Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.

Normothermic machine perfusion (NMP) has emerged as an innovative organ preservation technique. Developing an understanding for the donor organ immune cell composition and its dynamic changes during NMP is essential. We aimed for a comprehensive characterization of immune cell (sub)populations, cell trafficking and cytokine release during liver NMP. Single-cell transcriptome profiling of human donor livers prior to, during NMP and after transplantation shows an abundance of CXC chemokine receptor 1+/2+ (CXCR1+/CXCR2+) neutrophils, which significantly decreased during NMP. This is paralleled by a large efflux of passenger leukocytes with neutrophil predominance in the perfusate. During NMP, neutrophils shift from a pro-inflammatory state towards an aged/chronically activated/exhausted phenotype, while anti-inflammatory/tolerogenic monocytes/macrophages are increased. We herein describe the dynamics of the immune cell repertoire, phenotypic immune cell shifts and a dominance of neutrophils during liver NMP, which potentially contribute to the inflammatory response. Our findings may serve as resource to initiate future immune-interventional studies.

EpCAM is overexpressed in local and metastatic prostate cancer, suppressed by chemotherapy and modulated by MET-associated miRNA-200c/205.

BACKGROUND: Expression of epithelial cell adhesion molecule (EpCAM) is deregulated in epithelial malignancies. Beside its role in cell adhesion, EpCAM acts as signalling molecule with tumour-promoting functions. Thus, EpCAM is part of the molecular network of oncogenic receptors and considered an interesting therapeutic target. METHODS: Here, we thoroughly characterised EpCAM expression on mRNA and protein level in comprehensive tissue studies including non-cancerous prostate specimens, primary tumours of different grades and stages, metastatic lesions, and therapy-treated tumour specimens, as well as in prostate cancer cell lines. RESULTS: Epithelial cell adhesion molecule was overexpressed at mRNA and at protein level in prostate cancer tissues and cell lines. Altered EpCAM expression was an early event in prostate carcinogenesis with an upregulation in low-grade cancers and further induction in high-grade tumours and metastatic lesions. Interestingly, EpCAM was repressed upon induction of epithelial-to-mesenchymal transition (EMT) following chemotherapeutic treatment with docetaxel. Oppositely, re-induction of the epithelial phenotype through miRNAs miR-200c and miR-205, two inducers of mesenchymal-to-epithelial transition (MET), led to re-induction of EpCAM in chemoresistant cells. Furthermore, we prove that EpCAM cleavage, the first step of EpCAM signalling takes place in prostate cancer cells but in contrast to other cancer entities, EpCAM has no measurable impact on the proliferative behaviour of prostate cells, in vitro. CONCLUSIONS: In conclusion, our data confirm that EpCAM overexpression is an early event during prostate cancer progression. Epithelial cell adhesion molecule displays a dynamic, heterogeneous expression and associates with epithelial cells rather than mesenchymal, chemoresistant cells along with processes of EMT and MET.

Ovarian cancer stem cells.

