-N and hPRL Specific IFMAs for pituitary derived hGH-N and hPRL and hPL-A/B were established based on our own panel of well characterized monoclonal antibodies (MCA) (Staindl et al., 1987; Berger et al., 1988). IFMA technology provided the highest sensitivity and widest measuring range. They were performed in analogy to our assays for glycoprotein hormones (Madersbacher et al., 1993a,b). hPRL 81/541 (NIBSC, London, UK), hGH 66/217 (NIBSC) and hPL (NIADDK, NIH, Bethesda, MD) were used as hormone standard preparations. Ten mg of highly purified MCA, coded as INN-hPL37, INN-hGH-2 or INN-hPRL-9, respectively, were diluted in 100 ml PBS pH 7.2 and incubated for 2 h at 37°C in a microtiter plate (Nunc, Roskilde, Denmark). Remaining binding sites were blocked with 200 ml of 1% bovine serum albumin (BSA) in PBS for 30 min at 37°C. The plates were then washed three times with 200 ml per well with PBS containing 0.5 ml Tween 20 and 5 g of thiomersal/l. For the actual assay, we used an incubation volume of 100 ml/well and an assay buffer consisting of 50 mM Tris–HCl (pH 7.75), 9 g/l NaCl, 5 g BSA/l, 0.1 g/l Tween 40, 0.5 g/l bovine g-globulin and 20 mM diethylenetriaminepentaacid (Sigma-Aldrich, Milwaukee, WI). Graded amounts of the hormone standards or homogenized testicular tissue (1:2 in assay buffer) were allowed to react on a orbit shaker (500 rpm, 90 min, 20°C) followed by three washes and subsequently by incubation with 100 ng of europium-labeled detection MCA INN-hPL-5, INN-hGH-5 or INN-hPRL-1, respectively (30 min, 20°C, orbit shaker). After extensive washing, enhancement solution was added (0.1 mM potassium-phthalate, pH 3.2, containing 15 mmol of 2-naphtoyltrifluoroaceton, 50 mmol or tri-N-octylphosphine oxide, and l g of Triton X-100 per liter) and incubated for 5 min on a orbit shaker. Time-resolved fluorescence was measured for 1 s in a fluorometer (1232 Delfia-fluorometer; Wallac, Turku, Finland).
2.3. Isolation of Poly (A) + RNA Total RNA was extracted from 100 mg of homogenized tissue (Ultra-Turrax, Janke and Kunkel) by the
G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
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Fig. 1. PRL and GH/PL gene organisation and localization of the RT-PCR primers. Due to their common origin, the single gene for PRL and the five genes of the GH/PL gene-cluster have a similar exon/intron structure. Five exons, coded 1 – 5, are separated by four introns, A–D. The two PRL-specific primers are located in regions of exon 3 and exon 4/5 (Sabharwal et al., 1992), where PRL and GH/PL share only 30% of their nucleotide sequence. In contrast, due to high nucleotide sequence homology between the GH/PL genes (\ 90%), we used only a single pair of primers (Lytras et al., 1994), located in exons 3 and 4 at positions, where all five genes show sequence identity, thereby amplifying all major GH/PL gene products. If necessary, the generated 250 bp cDNA fragment can be distinguished by size from the longer 341 – 343 bp fragment of potentially amplified contaminating genomic DNA. To detect the alternative splice product of the GH-V gene, GH-V2, the 5%-non-selective GH/PL primer was combined with a GH-V specific primer, located in intron D at a position, where the PL-A gene shows nucleotide sequence divergence.
single step acid guanidinium thiocyanate phenol/chloroform method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). Extreme care was taken to avoid specimen contamination during the extraction procedure. Testes were homogenized prior to placentae and the pituitary, including intermitting washing steps (5 min) of the dispersing tool in 0.1 M NaOH and 0.5% SDS. Additionally, filtered pipette tips (Bio-Rad; Richmond, CA) were used. Integrity of RNA was assessed by analysis of 28S and 18S rRNA on ethidium bromide-stained 1% agarose gels (Boehringer-Mannheim, Mannheim, Germany). Poly (A) + RNA was isolated using a oligo(dT 30)-Cellulose (Serva, Heidelberg, Germany) according the manufacturer’s instructions.
2.4. Re6erse transcription Between 500– 1000 ng of poly (A) + RNA were diluted in 10 ml diethylpyrocarbonate (DEPC, SigmaAldrich) water in a 0.5 ml PCR tube (Perkin-Elmer Cetus, Norwalk, CT), denatured at 65°C for 10 min in a programmable thermal cycler (TC; Techne, Cambridge, UK), then rapidly cooled to 4°C and the reverse transcription performed in a final volume of 50 ml under the following reaction conditions: 50 mM Tris– HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each of dATP, dCTP, dGTP and dTTP (Promega, Madison, WI), 10 U human placental ribonuclease inhibitor (Promega), 200 pmol random hexamer oligonucleotide (Boehringer – Mannheim) and 50 U MMLV-Reverse Transcriptase (Promega). The reaction mix was incubated for 8 min at 20°C and 8 min at 25°C to achieve optimal hybridization between the RNA and the hexamer primers. The reverse transcriptase was then allowed to proceed at 37°C for 30 min.
This cycle was repeated four times to increase the amount of cDNA.
2.5. Polymerase chain reaction Each first strand reaction was adjusted to a volume of 70 ml and 7 ml (50–100 ng) were used for the PCR amplification. The PCR reaction was performed in a final volume of 25 ml under the following conditions: 10 mM Tris–HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 4 pmol each of the two oligonucleotide primers specific for each gene-product (PRL, GH/ PL, GH-V2) and 0.125 U Taq polymerase (Promega). After initial denaturation for 2 min at 95°C, 40 cycles of amplification were performed with 55 s denaturation at 95°C, 50 s annealing at either 58°C (PRL), 44°C (GH/PL) or 54°C (GH-V2) and 45 s extension at 73°C in a thermocycler. The last cycle had an elongation time of 5 min at 73°C. A set of primers amplifying cDNA of all members of the GH/PL gene cluster (Lytras et al., 1994) 5%-CAGAAGTATTCATTCCTGCA-3% located at position 737–756 (exon 3) and 5%-TTTGGATGCCTTCCTCTAG-3%, position 1060–1078 (exon 4, numbered according to the hGH-N gene sequence (Chen et al., 1989)), and a second set, specific for the alternative splice product GH-V2 (Fig. 1) 5%-CAGAAGTATTCATTCCTGCA-3% located at position 748–767 (exon 3) and 5%-TTTCTCCCCAGTCCCTGG-3%, position 1146–1165 (intron D) according to the sequence of hGH-V (Chen et al., 1989), were custom-made by Microsynth (Windisch, Switzerland). The antisense PCR primer for PRL (Sabharwal et al., 1992) was axon-spanning 5%-TTCAGGATGAACCTG-
G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
56 Table 1
PCR-primer set
Amplified gene product
DNA-fragment length (bp)
cDNA-RE-analyses: visible fragments (bp)
cDNA
genomic DNA
RsaI
HphI
XbaI
hGH/hPL sensea hGH/hPL antisense
hGH-N hGH-V hPL-A hPL-B hPL-L
250 250 250 250 250
342 341 342 343 341
190 Uncut Uncut Uncut 168
Uncut 87 207 207 164
Uncut Uncut 100 100 Uncut
hGH/hPL sense hGH-V2 antisense
hGH-V2
312
404
Uncut
171/87/54
Uncut
hPRL sense hPRL antisense
hPRL
276
−
n.d.
n.d.
