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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 10 3337-3341
Copyright © 1997 by The Endocrine Society

Original Studies

Selective Growth Hormone/Placental Lactogen Gene Transcription and Hormone Production in Pre- and Postmenopausal Human Ovaries1

P. Schwärzler2, G. Untergasser, M. Hermann, S. Dirnhofer, B. Abendstein, S. Madersbacher and P. Berger

Institute for Biomedical Aging Research, Austrian Academy of Sciences (P.S., G.U., M.H., S.D., S.M., P.B.); the Department of Obstetrics and Gynecology, Landeskrankenhaus, Bregenz; the Department of Pathology (S.D.) and the Institute for General and Experimental Pathology (P.B.), University of Innsbruck; and the Department of Obstetrics and Gynecology, Krankenhaus, Hall (B.A.), Innsbruck, Austria

Address all correspondence and requests for reprints to: Peter Berger, Ph.D., Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail: peter.berger{at}oeaw.ac.at


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In addition to effects of pituitary-derived gonadotropins, human GH modulates and regulates intraovarian reproductive processes in a dose-dependent manner via the endocrine GHRH/GH/insulin-like-growth-factor I (IGF-I) axis. Based on increasing evidence that ovarian regulation involves a complex system of putative para/autocrine factors, we investigated the possibility of gene-selective intraovarian GH/placental lactogen (PL) hormone production, with emphasis on differences between pre- and postmenopause. Analysis of both premenopausal (n = 8) and postmenopausal (n = 10) ovarian-derived messenger ribonucleic acid by reverse transcription-PCR, which amplifies all major gene products of the five-member GH/PL gene cluster GH-N, GH-V, PL-A/B, and PL-L, revealed specific transcripts in all specimens. Their share in gene selective expression by analytical restriction enzyme digestion was determined. The expression pattern of GH/PL messenger ribonucleic acid shows PL-A/B > GH-N, which sets it apart from those of pituitary and placenta.

Local production of the respective protein hormones was verified by two time-resolved immunofluorometric assays for human PL-A/B and GH-N; significant amounts of these hormones were detected in cytosolic extracts of premenopausal (n = 6; 555.5 ± 171 ng PL-A/B and 0.8 ± 0.6 ng GH-N/g tissue wet wt) and postmenopausal (n = 6; 5.2 ± 2.7 ng PL-A/B and 0.9 ± 0.6 ng GH-N/g tissue wet wt) ovaries. No difference was observed between pre- and postmenopausal ovarian GH-N contents, but PL values were 2–3 orders of magnitude lower in postmenopausal tissue (P < 0.001). Serum levels of healthy premenopausal (n = 21) and postmenopausal (n = 16) women were less than 0.02 ng PL/mL. In summary, ovarian-derived GH-N and PL-A/B synthesis correlates well with the established local cascade of GHRH, GHRH receptor, GH receptor, IGF-I, and IGF-I receptor as a putative para/autocrine regulator of ovarian reproductive function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REPEATED duplication of the human (h) GH/placental lactogen (PL) precursor gene has led to a gene cluster composed of five highly related genes, termed GH-N (normal), PL-L (like), PL-A, GH-V (variant), and PL-B, which share more than 90% nucleotide sequence homology (1). The GH-N gene, accounting for approximately 3% of the total messenger ribonucleic acid (mRNA), is highly expressed in somatotropic cells of the anterior pituitary gland, whereas the other four genes are expressed in fetal placental syncytiotrophoblastic cells (2). It is noteworthy that pituitary somatotrophs account for 35–45% of the total cellular mass; thus, GH is the most abundantly secreted pituitary-derived hormone. In contrast to the well established somatotropic functions of GH during childhood and puberty, its role in adults is still a matter of debate (3). Probands with no clinical evidence of pituitary pathology show decreasing serum levels of GH and insulin-like growth factor I (IGF-I) with advancing age, suggesting that these factors influence protein synthesis and subsequent levels of lean body and bone mass (4, 5). In female reproduction, GH is involved in regulating ovarian function through several mechanisms. A direct effect was postulated (6) and was confirmed by the identification and cellular localization of the GH receptor (GH-R) in the human ovary (7), whereas indirect effects might be mediated by hepatic production of IGF-I via the classical pathway or by increased intraovarian levels of IGF-I (8). The ovarian IGF-I system seems to act as a local amplifying mechanism for gonadotropin action (9), but its nature and regulation are more complex than previously thought (10). GH and IGF-I potentiate the action of FSH on aromatase activity, progesterone production, and LH receptor formation, suggesting an important role in controlling follicular growth and steroidogenesis (11). Independent from this synergistic effect, GH stimulates estradiol production by human granulosa cells (6), induces dose-dependent progesterone production in luteal cells, and acts synergistically with hCG (12). The use of GH for induction of folliculogenesis in women responding poorly to human menopausal gonadotropins raised serum levels of estradiol and increased the number of developed follicles and the number of collected, fertilized, and cleaved oocytes (13). hGH treatment of patients with hypogonadotropic hypogonadism significantly reduced the amount of human menopausal gonadotropin required for induction of ovulation, when assessed in randomized, double blind, placebo-controlled studies (14, 15).