Because of its semi-solid character in dissemination and growth, advanced ovarian cancer with its hundreds of peritoneal tumor nodules and plaques appears to be an excellent in vivo model for studying the cancer stem cell hypothesis. The most important obstacle, however, is to adequately define and isolate these tumor-initiating cells endowed with the properties of anoikis-resistance and unlimited self-renewal. Until now, no universal single marker or marker constellation has been found to faithfully isolate (ovarian) cancer stem cells. As these multipotent cells are known to possess highly elaborated efflux systems for cytotoxic agents, these pump systems have been exploited to outline putative stem cells as a side-population (SP) via dye exclusion analysis. Furthermore, the cells in question have been isolated via flow cytometry on the basis of cell surface markers thought to be characteristic for stem cells.In the Vienna variant of the ovarian cancer cell line A2780 a proof-of-principle model with both a stable SP and a stable ALDH1A1+ cell population was established. Double staining clearly revealed that both cell fractions were not identical. Of note, A2780V cells were negative for expression of surface markers CD44 and CD117 (c-kit). When cultured on monolayers of healthy human mesothelial cells, green-fluorescence-protein (GFP)-transfected SP of A2780V exhibited spheroid-formation, whereas non-side-population (NSP) developed a spare monolayer growing over the healthy mesothelium. Furthermore, A2780V SP was found to be partially resistant to platinum. However, this resistance could not be explained by over-expression of the "excision repair cross-complementation group 1" (ERCC1) gene, which is essentially involved in the repair of platinated DNA damage. ERCC1 was, nonetheless, over-expressed in A2780V cells grown as spheres under stem cell-selective conditions as compared to adherent monolayers cultured under differentiating conditions. The same was true for the primary ovarian cancer cells B-57.In summary our investigations indicate that even in multi-passaged cancer cell lines hierarchic government of growth and differentiation is conserved and that the key cancer stem cell population may be composed of small overlapping cell fractions defined by various arbitrary markers.

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells.

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.

The ageing male reproductive tract.

Ageing of the male reproductive system is characterized by changes in the endocrine system, hypogonadism, erectile dysfunction and proliferative disorders of the prostate gland. Stochastic damage accumulating within ageing leads to progressive dysregulation at each level of the hypothalamic-pituitary-gonadal (HPG) axis and in local auto/paracrine interactions, thereby inducing morphological changes in reproductive target organs, such as the prostate, testis and penis. Despite age-related changes in the HPG axis, endocrine functions are generally sufficient to maintain fertility in elderly men. Ageing of the male reproductive system can give rise to clinically relevant manifestations, such as benign prostatic hyperplasia (BPH), prostate cancer (PCa) and erectile dysfunction (ED). In this review, we discuss morphological/histological changes occurring in these organs and current views and concepts of the underlying pathology. Moreover, we emphasize the molecular/cellular pathways leading to reduced testicular/penile function and proliferative disorders of the prostate gland.

The antiapoptotic effect of IL-6 autocrine loop in a cellular model of advanced prostate cancer is mediated by Mcl-1.

Levels of the proinflammatory cytokine interleukin-6 (IL-6) are increased in therapy-resistant prostate cancer. IL-6 has been considered a positive growth factor in late-stage prostate cancer cells and a potential target for therapeutic interference. Effects of inhibition of IL-6 on cell survival were studied in LNCaP-IL6+ cells, a model system for advanced prostate cancer, which produce IL-6. We show that the autocrine IL-6 loop is responsible for resistance to apoptosis and increased cellular levels of myeloid cell leukemia-1 (Mcl-1) protein, an antiapoptotic member of the Bcl-2 family. Treatment of cells with a chimeric anti-IL-6 antibody (CNTO 328) led to the induction of apoptosis and downregulation of Mcl-1 protein levels. Specific knockdown of Mcl-1 gene expression by small interfering RNA also yielded an increase in apoptosis of LNCaP-IL-6+ cells. Vice versa, inactivation of IL-6 autocrine loop had no influence on apoptosis levels in the absence of Mcl-1, thus suggesting this molecule as a mediator of the survival action of IL-6. Mcl-1 protein regulation by the endogenous cytokine directly involved the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway. Our data support the concept of anti-IL-6 targeted therapy in therapy-resistant prostate cancer.

GAGEC1, a cancer/testis associated antigen family member, is a target of TGF-beta1 in age-related prostatic disease.

Transforming growth factor beta (TGF-beta) is a multi-functional cytokine that plays a fundamental role during embryonic development and tissue homeostasis in metazoans. Changes in TGF-beta signalling are implicated in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), two of the most common diseases affecting ageing males. GAGEC1 belongs to the GAGE-related family of cancer/testis associated antigens and in males is expressed only in prostate and testis. Previous reports demonstrate that GAGEC1 is up-regulated in symptomatic BPH and PCa. We demonstrate GAGEC1 up-regulation by TGF-beta1 in primary prostatic stromal and epithelial cells. Our data suggest that disease-associated increases in TGF-beta1 may account for the increase in GAGEC1 expression in BPH and PCa. Given its restricted spatial expression in males, GAGEC1 represents a promising target for therapeutic intervention of BPH and PCa.