234/42
n.d., not done; RE, restriction enzyme. −, per definition not amplified due to the exon-spanning antisense primer. a Radioactively labeled primer for RE-analyses
GCTGAC-3% (exons 4 and 5 position 504 – 483), only allowing for cDNA amplification (Fig. 1). The sense primer 5%-GGGTTCATTACCAAGGCCATC-3% was located in exon 3 (position 228 – 249) according to the hPRL sequence (Cooke et al., 1981). PCR products were run on a 2% agarose gel, containing 4 mg/100 ml ethidium bromide at 60 V for 2 h and visualized and photographed under ultra violet (UV)-light.
2.6. Radioacti6e restriction enzyme analysis of the GH/PL gene product To determine the organ-specific pattern of expression of the GH/PL gene cluster, PCR-derived cDNAs were radioactively labeled at the 5%-end and semiquantitatively analyzed by restriction enzymes. In a total volume of 10 ml, 60 pmol of the GH/PL sense primer were incubated with 3000 Ci/mmol (g-32P)ATP (Amersham, Little Chalfont, UK) and 8 U of T4 polynucleotide kinase (Promega) for 60 min at 37°C. cDNAs (1 ml) were subjected to a PCR of 10 cycles under the above-mentioned conditions but employing the radiolabeled primer. These PCR products were then incubated in parallel with the restriction enzymes RsaI, HphI and XbaI to discriminate between GH-N, PL-L, PL-A/B and GH-V gene products (Table 1). Digested fragments were run on polyacrylamide gels (6%), which were then dried and exposed to an Agfa RP-1 film (Agfa, Vienna, Austria) at −70°C for 1 h. Relative quantification of the gene products was done by densitometry (Scan-Pack 2.0, Biometra, Go¨ttingen, Germany).
3. Results
3.1. Expression of hGH/hPL genes in the human testis All specimens (20 testes, three placentae, one pituitary) yielded a positive cDNA signal of 250 bp in length (Fig. 2A). Testicular RNA, without reverse transcription, served as a negative control. Since the chosen set of primers non-selectively amplifies cDNA of all five GH/PL genes, randomly selected testicular-derived radioactively labeled PCR samples (n=5) were analyzed for specific GH/PL gene expression patterns by restriction enzyme digestion and compared with the classical organs of hGH/hPL production, i.e., the pituitary (n= 1) and the placenta (n= 3) (Fig. 2B). As expected, the radioactive 5%-labeled pituitary-derived PCR product was completely digested by RsaI, and the resulting single 190 bp fragment, characteristic for GH-N transcripts, was visualized by autoradiography following electrophoresis, where the 3% non-labeled fragments do not show. Neither HphI, specific for PL-A/B, GH-V and PL-L gene products, nor XbaI, specific for PL-A/B products, cut pituitary-derived cDNA (Fig. 2B). Conversely, in the three placentae no GH-N (190 bp) or PL-L (168 bp) cDNA fragments were present after RsaI digestion. HphI treatment resulted in complete conversion of the 250 bp fragment into a 207 bp PL-A/B-specific, and in one of three placentae, in a weak 87 bp GH-V-specific band (Fig. 2B). The XbaI digest confirmed the abundance of PL-A/B products corresponding to a single major band of 100 bp. In contrast to these classical sites of somatotrophic hormone production, all analyzed testes (n=5) re-
G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
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vealed a different, but consistent, pattern of GH/PL gene expression (Fig. 2B). The entire GH/PL gene-cluster is transcriptionally active. After RsaI restriction enzyme analysis of the 5% radioactively-labeled GH/PL PCRproduct, two bands became visible, 190 bp (GH-N) and of 168 bp (PL-L) in length. HphI digestion generated three fragments of 207 bp (PL-A/B), 164 bp (PL-L) and 87 bp (GH-V). The identity of PL-A/B cDNA was confirmed by XbaI digestion (100 bp fragment).
Fig. 3. GH-V gene alternative splice product GH-V2 in testicular tissue. (A) Oligonucleotide primers specific for GH-V2, located in exon 3 and intron D amplified a specific fragment of the predicted size of 312 bp from both, placental (plac) and testicular poly-(A) + RNAs (T3, T5, T6). Moreover, it could be shown that the choriocarcinoma cell lines Jar and Bewo alternatively splice GH-V RNA as well. cDNAs from pituitary (pit) and poly-(A) + RNA from testes without reverse transcription ( −RT) served as negative controls. The −RT yielded a weak signal of 404 bp as a result of a residual contamination of the RNA samples with genomic DNA, retaining intron C. (B) The specificity of the 312 bp PCR product of placenta and testis was proven by restriction enzyme digestion using XbaI, RsaI and HphI. It was not recognized by XbaI, specific for PL-A/B (PL-A2), or by RsaI, specific for GH-N and PL-L gene products. HphI generated three fragments of 171/87/54 bp in length, corresponding to the predicted pattern for GH-V2 cDNA. SM, 1 kb size-marker (Gibco-BRL, Paisley, UK) 506/396/344/298/220/200 bp
Fig. 2. Organ-specific GH/PL gene expression in human testes. (A) The RT-PCR was designed to amplify transcripts of all 5 GH/PL genes. Surprisingly, the different testes (T1–T5) showed a positive signal of the predicted size (250 bp), as did the control tissues pituitary (pit) and two placentae (P1, P2). Testicular total RNA without reverse transcription (− RT) yielded only a band of 341 – 343 bp as a result of genomic DNA contamination of the RNA sample. As a size marker (SM), we used pBR 327/Hinf with the fragment pattern: 517/453/298/222/154 bp. (B) Semi-quantification of organspecific GH/PL gene expression was done by radioactive restriction enzyme digestion. While in the pituitary exclusively GH-N was detected and abundant PL-A/B and very few GH-V PCR-products in the placenta, in human testes the five genes are expressed at different levels (PL-A/B\ GH-V ] PL-L=GH-N). Relative quantities were determined by densitometry. The 250 bp cDNA bands were excised from the 2% gel, 32P-labeled sense primer incorporated and the products analyzed by restriction enzyme digestion. The digests were run on a 6% polyacrylamide gel and autoradiographed. Whereas RsaI cuts GH-N and PL-L gene products into visible 190 bp and 168 bp 5%-fragments, respectively, XbaI digests PL-A and PL-B cDNAs, resulting in a 100 bp 5%-fragment. HphI generates a fragment pattern of 207 bp (PL-A/B), 164 bp (PL-L) and 87 bp (GH-V) of 5%-32P-labeled gene-products.
The alternative splice product GH-V2 includes intron D into the open reading frame. Three of 20 testicular cDNA samples revealed specific signals of 312 bp for this alternative splice product of the GH-V gene (Fig. 3A). Again, the specificity of the amplified cDNA fragment was proven by analytical digestion with RsaI, HphI and XbaI. XbaI was used to discriminate GH-V2 from the theoretically co-amplified alternative splice product of the PL-A gene (PL-A2), whereas digestion by HphI generated fragment pattern specific for GH-V2 (171/87/ 54 bp) in placentae and testes (Fig. 3B).
3.2. Testicular PRL gene expression Fourteen of twenty (70%) human testicular mRNAderived cDNA-samples in the RT-PCR showed a specific signal for PRL of 276 bp in length (Fig. 4). Its specificity was confirmed by digestion with the restriction endonuclease XbaI of the 276 bp band derived from pituitary, placenta and four randomly selected testicular cDNA samples (Fig. 4). This yielded two PRL-specific fragments of the predicted lengths, 234 and 42 bp.