Thus, although the ovary is undoubtedly a target for endocrine GH action, a local expression of a cascade of hormones and their receptors, [GH-RH (16), GHRH receptor (17), GH receptor (7), IGF-I (18, 19), and IGF-I receptor (20)] suggests that locally produced GH could be the missing link in intraovarian regulation of reproductive processes. Based on increasing evidence that ovarian regulation involves a complex system of autocrine and paracrine factors (21), we analyzed a possible organ-specific expression of the GH/PL gene cluster and eutopic GH/PL production in human ovaries, with particular focus on differences between pre- and postmenopausal specimens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects and tissue collection

Ovarian tissue collected prospectively during surgery for nonovarian diseases from previously untreated premenopausal (n = 8; age, 21–47 yr) and postmenopausal (n = 10; age, 54–79 yr) patients, a postmortem female pituitary (age, 72 yr), and human term placenta were snap-frozen in liquid nitrogen immediately after removal and stored at -80 C. Written informed consent was obtained from all patients before surgery. For immunological determination of GH-N- or PL-A/B-derived molecules, part of each ovarian sample (0.7–1.4 g) was homogenized (Ultra-Turrax, Janke and Kunkel, Stauffen, Germany) in 1.5 mL phosphate-buffered saline (PBS; pH 7.2) containing phenylmethylsulfonylfluoride (1 mmol/L; Merck, Darmstadt, Germany) as a protease inhibitor. After centrifugation at 13,000 x g for 20 min at 4 C, the supernatant was collected and stored at -20 C until analysis.

Immunofluorometric assays (IFMAs) for hPL-A/B and hGH-N

IFMAs specific for hGH-N or hPL-A/B were established based on our panel of well characterized mouse monoclonal antibodies (MCA) (22, 23) by a previously detailed method (24, 25). Unless stated otherwise, an incubation volume of 100 µL/well and an assay buffer consisting of 50 mmol/L Tris-HCl (pH 7.75), 9 g/L NaCl, 5 g BSA/L, 0.1 g/L Tween-40, 0.5 g/L bovine {gamma}-globulin, 0.1 g/L nonrelated MCA, and 20 mmol/L diethylenetriaminepentaacid (Sigma-Aldrich, Milwaukee, WI) was used. Briefly, 10 µg highly purified MCA, coded as INN(sbruck)-hGH-2 or INN-hPL-37, in PBS were incubated for 2 h at 37 C in a microtiter plate (Nunc, Roskilde, Denmark). The remaining binding sites were blocked with 200 µL 1% BSA in PBS for 30 min at 37 C. After washing the plates three times with 200 µL PBS containing 0.5 mL Tween-20 and 5 g thiomersal/L, graded amounts of the hormone standards hGH 66/217 (National Institute of Biological Standard Control, London, UK) and hPL (National Institute of Biological Standard Control) or homogenized ovarian tissue (1:2 in assay buffer) were added, and plates were incubated on an orbit shaker (500 rpm, 90 min, 20 C) followed by three washes and then the addition of 100 ng europium-labeled detection MCA, INN-hGH-5, or INN-hPL-5 (30 min at 20 C, orbit shaker). After extensive washing, enhancement solution was added and incubated for 5 min (orbit shaker). Time-resolved fluorescence was measured for 1 s in a fluorometer (Wallac, Turku, Finland).