TGF-beta cytokines increase senescence-associated beta-galactosidase activity in human prostate basal cells by supporting differentiation processes, but not cellular senescence.

The family of transforming growth factors betas (TGF-betas) comprises molecules involved in growth inhibition, stress-induced premature senescence, epithelial mesenchymal transition and differentiation processes. The aim of this study was to clarify the effect of long term exposure of human prostate basal cells to TGF-betas, which are found in high concentrations in prostatic fluid and areas of benign prostatic hyperplasia (BPH). Basal cell cultures established from prostate explants (n=3) were either grown into cellular senescence, or stimulated with TGF-beta1, beta2 and beta3. Similar to cellular senescence, TGF-beta stimulation resulted in an increase of SA-beta galactosidase (SA-beta-gal) activity, flattened and enlarged cell morphology, and down-regulation of the inhibitor of differentiation Id-1. TGF-beta-treated prostate epithelial cells neither showed terminal growth arrest nor induction of important senescence-relevant genes, such as p16(INK4A), IFI-6-16, IGFBP-3 or Dkk-3. Cells stained positive for cytokeratins 8/18, but did not express other lumenal markers, such as prostate-specific antigen and androgen-receptors. TGF-betas increased also the expression of the mesenchymal marker vimentin, indicating that basal epithelial cells underwent differentiation with lumenal and mesenchymal features. In contrast, in vitro-differentiated neuroendocrine-like cells from prostate organoide cultures, expressing chromogranin A and cytokeratin 18, strongly stained positive for SA-beta-gal. Thus, SA-beta-gal activity is not only a marker for senescence, but also for differentiation of human prostate epithelial cells. With regard to the in vivo situation, in addition to cellular senescence, TGF-beta could contribute to the increased number of SA-beta-gal positive epithelial cells in BPH.

Seminal plasma factors induce in vitro PRL secretion in smooth muscle cells of the human prostate.

Next to the sex steroid hormone T, PRL has been shown to influence prostatic function and development. Transgenic mice overexpressing the rat PRL gene develop dramatic enlargements of the prostate gland. Proliferation and secretory activities of epithelial cells are stimulated by PRL in rodents and men. Low concentrations of human PRL (hPRL) and hPRL receptors have been observed in human prostatic epithelial cells (ECs). The aim of this study was to compare regulation of the in vitro hPRL secretion in prostatic ECs and stromal smooth muscle cells (SMCs) after stimulation with seminal plasma (SMP), containing a variety of prostatic factors. SMCs released up to 1 ng hPRL/ml (i.e., approximately 500-fold more than unstimulated SMCs and ECs). Quantification of PRL mRNA by highly sensitive quantitative RT-PCR revealed that hPRL gene expression increased 5-fold within 24 h of SMP incubation. Sex steroids (dihydrotestosterone, progesterone, 17beta-estradiol), prostaglandins (PGE-1, PGE-2), and cAMP-stimulating substances (forskolin) were not responsible for induction of hPRL. Compared with endometrial SMCs, regulation of prostatic hPRL secretion was independent of progesterone and cAMP. HPLC analysis of human SMP revealed that the common action of at least two different proteins and a low molecular cofactor is required. We concluded that prostatic ECs secrete proteins acting synergistically with low-molecular-weight cofactors to induce differentiation and hPRL release in SMCs. Age-related increases in SMC-derived hPRL might contribute to the development of benign hyperplasia of the prostate.

A low-molecular-weight fraction of human seminal plasma activates adenylyl cyclase and induces caspase 3-independent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential and Bcl-2/Bax ratio.