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G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
3.3. hPL-A/B, hGH-N and hPRL in cytosolic testicular homogenates Sensitivities of the IFMAs for PRL, GH-N and PL-A/B were 2.4 pg/ml, the non-specific signals of the zero-standard 1424 9227, 668 9 93 and 658 9 107 counts per second (cps), cross-reactivities with the complementary protein hormones less than 0.1% and the intra- and inter-assay coefficients of variation less than 10%, as determined on three consecutive days. Concerning the IFMA for hPL the within-assay precision of the standard (n = 6) averaged at 8% (range: 2.1–16%). Three pregnancy sera containing high levels of hPL ranging from 1.2 to 2.9 ng/ml were serially diluted in assay buffer. The respective serial dilutions ran in parallel to the hormone standard over three orders of magnitude and revealed a linearity of 0.998– 0.999 (mean 0.999). Recovery studies were performed by adding 2.5 ng hPL/ml and 10 ng hPL/ml to eight sera containing 0.09 – 3.4 ng hPL/ml. The respective recovery values ranged from 102 to 125% (mean 110%). In addition, testicular cytosolic and placental extracts were spiked with 2.5 and 10 ng/ml, yielding recovery
Fig. 4. Expression of PRL gene in human testicular tissue. Poly (A) + RNA from different testes (T1–T4) and total RNA from pituitary and placenta were used as template for RT-PCR. Due to the exonspanning antisense PRL-specific primer, a 276 bp PRL cDNA fragment, but no genomic DNA, was selectively amplified. Restriction enzyme digestion with XbaI of this 276 bp PCR fragment yielded two bands at 234 and 42 bp, identical to that of the pituitary derived PRL gene-product. Testicular mRNA of the respective samples without reverse transcription ( −RT) served as a negative control. pBR 327/HinfI was used as size marker (SM): 517/453/298/222/154 bp.
values of 109–118% (mean 111%). Homogenized subcapsular testicular tissue (n= 11) was extracted and analyzed in duplicate in all three assays. Cytosolic hPRL concentrations corresponded to 58.7 ng/g tissue wet weight (tww; mean 9 S.D. 37.1 ng/g; range 14–160 ng), hGH-N concentrations to 5.1 ng/g tww (mean9 S.D. 3.0 ng/g; range: 2.7–12.5. ng/g) and hPL-A/B concentrations to 1.0 ng/g tww (mean9 S.D. 0.74 ng/g; range: 0.24–2.2 ng/g). Most importantly, normal serum values of an age-matched elderly male collective (n= 18; age: 72 9 3 years) were lower than 0.02 ng hPL/ml.
4. Discussion Three cell types of the human testis, Leydig, peritubular and Sertoli cells, regulated by the concerted action of gonadotropins, provide the hormonal milieu and essential factors for germ cell development and functional intra-testicular cell-cell interactions (Skinner, 1991). Pituitary-derived LH and FSH are definitely the primary hormonal support for Leydig and Sertoli cell function. The most important local regulatory hormone is testosterone, synthesized by Leydig cells, which acts as the major paracrine stimulator of germ cell development (Dufau, 1988). It has been shown that glycoprotein hormones, like LHb and CGb (Berger et al., 1994) and GHRH (Berry et al., 1992; Ciampani et al., 1992), are eutopically produced in the human testis. Increasing evidence suggests that locally synthesized factors like IGF-I (Skinner, 1991), GHRH (Berry et al., 1992; Ciampani et al., 1992), oxytocin, activin, b-endorphin, enkephalins and dynorphins (Skinner, 1991) may act in an auto/paracrine manner on testicular physiology. In this study, we demonstrated the production of GH/PL and PRL in the human testis at the mRNA and protein levels. In the pituitary, as expected and confirmed by us, the GH-N gene is the only transcriptionally active gene (Nachtigal et al., 1993), whereas the other four genes are repressed by binding of pituitary-specific factor (PSF) to 5%-located p-sequences (Nachtigal et al., 1993). Conversely, under control of the PL transcriptional enhancer (Walker et al., 1990), PL-A/B/L and the GH-V genes are transcribed in the placenta (Chen et al., 1989), but GH-N is not. PL-A is expressed at a 5-fold higher level than PL-B at term (MacLeod et al., 1992), but both mRNAs encode for an identical 22 K PL protein. GH-V gene transcription increases in parallel with that of PL, but at levels at least two orders of magnitude lower (Lytras et al., 1994). The proportion of alternatively spliced GH-V transcripts retaining intron D (GH-V2) is similarly regulated, increasing 3-fold during gestation up to a total of 15% of GH-V at term (MacLeod et al., 1992). PL-L is transcribed at very low levels in the placenta (Chen et al., 1989; Lytras et al.,
G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
1994) and these transcripts undergo two distinct and developmentally unaltered splicing pathways between exon 2 and 3 (Chen et al., 1989; Press et al., 1994). With our methodology, simultaneously amplifying transcripts of the entire gene-cluster, we could find only a weak GH-V, and no PL-L, gene expression in the term placentae (n=3). In a previous report (Lytras et al., 1994), based on a similar RT-PCR quantification of placental GH/PL mRNA (n =3), the proportions of GH-V (4.2%) and PL-L ( B1%) PCR-products, were higher compared with our results. In contrast to pituitary and placenta, the human testis has its own specific differential expression pattern of the GH/PL gene-cluster: PL-A/B \GH-V ] PL-L= GH-N. Moreover, we were able to demonstrate production of not only GH-V, but its alternative splice product GH-V2. Selective GH-N and PL-A/B transcription in the human ovary (unpublished data) adds further to the concept of organ specific GH/PL gene expression patterns. Due to GH/PL sense primer localisation at the beginning of exon 3 (Fig. 1), this particular PCR amplifies only the major products of the five GH/PL genes encoding for the 22 K protein form, not the 20 K GH-N, which misses the first 45 bp of exon 3, or the 20 K PL-L, truncated at the first 73 bp of exon 3. The amplified alternative splice product of the PL-L gene uses a cryptic splice acceptor site located 4 bp 3% from the position of the splice acceptor site of exon 3 utilized in the other genes of the cluster (Chen et al., 1989; MacLeod et al., 1992; Press et al., 1994). In the testis, the ratio of GH-V and PL-L to PL-A/B genetranscripts is higher than in the placenta, indicating a different regulation of GH/PL gene transcription. With highly specific and sensitive IFMAs, we detected hPL-A/B, hGH-N and hPRL in homogenized testicular tissue (1.0 ng/g, 5.1 ng/g and 58.7 ng/g tww). GH-V, PL-L and variants like GH-V2, PL-A2 and PL-L% (MacLeod et al., 1992) could not be investigated due to the lack of appropriate antibodies. As generally accepted and shown for the human B-cell Burkitt lymphoma cell line (Ramos) by RT-PCR and sequencing of the cDNA fragment human lymphocytes produce exclusively GH-N (Lytras et al., 1993). Thus minor amounts of testicular GH-N but not of PL-A/B might originate from few contaminating lymphocytes. Most noteworthy, a positive concentration gradient exists between cubital vein serum ( B 0.02 ng hPL/ml) of age-matched elderly probands to the testis (1 ng hPL/g tww), confirming testicular hPL synthesis. In addition to the villious syncytiotrophoblast, trophoblastic and non-trophoblastic germ cell neoplasms can ectopically produce hPL as well as the established marker hCG (Frantz et al., 1965; Samaan et al., 1966; Weintraub and Rosen, 1971). We observed that the healthy testis is a source of eutopically-synthesized protein hormones. The biological roles and functions of
59
GH-V, PL-A/B/L and their alternative splice products are still a matter of debate, and only the effects of exogenous administration of hGH-N on the testicular steroidogenesis in panhypopituitary patients are well known (Balducci et al., 1993; Chatelain et al., 1991). In these individuals, hGH-N augments hCG-triggered testosterone production and spermatogenesis. Moreover, GH and IGF-I increase the numbers of testicular LH-receptors and steroidogenic responsiveness in GHdeficient dwarf mice (Chatelain et al., 1991). PRL also exerts modulatory effects on the Leydig cells, which express the PRL-receptor on their surface (Bouhdiba et al., 1989), by regulating the number of LH-receptors (Zipf et al., 1978; Dufau, 1988). The occurrence of ectopic hPL-production in testicular germ cell neoplasms lead to the hypothesis that developing germ cells of normal testes could produce these protein hormones. GHRH localized in early sperm stages and mature sperm cells (Berry et al., 1992) might act as an auto/ paracrine releasing-factor for GH/PL gene-products. The binding of these molecules to human testicular GH-receptor, presumably present on Sertoli cells (Lobie et al., 1990) could, in turn, support spermatogenesis via IGF-I, produced by this cell type (Skinner, 1991). The occurrence of eutopically-produced PRL and GH/ PL gene-products in the testis supports the view that they may have an auto/paracrine effect on the testosterone production of Leydig cells and/or are involved in the process of spermatogenesis.