Isolation of total and polyadenylated [poly(A)+] RNA

RNA extraction was performed using the single step acid guanidinium thiocyanate phenol-chloroform method as described by Chomczynski and Sacchi (26). The integrity of the extracted RNA samples was verified by analysis of 28S and 18S ribosomal RNA on 0.7 mol/L formaldehyde, 1% agarose, and 0.01% ethidium bromide gels (Boehringer Mannheim, Mannheim, Germany). To remove residual DNA cross-contaminants, total RNA was treated with deoxyribonuclease RQ1 (Promega, Madison, WI) at 37 C for 15 min. Poly(A)+ RNA was prepared by the oligo(deoxythymidine)-cellulose affinity chromatography method according to the manufacturer’s instructions.

Reverse transcription-PCR (RT-PCR)

RT-PCR to detect the GH/PL complementary DNA (cDNA) was performed essentially as described previously (27). Briefly, poly(A)+-enriched RNA samples (0.5–1 µg/reaction) from normal human pituitary, placenta, and ovaries were reverse transcribed in a final volume of 50 µL using 200 pmol random hexamer oligonucleotide (Boehringer Mannheim) and 50 U Moloney murine leukemia virus-reverse transcriptase (Promega).

PCR amplifications were carried out for 40 cycles in 50-µL volumes with 1.25 U Red Hot polymerase (Advanced Biotechnology, Epsom, UK). Temperatures for annealing, elongation, and denaturation were 48 (40 s), 73 (45 s), and 95 C (60 s), respectively. A single pair of custom-made oligonucleotide primers (Microsynth, Windisch, Switzerland), located in sequences identical to all five members of the GH/PL gene cluster (Fig. 1Go; exon 3, position 737–756 according to the hGH-N gene, 5'-CAGAAGTATTCATTCCTGCA-3'; and exon 4, position 1060–1078, 5'-TTTGGATGCCTTCCTCTAG-3') (28) was designed to nonselectively amplify all major GH/PL transcripts. cDNA fragments were electrophoretically separated in 2% ethidium bromide-stained agarose gels with reference to a mol wt marker (100-bp DNA ladder; Advanced Biotechnology).



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  		Figure 1. A, GH/PL gene structure and primer localization for PCR. The five genes display more than 90% nucleotide sequence identity in their coding and flanking regions. All contain five exons, coded I–V (continuous lines), separated by four introns, A–D (dotted lines). B, GH/PL gene expression in human ovaries by PCR-amplified cDNA in an ethidium bromide-stained 2% agarose gel. The specific cDNA fragment of 250 bp in length was consistently observed in each (n = 18) of the premenopausal (lanes 1–3) and postmenopausal (lanes 4–6) ovarian specimens. Pituitary-derived cDNA (pit) and mock-transcribed ovarian total RNA (-RT) served as positive and negative controls. Mol wt markers (SM) corresponded to sizes from 100–1000 bp.

 
Gene assignment of the GH/PL cDNA fragments

PCR-generated cDNA fragments were excised (Silica Beads, Merck, Darmstadt, Germany) from agarose gels and radiolabeled by 10 cycles of PCR amplification, as described above, using the 32P-labeled sense primer in combination with the unlabeled antisense primer. The GH/PL oligonucleotide sense primer (60 pmol) was end labeled with 8 U T4 polynucleotide kinase (Promega) using 3000 Ci/mmol [{gamma}-32P]ATP (Amersham, Little Chalfont, UK) for 60 min at 37 C. To distinguish among cDNA derived from the five different GH/PL-genes, these 5' radioactively labeled cDNA fragments were subjected to a series of digestions with restriction endonucleases HphI (Advanced Biotechnology), RsaI (Promega), XbaI (Promega), and AvaII (Promega), either separately or simultaneously. The resulting DNA fragments were then analyzed by electrophoresis in polyacrylamide gels (6%) and visualized by exposure to Agfa RP-1 film (Agfa, Vienna, Austria) at -70 C for 3–7 h (Fig. 2Go).