The majority of elderly men are affected by benign and malign diseases of the prostate that are governed by endocrine factors and local stromal/epithelial and luminal/epithelial interactions. Prostate epithelial cells secrete numerous factors into the seminal plasma (SMP) that are thought to be responsible for nutrition, accurate pH, and ionic environment of sperm. Our hypothesis assumes that prostatic factors responsible for optimal fertility might have retrograde influences on epithelial cell growth, differentiation, and function. SMP was analyzed for proteins and other biologically active substances by size exclusion high-performance liquid chromatography. Each fraction was investigated for its effect on cell growth and death. A low molecular mass fraction (2-4 kDa) was responsible for inducing apoptosis in proliferating prostate epithelial cells. Signal transduction was mediated by the production of cAMP; no significant changes in tyrosine phosphorylation of membrane receptors were observed. Mechanisms of apoptosis, i.e., caspase- and mitochondria-dependent pathways, were investigated in prostate epithelial cells by caspase activity assays, annexin/propidium iodide staining, changes in mitochondrial potential, p53, Par-4, Bax, and Bcl-2 protein levels. SMP induced p53- and Bcl-2-dependent apoptosis without activation of caspase-3. Obviously, SMP contains protective factors that help eliminate degenerated cells and control epithelial renewal. Age-related changes in the composition of SMP or the susceptibility of epithelial cells might, therefore, contribute to proliferative prostatic diseases

High levels of zinc ions induce loss of mitochondrial potential and degradation of antiapoptotic Bcl-2 protein in in vitro cultivated human prostate epithelial cells.

Prostate epithelial cells contain the highest levels of zinc among all organs and tissues in the human body. Zinc is accumulated primarily in the mitochondria, where it is responsible for inhibition of mitochondrial aconitase activity, thereby increasing citrate production. The present study was designed to clarify the role of zinc for human prostate epithelial cell growth and apoptosis. Apoptosis of in vitro cultivated human prostate epithelial cells exposed to ZnCl(2) was analyzed by determination of phospholipid membrane asymmetry, nuclear fragmentation, DNA strand breaks, changes of mitochondrial potential and cellular pro/antiapoptotic proteins. Zinc induced apoptosis without involvement of p53 by decreasing mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 protein levels in proliferating epithelial cells. Thus, the high local concentrations of zinc ions in the prostatic lumen seem to be necessary to regulate proliferative activities and to enforce epithelial differentiation processes.

Aging of the male reproductive system.

Reproductive and sexual physiology, changes in body composition and mental performance in the aging male cannot simply be reduced to presumptive hypogonadism defined by low androgen serum levels or by decreasing levels of growth hormone (GH) and melatonin. Morphological changes in organs at different regulatory levels of hormonal networks governing, for example reproduction, such as diminished hypothalamic pulse generator mass, focal degeneration and loss of Leydig cells in testicular tissue, lead to diminished reserve capacities in production and to loss of coordinated pulsatile release of hypothalamic neuropeptides (e.g. gonadotropin releasing hormone, GnRH) and consequently diminished release of pituitary protein and glycoprotein hormones and testicular steroid hormones. Owing to presumptive alterations in feedback sensitivity, decreased testosterone levels do not necessarily upregulate pituitary LH secretion. Alternatively, increased serum levels of LH and FSH can be observed in old men either because of primary hypogonadism or to decreased hypothalamic opioid tone. In general, endocrine functions are sufficient to maintain fertility in elderly men because, except for sperm motility, quantitative and qualitative functional semen parameters are apparently not affected by age. Nevertheless, reduced endocrine and organic functions might become critical at different levels, with high inter-individual variability, of the hypothalamo/pituitary/gonadal-axis. One of the most intriguing organic manifestations of male aging is benign prostatic hyperplasia (BPH), the pathologic prevalence of which closely matches age. Age-associated changes in the endocrine system and in local networks of epithelial, stromal and luminal factors may play important roles in BPH development.