Acknowledgements The authors thank Miss Regine Gerth for her excellent technical assistance and the NIADDK (NIH, Bethesda) for their generous supply of GH, PRL and PL.
References Balducci, R., Toscano, V., Mangiantini, A., Bianchi, P., Guglielmi, R., Boscherini, B., 1993. The effect of growth hormone administration on testicular response during gonadotropin therapy in subjects with combined gonadotropin and growth hormone deficiencies. Acta. Endocrinol. 128, 19 – 23. Bartke, A., 1980. Role of prolactin in reproduction in male mammals. Fed. Proc. 39, 2577 – 2581. Berger, P., Kranewitter, W., Madersbacher, S., Gerth, R., Geley, S., Dirnhofer, S., 1994. Eutopic production of human chorionic gonadotropin b (hCGb) and luteinizing hormone b (hLHb) in the human testis. FEBS Lett. 343, 229 – 233. Berger, P., Staindl, B., Wick, G. (1988) Antigenic features of the human protein hormones elucidated by monoclonal antibodies. In: Paul, I.P., McCurden, A.B., Schuetz, P.W. (Eds.) The Influence of New Technology in the Medical Practice. Macmillian Press, London, pp. 212 – 219. Berry, S.A., Srivastava, C.H., Rubin, L.R., Phipps, R.W., Pescovitz, O.H., 1992. Growth hormone-releasing hormone-like messenger
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ribonucleic acid and immunoreactive peptide are present in human testis and placenta. J. Clin. Endocrinol. Metab. 75, 281 – 284. Bouhdiba, M., Leroy-Martin, B., Peyrat, J.P., Sant-Pol, P., Dijane, J., Leonardelli, J., 1989. Immunohistochemical detection of prolactin and its receptor in human testis. Andrologia 21, 223 – 228. Chatelain, P.G., Sanchez, P., Saez, J.M., 1991. Growth hormone and insulin-like growth factor I treatment increase testicular luteinizing hormone receptors and steroidogenic responsiveness of growth hormone deficient dwarf mice. Endocrinology 128, 1857 – 1862. Chen, E.Y., Liao, Y.C., Smith, D.H., Barrena-Saldana, H.A., Gelinas, R.E., Seeburg, P.H., 1989. The growth hormone locus: nucleotide sequence, biology and evolution. Genomics 4, 479 – 497. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–157. Ciampani, T., Fabbri, A., Isidori, A., Dufau, M.L., 1992. Growth hormone-releasing hormone is produced by rat Leydig cell in culture and acts as a positive regulator of Leydig cell function. Endocrinology 131, 2785–2792. Cooke, N.E., Coit, D., Shine, J., Baxter, J.D., Martial, J.A., 1981. Human Prolactin cDNA structural analysis and evolutionary comparisons. J. Biol. Chem. 256, 4007–4016. Dufau, M.L., 1988. Endocrine regulation and communicating functions of the Leydig cell. Annu. Rev. Physiol. 50, 483 – 508. Frantz, A.G., Rabkin, M.T., Friesen, H., 1965. Human placental lactogen in choriocarcinoma of the male. Measurement by Radioimmunoassay.. J. Clin. Endocrinol. Metab. 25, 1136 – 1139. Handwerger, S., 1991. Clinical counterpoint: The physiology of placental lactogen in human pregnancy. Endocr. Rev. 12, 329 – 336. Lytras, A., Bock, M.E., Dodd, J.G., Cattini, P.A., 1994. Detection of placental growth hormone variant and chorionic somatomammotropin ribonucleic acid expression in human trophoblastic neoplasms by reverse transcriptase-polymerase chain reaction. Endocrinology 134, 2461–2467. Lytras, A., Quan, N., Vrontakis, M.E., Shaw, J.E., Cattini, P.A., Friesen, H.G., 1993. Growth hormone expression in human Burkitt lymphoma serum-free Ramos cell line. Endocrinology 132, 620 – 628. Lobie, P.E., Breipohl, W., Aragon, J.G., Waters, M.J., 1990. Cellular localisation of the growth hormone receptor/binding protein in the male and female reproduction systems. Endocrinology 126, 2214 – 2221. MacLeod, J.N., Lee, A.K., Liebhaber, S.A., Cooke, N.E., 1992. Developmental control and alternative splicing of the placentally expressed transcripts from the human growth hormone gene
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cluster. J. Biol. Chem. 267, 14219 – 14226. Madersbacher, S., Shu-Chen, T., Schwarz, S., Dirnhofer, S., Wick, G., Berger, P., 1993a. Time-resolved immunofluorometry and other frequently used immunoassay types for follicle stimulating hormone compared by using identical monoclonal antibodies. Clin. Chem. 39, 229 – 233. Madersbacher, S., Stulnig, T., Huber, L.A., Scho¨nitzer, D., Dirnhofer, S., Wick, G., Berger, P., 1993b. Serum glycoprotein hormones and their free a-subunit in a healthy elderly population selected according to the SENIEUR protocol. Mech. Ageing Dev. 71, 223 – 233. Meiters, J. (1988) Biological functions of prolactinin mammals. In: Hoshino, K., (Ed.), Prolactin gene family and its receptors. Elsevier, Amsterdam, pp. l23 – 130. Nachtigal, N.W., Nickel, B.E., Cattini, P.A., 1993. Pituitary-specific repression of placental members of the human growth hormone gene family. J. Biol. Chem. 268, 8473 – 8479. Press, A.M., Cooke, N.E., Liebhaber, S.A., 1994. Complex alternative splicing partially inactivates the human chorionic somatomammotropin-like (hCS-L) gene. J. Biol. Chem. 269, 23220 – 23229. Sabharwal, P., Glaser, R., Lafuse, W., Varma, S., Liu, Q., Arkins, S., Kooijman, R., Kutz, L., Kelley, K.W., Malarkey, W.B., 1992. Prolactin synthesized and secreted by human peripheral blood mononuclear cells: An autocrine growth factor for lymphoproliferation. Proc. Natl. Acad. Sci. USA 89, 7713 – 7716. Samaan, N., Yen, S.C.C., Friesen, H., Pearson, O.H., 1966. Serumplacental lactogen levels during pregnancy and in trophoblastic disease. J. Clin. Endocrinol. Metab. 26, 1303 – 1308. Skinner, M.K., 1991. Cell-cell interaction in the testis. Endocr. Rev. 12, 45 – 77. Staindl, B., Berger, P., Kofler, R., Wick, G., 1987. Monoclonal antibodies against human, bovine and rat prolactin: Epitope mapping of human prolactin and development of a two-site immunoradiometric assay. J. Endocrinol. 114, 311 –318. Walker, W.H., Fritzpatrick, S.L., Saunders, G.F., 1990. Human placental lactogen transcriptional enhancer. J. Biol. Chem. 265, 12940 – 12948. Wallis, M., 1992. The expanding growth hormone/prolactin family. J. Mol. Endocrinol. 9, 185 – 188. Weintraub, B.D., Rosen, S.W., 1971. Ectopic production of human chorionic somatomammotropin by nontrophoblastic cancers. J. Clin. Endocrinol. Metab. 32, 94 – 101. Zipf, W.B., Payne, A.H., Kelch, R.P., 1978. Prolactin, growth hormone, and luteinizing hormone in the maintenance of testicular luteinizing hormone receptor. Endocrinology 103, 595–600.