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  		Figure 2. Ovarian GH/PL gene selective expression pattern. A, Restriction map of the radioactively labeled GH/PL-derived 250-bp cDNA fragment. The scissors indicate the specific endonuclease restriction sites of HphI (GH-V, PL-L, and PL-A/B), XbaI (PL-A/B), and RsaI (PL-L and GH-N), yielding the fragment pattern shown below. B, Digestion of evenly amplified cDNA fragments derived from all major transcripts of the whole GH/PL gene cluster yields a fragment pattern corresponding to gene-specific expression. RsaI produces a GH-N-specific 190 bp fragment in pituitary- and ovarian-derived, but not placental-derived, cDNA, indicating the presence of GH-N, whereas the digestion products of HphI and XbaI, 207- and 100-bp fragments, respectively, in ovary- and placenta-derived cDNA confirms the presence of PL-A/B transcripts. A portion of ovary-derived cDNA remained undigested even after simultaneous incubation with all three restriction enzymes, but was completely digested with AvaII, the restriction site of which is located at position 22 of the 250-bp cDNA fragment (data not shown), proving it to be a specific derivative of the GH/PL gene cluster.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selective GH/PL gene expression in the human ovary

Eutopic expression of major products of the GH/PL gene cluster was investigated by RT-PCR. Normal human pituitary (n = 1) and human term placenta (n = 1) revealed the expected cDNA fragment derived from GH/PL transcripts (250 bp). Interestingly, in each case, pre- (n = 8) and postmenopausal (n = 10) ovarian specimens yielded intense GH/PL signals. Mock-transcribed ovarian RNA without RT was analyzed in the same manner as a negative control (Fig. 1Go).

To discriminate between products of the five GH/PL genes, restriction endonuclease digestion was performed with the 5' radioactively labeled PCR product. Ovarian-derived premenopausal (n = 8) and postmenopausal (n = 10) cDNA both displayed a consistent pattern of GH/PL gene expression that differed from those of pituitary and placenta; digestion with RsaI produced a 190-bp fragment, indicating the presence of GH-N, whereas the digestion product of HphI and XbaI, 207- and 100-bp fragments, respectively, indicated the presence of PL-A/B (Fig. 2Go).

IFMA for hGH-and hPL assessment

IFMA sensitivities for GH-N and PL-A/B were 2.4 and 0.6 pg/mL, cross-reactions with the complementary protein hormones were less than 0.1%, and intra- and interassay coefficients were determined on 3 consecutive days to be less than 10%.

PL-A/B and GH-N were consistently present in pre- (n = 6) and postmenopausal (n = 6) ovarian tissue. Intraovarian concentrations of PL-A/B in pre- and postmenopausal ovaries was 555.5 ± 171 ng/g tissue wet wt (tww; range, 287–791 ng/g) and 5.1 ± 2.7 ng/g tww (range, 2.0–8.4 ng/g), respectively, and the lower concentrations of GH-N ranged from 0.23–1.8 ng/g (mean, 0.77 ± 0.67 ng/g) in premenopausal to 0.45–2.0 ng/g (mean, 0.94 ± 0.58 ng/g) in postmenopausal ovaries (Fig. 3Go). Serum values of healthy pre- (n = 21) and postmenopausal (n = 16) collectives were less than 20 pg/mL.