An unusual member of the human growth hormone/placental lactogen (GH/PL) family, the testicular alternative splicing variant hPL-A2: recombinant expression revealed a membrane-associated growth factor molecule.

The human growth hormone/placental lactogen (GH/PL) gene cluster consists of five highly-related genes (GH-N, GH-V, PL-L, PL-A, PL-B). This evolutionarily young gene cluster codes for an array of mRNAs and proteins, such as the major 22 k forms (hGH-N/V, identical PL-A and B), 20 k and 17.5 k hGH-N and the recently described 25 k hGH-Delta4, a presumably chimeric molecule. In addition, two longer alternatively spliced, (intron D retaining) mRNAs isoforms, termed PL-A2 and GH-V2, have been described in placenta and testis. To elucidate the role of hPL-A2 in male reproduction and pregnancy, testicular PL-A2 cDNA was cloned in a complementary overlapping 2-way RT-PCR approach to analyze translation, localization and structure/function of this unusual member of the GH/PL growth factor family. Analysis of insect mRNA revealed that intron D-retaining PL-A2 cDNA was expressed without splicing in the baculovirus expression system. Thus, PL-A2 mRNA does not represent a nuclear intermediate splicing product simply co-isolated with the mature RNA, but is a stable mRNA isoform generated by placental/testis-specific splicing factors. Recombinant protein was present in whole cell extracts, and no secreted protein was detected in the supernatant. Immunologically, the N-terminus of the 230 amino acid protein is similar to 22 k hPL-A/B, as determined by hPL-specific monoclonal antibodies. In contrast, the C-terminus shares a hydrophobic region presumably responsible for membrane insertion. By the use of confocal microscopy recombinant hPL-A2 was localized in the cell membrane. Thus, hPL-A2 might exert its function by modulating GH/PL actions or act as an independent growth-regulatory molecule itself and its functions in male reproduction and embryonic development remain to be investigated.

The testis-specific expression pattern of the growth hormone/placental lactogen (GH/PL) gene cluster changes with malignancy.

Growth hormone (GH) and placental lactogen (PL) gene transcription patterns in testicular germ cell tumors (GCT) and normal testicular tissue were comparatively investigated to identify GH/PL gene products associated with the development of GCT. This was done by nondiscriminative reverse transcriptase-polymerase chain reaction (RT-PCR), amplifying all major transcripts of any of the 5 GH/PL genes--GH-N(ormal), GH-V(ariant), PL-A, PL-B, PL-L(ike)--and subsequent analytical restriction enzyme analyses of 5'-end radioactively labeled cDNA. Surprisingly, all nonseminomatous GCT (NSGCT; n = 9) expressed GH-N, PL-A/B, and PL-L transcripts (9 of 9). Seminoma (n = 7) showed a distinctly unique pattern of GH-N and PL-A/B. GH-V products, which are hallmarks of the normal healthy testis, were not detected in any testicular cancer specimen (0 of 16). The fact that both seminomatous and NSGCT showed alterations in the same gene cluster indicates a pathogenetic relationship. Two choriocarcinoma cell lines of conceptus origin, BeWo and JAR, clearly differing from the male counterparts, exhibited a placental-derived pattern of PL-A/B and GH-V. Obviously, profound differences exist between conceptus and male germ cell GH/PL gene cluster transcription. In summary, the unique testicular pattern of GH/PL gene expression changes significantly and in directed ways with malignancy. Loss of GH-V gene expression in testicular GCT compared with normal testis and loss (seminoma) or mutation (NSGCT) of PLL gene products might have significance in terms of the relationship between these tumors and for testicular GCT development.

Proliferative disorders of the aging human prostate: involvement of protein hormones and their receptors.