Organ-specific expression pattern of the human growth hormone/placental lactogen gene-cluster in the testis G. Untergasser a, W. Kranewitter a, P. Schwa¨rzler a, S. Madersbacher c, S. Dirnhofer a,b, P. Berger a,d,* a
Institute for Biomedical Aging Research of the Austrian Academy of Sciences, A-6020 Innsbruck, Austria b Department of Pathology, Uni6ersity of Innsbruck, A-6020 Innsbruck, Austria c Department of Urology, Uni6ersity of Vienna, Vienna, Austria d Institute for General and Experimental Pathology, Uni6ersity of Innsbruck, A-6020 Innsbruck, Austria Received 14 December 1996; accepted 24 March 1997
Abstract In addition to testosterone, the essential paracrine factor for spermatogenesis, a number of potential auto/paracrine regulatory substances such as b-endorphins, enkephalins, chorionic gonadotropin b, growth hormone-releasing hormone (GHRH) and insulin-like growth factor I (IGF-I) have been identified in the testis of various mammalian species. The latter findings prompted us to investigate a possible eutopic production of GH, placental lactogen (PL) and PRL in human testes. Specific expression of testicular GH/PL mRNA (n=20) was shown by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of primers designed to non-selectively amplify any transcript of the five GH/PL genes (GH-N, GH-V, PL-A, PL-B, PL-L). In contrast to the classical sites of production, the pituitary (exclusively GH-N transcripts) and the placenta (PL-A/B \99%, GH-VB1%), radioactive semiquantitative restriction enzyme analysis of the PCR-products revealed, that the testis has its own organ-specific pattern of GH/PL gene expression: PL-A/B \ GH-V ]PL-L =GH-N. All three organs express the single PRL gene, and testis and placenta show the alternative splice variant GH-V2. Immunological analyses by immunofluorometric assays for hPL-A/B, hGH-N and hPRL, demonstrated significant amounts of protein hormones in all testicular cytosolic homogenates (means: hPL 1.0 ng/g, hGH 5.1 ng/g and hPRL 58.7 ng/g tissue wet weight). Most noteworthy, hPL serum levels in an elderly age-matched healthy subjects (n=18) were B 0.02 ng/ml. The concept of purely endocrine functions of placental and pituitary-derived GH/PL needs to be reassessed, since human testicular synthesis of these molecules suggest auto/paracrine functions in the male reproductive tract. © 1997 Elsevier Science Ireland Ltd. Keywords: Growth hormone; Placental lactogen; Prolactin; Human testis; Eutopic production
1. Introduction The genes coding for the human (h) protein hormones prolactin (hPRL) and for the growth hormone/ placental lactogen (hGH/hPL) gene-cluster segregated from a common ancestral gene approximately 400 million years ago (Wallis, 1992). The members of this protein hormone family share genetic and subsequent * Corresponding author. Tel.: +43 512 58391924; fax: + 43 512 5839198; e-mail: [email protected]
structural, immunological and biological features. Located on chromosome 17 q22–24, the GH/PL genecluster is comprised of five highly related genes (GH-N, PL-L, PL-A, GH-V and PL-B), which evolved through a series of duplication events approx. ten million years ago, and share more than 90% nucleotide sequence identity in their coding and flanking regions (Chen et al., 1989). Despite these similarities, they are expressed in an organ-specific manner at high levels either in the anterior pituitary (GH-N) or as the other four genes, in the syncytiotrophoblast of the human placenta.
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These placental hormones and alternative splice products of the GH-V and PL-A genes, GH-V2 and PL-A2, containing intron D, are coordinately induced during fetal development, increasing between weeks 12 and 20 of gestation and then plateauing through term (MacLeod et al., 1992). Their function during pregnancy is still a matter of debate, but they may have an important influence on the carbohydrate- and lipidmetabolism of mother and fetus. In addition, PL stimulates development and cell division of mammary gland epithelial cells (Handwerger, 1991). With primary support from the pituitary-derived gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH), the three major somatic cell types of the human testis (Leydig, peritubular and Sertoli cells) provide the hormonal milieu and essential factors for germ cell development and intra-testicular cell-cell interactions. Supporting the actions of gonadotropins, pituitary-derived GH and PRL play a modulatory role in the maintenance of testicular function (Zipf et al., 1978; Balducci et al., 1993; Chatelain et al., 1991). GH increases the numbers of testicular luteinizing hormone receptors (LH-R), and choriogonadotropin (hCG)-stimulated testosterone production in men and mice (Balducci et al., 1993; Chatelain et al., 1991). Evidence for an important role of PRL is seen in patients with hyperprolactinemia, who can be infertile, due to down-regulation of the LH-R on Leydig cells via the PRL-receptor (Zipf et al., 1978; Meiters, 1988; Bartke, 1980). Recently, it has been shown that, in the human testis, GH-releasing hormone (GHRH) (Berry et al., 1992; Ciampani et al., 1992) and the glycoprotein hormone subunits hCGb and LHb are transcribed and present in low amounts (Berger et al., 1994). Increasing evidence that locally produced factors including b-endorphin, enkephalins, dynorphins, oxytocin, activin, LH-releasing hormone (LHRH), GHRH, insulin-like growth factor I (IGF-I) (Skinner, 1991) and CGb might act in auto/paracrine fashions prompted us to investigate a possible eutopic expression of protein hormones in the human testis. 2. Materials and methods
2.1. Samples Testicular tissue (n =20) from previously untreated prostatic cancer patients undergoing subcapsular orchiectomy (age range, 68 – 83 years), one post-mortem pituitary (female, age 72 years; reason of death, cardiac failure), Jar and Bewo choriocarcinoma cells (106 cells) and human term placentae (n =3) were snap frozen in liquid nitrogen and kept at − 70°C until used for RNA analysis in the reverse transcription polymerase-chainreaction (RT-PCR).
One gram of testicular tissue (n = 11) was homogenized on ice in 5 ml phosphate buffer saline (PBS) (Ultra-Turrax, Janke and Kunkel, Stauffen, Germany). After centrifugation (10 000 × g, 20 min, 4°C) the aqueous cytosolic extract was stored at −20°C until analyses for hGH/hPRL/hPL content by immunofluorometric assays (IFMA). Phenylmethane-sulfonyl fluoride (Merck, Darmstadt, Germany) was added as protease inhibitor (1 mM).