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  		Figure 3. Eutopic ovarian PL-A/B and GH-N production (nanograms of hormone per g tww) was verified by time-resolved IFMAs. The concentration of PL-A/B in premenopausal (n = 6) tissue (mean, 555 ± 171 ng/g tww) differs significantly (P < 0.001) from that in postmenopausal tissue (n = 6; mean, 5.1 ± 2.7 ng/g tww), whereas the assessment of GH-N yielded no statistical difference (mean, 0.7 ± 0.68 vs. 0.9 ± 0.58 ng/g tww). Note the logarithmic scale for hormone values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GH is believed to exert effects on ovarian physiology by endocrine mechanisms. GH replacement therapy induces puberty in GH-deficient children with pubertal delay, and cotreatment with GH augments the gonadal response to gonadotropin stimulation in patients with hypogonadotropic hypogonadism (15, 29). Conversely, precocious puberty is possible in the absence of circulating GH (30), and it should be noted that clinical observations of successful ovulation and conception have been reported in women with Laron-type dwarfism, a condition characterized by GH resistance and consequent systemic IGF deficiency (31). These data suggest that GH plays a more regulatory and modulatory than obligatory role in reproduction.

The classical concepts of endocrine ovarian regulation, which regards the follicle as the basic functional unit, with cyclic events being controlled by the concerted action of gonadotropins, have recently been extended by identification of a complex system of local nonsteroidal regulators. In view of the rapid accumulation of new data and suggestions of additional local regulatory mechanisms, in most cases by deducing intragonadal functions from in vitro model systems, Tsafriri and co-workers put forward criteria for defining intraovarian hormones: 1) these substances are present in the ovary without evidence of extraovarian origin; 2) receptors for these hormones have to be expressed within the gonad; and 3) a biological effect can be demonstrated on the ovary in situ and on ovarian cell preparations in vitro (32). Although GH fulfills the requirements for the second and third criteria, (7), the first has not been met to date. Herein, we demonstrate the presence of mRNA for GH/PL gene products by RT-PCR in the intact human ovary. Furthermore, analysis of gene products by radioactive restriction enzyme digestion revealed a specific expression pattern of GH/PL gene cluster that differed from those in pituitary and placenta. The majority of transcripts were derived from the PL-A and PL-B genes, whereas the GH-N gene expressed at lower levels. However, the biological significance of this pattern and the function of the molecular mechanisms in regulating differential gene expression remain to be elucidated.

Although attention was paid to a possible differential regulation associated with pre- or postmenopausal status, within the limits of our techniques, we observed no age-related switch in the GH/PL gene expression.

Significant amounts of the expected hGH-N, particularly hPL-A/B proteins, were detected in homogenized ovarian specimen by highly specific and selective IFMAs. hPL protein was present far more than GH-N, which parallels the higher expression of the PL-A/B genes compared to the GH-N gene. In keeping with the profound changes in hormone secretion profiles caused by menopause, the concentrations of hPL in pre- and postmenopausal specimens differed significantly (100- to 1000-fold), suggesting the relevance of hPL to female reproductive physiology. Thus, we consider the ovary as a site of eutopic GH/PL production, which is supported by demonstration of intraovarian PL-A/B and GH-N mRNA and their corresponding proteins and by the lack of significant hPL serum values in healthy pre- and postmenopausal women. Furthermore, hGH was previously found in surgically removed human ovaries (n = 7) at concentrations ranging from 50–51,000 ng/g tissue (33).

Current and future knowledge of the role of GH/PL in ovarian physiology may lead to its use as a therapeutical tool in the management of irregular bleeding and disorders of ovulation, and the potential of GH/PL as a growth factor in the wide spectrum of agents involved in ovarian-tumor development warrants further investigation.


   Acknowledgments
 
We are grateful to Dr. G. Daxenbichler, Innsbruck University, and Dr. H. Concin, Landeskrankenhaus Bregenz, for generously providing tissue samples. The excellent technical assistance of Regine Gerth and Thomas Öttl is gratefully acknowledged.


   Footnotes
 
1 This work was supported in part by a grant from the Vorarlberger Landesregierung. Back

2 Recipient of a Hans and Blanca Moser Foundation Basic Science Award. Back

Received November 14, 1996.

Revised May 28, 1997.

Accepted June 18, 1997.


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 Introduction
 Materials and Methods
 Results
 Discussion
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