2.2. Immunofluorometric assays for hPL-A/B, hGH-N and hPRL Specific IFMAs for pituitary derived hGH-N and hPRL and hPL-A/B were established based on our own panel of well characterized monoclonal antibodies (MCA) (Staindl et al., 1987; Berger et al., 1988). IFMA technology provided the highest sensitivity and widest measuring range. They were performed in analogy to our assays for glycoprotein hormones (Madersbacher et al., 1993a,b). hPRL 81/541 (NIBSC, London, UK), hGH 66/217 (NIBSC) and hPL (NIADDK, NIH, Bethesda, MD) were used as hormone standard preparations. Ten mg of highly purified MCA, coded as INN-hPL37, INN-hGH-2 or INN-hPRL-9, respectively, were diluted in 100 ml PBS pH 7.2 and incubated for 2 h at 37°C in a microtiter plate (Nunc, Roskilde, Denmark). Remaining binding sites were blocked with 200 ml of 1% bovine serum albumin (BSA) in PBS for 30 min at 37°C. The plates were then washed three times with 200 ml per well with PBS containing 0.5 ml Tween 20 and 5 g of thiomersal/l. For the actual assay, we used an incubation volume of 100 ml/well and an assay buffer consisting of 50 mM Tris–HCl (pH 7.75), 9 g/l NaCl, 5 g BSA/l, 0.1 g/l Tween 40, 0.5 g/l bovine g-globulin and 20 mM diethylenetriaminepentaacid (Sigma-Aldrich, Milwaukee, WI). Graded amounts of the hormone standards or homogenized testicular tissue (1:2 in assay buffer) were allowed to react on a orbit shaker (500 rpm, 90 min, 20°C) followed by three washes and subsequently by incubation with 100 ng of europium-labeled detection MCA INN-hPL-5, INN-hGH-5 or INN-hPRL-1, respectively (30 min, 20°C, orbit shaker). After extensive washing, enhancement solution was added (0.1 mM potassium-phthalate, pH 3.2, containing 15 mmol of 2-naphtoyltrifluoroaceton, 50 mmol or tri-N-octylphosphine oxide, and l g of Triton X-100 per liter) and incubated for 5 min on a orbit shaker. Time-resolved fluorescence was measured for 1 s in a fluorometer (1232 Delfia-fluorometer; Wallac, Turku, Finland).
2.3. Isolation of Poly (A) + RNA Total RNA was extracted from 100 mg of homogenized tissue (Ultra-Turrax, Janke and Kunkel) by the
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Fig. 1. PRL and GH/PL gene organisation and localization of the RT-PCR primers. Due to their common origin, the single gene for PRL and the five genes of the GH/PL gene-cluster have a similar exon/intron structure. Five exons, coded 1 – 5, are separated by four introns, A–D. The two PRL-specific primers are located in regions of exon 3 and exon 4/5 (Sabharwal et al., 1992), where PRL and GH/PL share only 30% of their nucleotide sequence. In contrast, due to high nucleotide sequence homology between the GH/PL genes (\ 90%), we used only a single pair of primers (Lytras et al., 1994), located in exons 3 and 4 at positions, where all five genes show sequence identity, thereby amplifying all major GH/PL gene products. If necessary, the generated 250 bp cDNA fragment can be distinguished by size from the longer 341 – 343 bp fragment of potentially amplified contaminating genomic DNA. To detect the alternative splice product of the GH-V gene, GH-V2, the 5%-non-selective GH/PL primer was combined with a GH-V specific primer, located in intron D at a position, where the PL-A gene shows nucleotide sequence divergence.
single step acid guanidinium thiocyanate phenol/chloroform method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). Extreme care was taken to avoid specimen contamination during the extraction procedure. Testes were homogenized prior to placentae and the pituitary, including intermitting washing steps (5 min) of the dispersing tool in 0.1 M NaOH and 0.5% SDS. Additionally, filtered pipette tips (Bio-Rad; Richmond, CA) were used. Integrity of RNA was assessed by analysis of 28S and 18S rRNA on ethidium bromide-stained 1% agarose gels (Boehringer-Mannheim, Mannheim, Germany). Poly (A) + RNA was isolated using a oligo(dT 30)-Cellulose (Serva, Heidelberg, Germany) according the manufacturer’s instructions.
2.4. Re6erse transcription Between 500– 1000 ng of poly (A) + RNA were diluted in 10 ml diethylpyrocarbonate (DEPC, SigmaAldrich) water in a 0.5 ml PCR tube (Perkin-Elmer Cetus, Norwalk, CT), denatured at 65°C for 10 min in a programmable thermal cycler (TC; Techne, Cambridge, UK), then rapidly cooled to 4°C and the reverse transcription performed in a final volume of 50 ml under the following reaction conditions: 50 mM Tris– HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each of dATP, dCTP, dGTP and dTTP (Promega, Madison, WI), 10 U human placental ribonuclease inhibitor (Promega), 200 pmol random hexamer oligonucleotide (Boehringer – Mannheim) and 50 U MMLV-Reverse Transcriptase (Promega). The reaction mix was incubated for 8 min at 20°C and 8 min at 25°C to achieve optimal hybridization between the RNA and the hexamer primers. The reverse transcriptase was then allowed to proceed at 37°C for 30 min.
This cycle was repeated four times to increase the amount of cDNA.
2.5. Polymerase chain reaction Each first strand reaction was adjusted to a volume of 70 ml and 7 ml (50–100 ng) were used for the PCR amplification. The PCR reaction was performed in a final volume of 25 ml under the following conditions: 10 mM Tris–HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 4 pmol each of the two oligonucleotide primers specific for each gene-product (PRL, GH/ PL, GH-V2) and 0.125 U Taq polymerase (Promega). After initial denaturation for 2 min at 95°C, 40 cycles of amplification were performed with 55 s denaturation at 95°C, 50 s annealing at either 58°C (PRL), 44°C (GH/PL) or 54°C (GH-V2) and 45 s extension at 73°C in a thermocycler. The last cycle had an elongation time of 5 min at 73°C. A set of primers amplifying cDNA of all members of the GH/PL gene cluster (Lytras et al., 1994) 5%-CAGAAGTATTCATTCCTGCA-3% located at position 737–756 (exon 3) and 5%-TTTGGATGCCTTCCTCTAG-3%, position 1060–1078 (exon 4, numbered according to the hGH-N gene sequence (Chen et al., 1989)), and a second set, specific for the alternative splice product GH-V2 (Fig. 1) 5%-CAGAAGTATTCATTCCTGCA-3% located at position 748–767 (exon 3) and 5%-TTTCTCCCCAGTCCCTGG-3%, position 1146–1165 (intron D) according to the sequence of hGH-V (Chen et al., 1989), were custom-made by Microsynth (Windisch, Switzerland). The antisense PCR primer for PRL (Sabharwal et al., 1992) was axon-spanning 5%-TTCAGGATGAACCTG-
G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
56 Table 1
PCR-primer set
Amplified gene product
DNA-fragment length (bp)
cDNA-RE-analyses: visible fragments (bp)
cDNA
genomic DNA
RsaI
HphI
XbaI
hGH/hPL sensea hGH/hPL antisense
hGH-N hGH-V hPL-A hPL-B hPL-L
250 250 250 250 250
342 341 342 343 341
190 Uncut Uncut Uncut 168
Uncut 87 207 207 164
Uncut Uncut 100 100 Uncut
hGH/hPL sense hGH-V2 antisense
hGH-V2
312
404
Uncut
171/87/54
Uncut
hPRL sense hPRL antisense
hPRL
276
−
n.d.
n.d.
234/42
n.d., not done; RE, restriction enzyme. −, per definition not amplified due to the exon-spanning antisense primer. a Radioactively labeled primer for RE-analyses
GCTGAC-3% (exons 4 and 5 position 504 – 483), only allowing for cDNA amplification (Fig. 1). The sense primer 5%-GGGTTCATTACCAAGGCCATC-3% was located in exon 3 (position 228 – 249) according to the hPRL sequence (Cooke et al., 1981). PCR products were run on a 2% agarose gel, containing 4 mg/100 ml ethidium bromide at 60 V for 2 h and visualized and photographed under ultra violet (UV)-light.
2.6. Radioacti6e restriction enzyme analysis of the GH/PL gene product To determine the organ-specific pattern of expression of the GH/PL gene cluster, PCR-derived cDNAs were radioactively labeled at the 5%-end and semiquantitatively analyzed by restriction enzymes. In a total volume of 10 ml, 60 pmol of the GH/PL sense primer were incubated with 3000 Ci/mmol (g-32P)ATP (Amersham, Little Chalfont, UK) and 8 U of T4 polynucleotide kinase (Promega) for 60 min at 37°C. cDNAs (1 ml) were subjected to a PCR of 10 cycles under the above-mentioned conditions but employing the radiolabeled primer. These PCR products were then incubated in parallel with the restriction enzymes RsaI, HphI and XbaI to discriminate between GH-N, PL-L, PL-A/B and GH-V gene products (Table 1). Digested fragments were run on polyacrylamide gels (6%), which were then dried and exposed to an Agfa RP-1 film (Agfa, Vienna, Austria) at −70°C for 1 h. Relative quantification of the gene products was done by densitometry (Scan-Pack 2.0, Biometra, Go¨ttingen, Germany).
3. Results
3.1. Expression of hGH/hPL genes in the human testis All specimens (20 testes, three placentae, one pituitary) yielded a positive cDNA signal of 250 bp in length (Fig. 2A). Testicular RNA, without reverse transcription, served as a negative control. Since the chosen set of primers non-selectively amplifies cDNA of all five GH/PL genes, randomly selected testicular-derived radioactively labeled PCR samples (n=5) were analyzed for specific GH/PL gene expression patterns by restriction enzyme digestion and compared with the classical organs of hGH/hPL production, i.e., the pituitary (n= 1) and the placenta (n= 3) (Fig. 2B). As expected, the radioactive 5%-labeled pituitary-derived PCR product was completely digested by RsaI, and the resulting single 190 bp fragment, characteristic for GH-N transcripts, was visualized by autoradiography following electrophoresis, where the 3% non-labeled fragments do not show. Neither HphI, specific for PL-A/B, GH-V and PL-L gene products, nor XbaI, specific for PL-A/B products, cut pituitary-derived cDNA (Fig. 2B). Conversely, in the three placentae no GH-N (190 bp) or PL-L (168 bp) cDNA fragments were present after RsaI digestion. HphI treatment resulted in complete conversion of the 250 bp fragment into a 207 bp PL-A/B-specific, and in one of three placentae, in a weak 87 bp GH-V-specific band (Fig. 2B). The XbaI digest confirmed the abundance of PL-A/B products corresponding to a single major band of 100 bp. In contrast to these classical sites of somatotrophic hormone production, all analyzed testes (n=5) re-
G. Untergasser et al. / Molecular and Cellular Endocrinology 130 (1997) 53–60
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vealed a different, but consistent, pattern of GH/PL gene expression (Fig. 2B). The entire GH/PL gene-cluster is transcriptionally active. After RsaI restriction enzyme analysis of the 5% radioactively-labeled GH/PL PCRproduct, two bands became visible, 190 bp (GH-N) and of 168 bp (PL-L) in length. HphI digestion generated three fragments of 207 bp (PL-A/B), 164 bp (PL-L) and 87 bp (GH-V). The identity of PL-A/B cDNA was confirmed by XbaI digestion (100 bp fragment).
Fig. 3. GH-V gene alternative splice product GH-V2 in testicular tissue. (A) Oligonucleotide primers specific for GH-V2, located in exon 3 and intron D amplified a specific fragment of the predicted size of 312 bp from both, placental (plac) and testicular poly-(A) + RNAs (T3, T5, T6). Moreover, it could be shown that the choriocarcinoma cell lines Jar and Bewo alternatively splice GH-V RNA as well. cDNAs from pituitary (pit) and poly-(A) + RNA from testes without reverse transcription ( −RT) served as negative controls. The −RT yielded a weak signal of 404 bp as a result of a residual contamination of the RNA samples with genomic DNA, retaining intron C. (B) The specificity of the 312 bp PCR product of placenta and testis was proven by restriction enzyme digestion using XbaI, RsaI and HphI. It was not recognized by XbaI, specific for PL-A/B (PL-A2), or by RsaI, specific for GH-N and PL-L gene products. HphI generated three fragments of 171/87/54 bp in length, corresponding to the predicted pattern for GH-V2 cDNA. SM, 1 kb size-marker (Gibco-BRL, Paisley, UK) 506/396/344/298/220/200 bp
Fig. 2. Organ-specific GH/PL gene expression in human testes. (A) The RT-PCR was designed to amplify transcripts of all 5 GH/PL genes. Surprisingly, the different testes (T1–T5) showed a positive signal of the predicted size (250 bp), as did the control tissues pituitary (pit) and two placentae (P1, P2). Testicular total RNA without reverse transcription (− RT) yielded only a band of 341 – 343 bp as a result of genomic DNA contamination of the RNA sample. As a size marker (SM), we used pBR 327/Hinf with the fragment pattern: 517/453/298/222/154 bp. (B) Semi-quantification of organspecific GH/PL gene expression was done by radioactive restriction enzyme digestion. While in the pituitary exclusively GH-N was detected and abundant PL-A/B and very few GH-V PCR-products in the placenta, in human testes the five genes are expressed at different levels (PL-A/B\ GH-V ] PL-L=GH-N). Relative quantities were determined by densitometry. The 250 bp cDNA bands were excised from the 2% gel, 32P-labeled sense primer incorporated and the products analyzed by restriction enzyme digestion. The digests were run on a 6% polyacrylamide gel and autoradiographed. Whereas RsaI cuts GH-N and PL-L gene products into visible 190 bp and 168 bp 5%-fragments, respectively, XbaI digests PL-A and PL-B cDNAs, resulting in a 100 bp 5%-fragment. HphI generates a fragment pattern of 207 bp (PL-A/B), 164 bp (PL-L) and 87 bp (GH-V) of 5%-32P-labeled gene-products.
The alternative splice product GH-V2 includes intron D into the open reading frame. Three of 20 testicular cDNA samples revealed specific signals of 312 bp for this alternative splice product of the GH-V gene (Fig. 3A). Again, the specificity of the amplified cDNA fragment was proven by analytical digestion with RsaI, HphI and XbaI. XbaI was used to discriminate GH-V2 from the theoretically co-amplified alternative splice product of the PL-A gene (PL-A2), whereas digestion by HphI generated fragment pattern specific for GH-V2 (171/87/ 54 bp) in placentae and testes (Fig. 3B).
3.2. Testicular PRL gene expression Fourteen of twenty (70%) human testicular mRNAderived cDNA-samples in the RT-PCR showed a specific signal for PRL of 276 bp in length (Fig. 4). Its specificity was confirmed by digestion with the restriction endonuclease XbaI of the 276 bp band derived from pituitary, placenta and four randomly selected testicular cDNA samples (Fig. 4). This yielded two PRL-specific fragments of the predicted lengths, 234 and 42 bp.
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3.3. hPL-A/B, hGH-N and hPRL in cytosolic testicular homogenates Sensitivities of the IFMAs for PRL, GH-N and PL-A/B were 2.4 pg/ml, the non-specific signals of the zero-standard 1424 9227, 668 9 93 and 658 9 107 counts per second (cps), cross-reactivities with the complementary protein hormones less than 0.1% and the intra- and inter-assay coefficients of variation less than 10%, as determined on three consecutive days. Concerning the IFMA for hPL the within-assay precision of the standard (n = 6) averaged at 8% (range: 2.1–16%). Three pregnancy sera containing high levels of hPL ranging from 1.2 to 2.9 ng/ml were serially diluted in assay buffer. The respective serial dilutions ran in parallel to the hormone standard over three orders of magnitude and revealed a linearity of 0.998– 0.999 (mean 0.999). Recovery studies were performed by adding 2.5 ng hPL/ml and 10 ng hPL/ml to eight sera containing 0.09 – 3.4 ng hPL/ml. The respective recovery values ranged from 102 to 125% (mean 110%). In addition, testicular cytosolic and placental extracts were spiked with 2.5 and 10 ng/ml, yielding recovery
Fig. 4. Expression of PRL gene in human testicular tissue. Poly (A) + RNA from different testes (T1–T4) and total RNA from pituitary and placenta were used as template for RT-PCR. Due to the exonspanning antisense PRL-specific primer, a 276 bp PRL cDNA fragment, but no genomic DNA, was selectively amplified. Restriction enzyme digestion with XbaI of this 276 bp PCR fragment yielded two bands at 234 and 42 bp, identical to that of the pituitary derived PRL gene-product. Testicular mRNA of the respective samples without reverse transcription ( −RT) served as a negative control. pBR 327/HinfI was used as size marker (SM): 517/453/298/222/154 bp.
values of 109–118% (mean 111%). Homogenized subcapsular testicular tissue (n= 11) was extracted and analyzed in duplicate in all three assays. Cytosolic hPRL concentrations corresponded to 58.7 ng/g tissue wet weight (tww; mean 9 S.D. 37.1 ng/g; range 14–160 ng), hGH-N concentrations to 5.1 ng/g tww (mean9 S.D. 3.0 ng/g; range: 2.7–12.5. ng/g) and hPL-A/B concentrations to 1.0 ng/g tww (mean9 S.D. 0.74 ng/g; range: 0.24–2.2 ng/g). Most importantly, normal serum values of an age-matched elderly male collective (n= 18; age: 72 9 3 years) were lower than 0.02 ng hPL/ml.
4. Discussion Three cell types of the human testis, Leydig, peritubular and Sertoli cells, regulated by the concerted action of gonadotropins, provide the hormonal milieu and essential factors for germ cell development and functional intra-testicular cell-cell interactions (Skinner, 1991). Pituitary-derived LH and FSH are definitely the primary hormonal support for Leydig and Sertoli cell function. The most important local regulatory hormone is testosterone, synthesized by Leydig cells, which acts as the major paracrine stimulator of germ cell development (Dufau, 1988). It has been shown that glycoprotein hormones, like LHb and CGb (Berger et al., 1994) and GHRH (Berry et al., 1992; Ciampani et al., 1992), are eutopically produced in the human testis. Increasing evidence suggests that locally synthesized factors like IGF-I (Skinner, 1991), GHRH (Berry et al., 1992; Ciampani et al., 1992), oxytocin, activin, b-endorphin, enkephalins and dynorphins (Skinner, 1991) may act in an auto/paracrine manner on testicular physiology. In this study, we demonstrated the production of GH/PL and PRL in the human testis at the mRNA and protein levels. In the pituitary, as expected and confirmed by us, the GH-N gene is the only transcriptionally active gene (Nachtigal et al., 1993), whereas the other four genes are repressed by binding of pituitary-specific factor (PSF) to 5%-located p-sequences (Nachtigal et al., 1993). Conversely, under control of the PL transcriptional enhancer (Walker et al., 1990), PL-A/B/L and the GH-V genes are transcribed in the placenta (Chen et al., 1989), but GH-N is not. PL-A is expressed at a 5-fold higher level than PL-B at term (MacLeod et al., 1992), but both mRNAs encode for an identical 22 K PL protein. GH-V gene transcription increases in parallel with that of PL, but at levels at least two orders of magnitude lower (Lytras et al., 1994). The proportion of alternatively spliced GH-V transcripts retaining intron D (GH-V2) is similarly regulated, increasing 3-fold during gestation up to a total of 15% of GH-V at term (MacLeod et al., 1992). PL-L is transcribed at very low levels in the placenta (Chen et al., 1989; Lytras et al.,
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1994) and these transcripts undergo two distinct and developmentally unaltered splicing pathways between exon 2 and 3 (Chen et al., 1989; Press et al., 1994). With our methodology, simultaneously amplifying transcripts of the entire gene-cluster, we could find only a weak GH-V, and no PL-L, gene expression in the term placentae (n=3). In a previous report (Lytras et al., 1994), based on a similar RT-PCR quantification of placental GH/PL mRNA (n =3), the proportions of GH-V (4.2%) and PL-L ( B1%) PCR-products, were higher compared with our results. In contrast to pituitary and placenta, the human testis has its own specific differential expression pattern of the GH/PL gene-cluster: PL-A/B \GH-V ] PL-L= GH-N. Moreover, we were able to demonstrate production of not only GH-V, but its alternative splice product GH-V2. Selective GH-N and PL-A/B transcription in the human ovary (unpublished data) adds further to the concept of organ specific GH/PL gene expression patterns. Due to GH/PL sense primer localisation at the beginning of exon 3 (Fig. 1), this particular PCR amplifies only the major products of the five GH/PL genes encoding for the 22 K protein form, not the 20 K GH-N, which misses the first 45 bp of exon 3, or the 20 K PL-L, truncated at the first 73 bp of exon 3. The amplified alternative splice product of the PL-L gene uses a cryptic splice acceptor site located 4 bp 3% from the position of the splice acceptor site of exon 3 utilized in the other genes of the cluster (Chen et al., 1989; MacLeod et al., 1992; Press et al., 1994). In the testis, the ratio of GH-V and PL-L to PL-A/B genetranscripts is higher than in the placenta, indicating a different regulation of GH/PL gene transcription. With highly specific and sensitive IFMAs, we detected hPL-A/B, hGH-N and hPRL in homogenized testicular tissue (1.0 ng/g, 5.1 ng/g and 58.7 ng/g tww). GH-V, PL-L and variants like GH-V2, PL-A2 and PL-L% (MacLeod et al., 1992) could not be investigated due to the lack of appropriate antibodies. As generally accepted and shown for the human B-cell Burkitt lymphoma cell line (Ramos) by RT-PCR and sequencing of the cDNA fragment human lymphocytes produce exclusively GH-N (Lytras et al., 1993). Thus minor amounts of testicular GH-N but not of PL-A/B might originate from few contaminating lymphocytes. Most noteworthy, a positive concentration gradient exists between cubital vein serum ( B 0.02 ng hPL/ml) of age-matched elderly probands to the testis (1 ng hPL/g tww), confirming testicular hPL synthesis. In addition to the villious syncytiotrophoblast, trophoblastic and non-trophoblastic germ cell neoplasms can ectopically produce hPL as well as the established marker hCG (Frantz et al., 1965; Samaan et al., 1966; Weintraub and Rosen, 1971). We observed that the healthy testis is a source of eutopically-synthesized protein hormones. The biological roles and functions of
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GH-V, PL-A/B/L and their alternative splice products are still a matter of debate, and only the effects of exogenous administration of hGH-N on the testicular steroidogenesis in panhypopituitary patients are well known (Balducci et al., 1993; Chatelain et al., 1991). In these individuals, hGH-N augments hCG-triggered testosterone production and spermatogenesis. Moreover, GH and IGF-I increase the numbers of testicular LH-receptors and steroidogenic responsiveness in GHdeficient dwarf mice (Chatelain et al., 1991). PRL also exerts modulatory effects on the Leydig cells, which express the PRL-receptor on their surface (Bouhdiba et al., 1989), by regulating the number of LH-receptors (Zipf et al., 1978; Dufau, 1988). The occurrence of ectopic hPL-production in testicular germ cell neoplasms lead to the hypothesis that developing germ cells of normal testes could produce these protein hormones. GHRH localized in early sperm stages and mature sperm cells (Berry et al., 1992) might act as an auto/ paracrine releasing-factor for GH/PL gene-products. The binding of these molecules to human testicular GH-receptor, presumably present on Sertoli cells (Lobie et al., 1990) could, in turn, support spermatogenesis via IGF-I, produced by this cell type (Skinner, 1991). The occurrence of eutopically-produced PRL and GH/ PL gene-products in the testis supports the view that they may have an auto/paracrine effect on the testosterone production of Leydig cells and/or are involved in the process of spermatogenesis.
Acknowledgements The authors thank Miss Regine Gerth for her excellent technical assistance and the NIADDK (NIH, Bethesda) for their generous supply of GH, PRL and PL.
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