The majority of elderly men is affected by benign and malignant diseases of the prostate. Both proliferative disorders, i.e., benign hyperplasia of the prostate (BPH) and prostate cancer (PCa)-which has recently emerged as the most common male malignancy in industrialized countries-seem to be governed by endocrine factors such as sex steroid hormones, but auto/paracrine factors are involved as well. Age-related changes in levels and ratios of endocrine factors as androgens, estrogens, gonadotropins, and prolactin (PRL) and changes in the balance between auto/paracrine growth-stimulatory and growth-inhibitory factors such as insulin-like growth factors (IGFs), epidermal growth factor (EGF), nerve growth factor (NGF), IGF-binding proteins (IGFBPs), and transforming growth factor beta (TGFbeta) are meant to be responsible for abnormal prostatic growth. We investigated the existence of putative local regulatory circuits involving the protein hormones, human growth hormone (hGH), human placental lactogen (hPL), and hPRL, and their corresponding receptors in prostatic tissue specimens (transurethral resections of the prostate, TURP; n = 11), in the prostatic cancer cell lines PC3, Du145, LnCap, a virus-transformed BPH cell line (BPH-1), and in a normal healthy prostate by RT-PCRs and highly specific and sensitive immunofluorometric assays (IFMA). Neither hPRL nor hGH was detected at the mRNA or protein levels in prostatic tissue and cell lines, with the exception of 2 of 11 prostatic TURP-samples, which showed weak expression of the PL-A/B genes. PRL- and GH-receptors were expressed in all normal and pathological prostatic specimens. Surprisingly, PRL-receptor expression was not detectable in prostatic cancer cell lines. The trophic effects of exogenous hGH, hPL, and hPRL were investigated by cell proliferation assays (WST-I) in prostatic primary cell cultures and PCa cell lines. hGH significantly (p < 0.005) increased cell proliferation up to 138+/-3.2% (1 nM hGH), while hPL and hPRL revealed only moderate effects. Our data suggest that local auto/paracrine networks of protein hormone actions are not involved in the pathology of BPH or prostatic cancer. On the other hand, systemic pituitary-derived hGH can increase the proliferative response of BPH and PCa, acting directly on the target organ prostate, via the hGH-R. In this case, envisaged GH substitution in elderly people must be viewed at with caution because age-related declines in GH/IGF-I could act as a protective mechanism against abnormal cell growth.

Complex alternative splicing of the GH-V gene in the human testis.

The human growth hormone variant (GH-V) gene is expressed during pregnancy in the syncytiotrophoblast and, as shown recently, in the normal human testis. In addition to the classical transcript encoding for the 22 K major form, intron D-retaining processed mRNAs (GH-V2) have also been described in both tissues. In the present study we analyzed testicular GH-V RNA alternative splicing patterns, a major source of GH variability. We observed three types of GH-V-derived mRNAs by reverse transcription-polymerase chain reaction amplification of GH/placental lactogen mRNA, subsequent cloning into appropriate vectors, vector amplification, restriction-endonuclease map-analysis and double-strand sequencing of GH-V clones. Apart from the conventional splice product encoding classical hGH-V (22K, 191 amino acids (aa)) and intron D-retaining mRNA GH-V2 (230aa), we detected an additional GH-V mRNA variant, GH-Vdelta4, utilizing a competitive splice-donor site 4 bp 5'of the conventional exon 4/intron D splice-donor site, but retaining the genuine intron D/exon 5 splice-acceptor site. This mRNA encodes a putative 25 K protein of 219 amino acids in length, having the first 124 amino acids and, thus, two and a half structural alpha-helices in common with hGH-V.hGH-Vdelta4 has lost the N-glycosylation site at Asn 140 of hGH-V, but acquires a novel site at position 148 as well as a cystein-rich domain in the 65 carboxyl-terminal amino acids, potentially involved in multiple disulfide-bridge formation. Tissue specificity and possible functions for testicular physiology remain to be investigated.

Reassessment of the role of human placental lactogen in physiological non-pregnant and pathological conditions.

The recent demonstration of ectopic production of human placental lactogen (PL) in the human testis and ovary prompted us to reassess its role under non-pregnant physiological and pathological conditions. Possible physiological hPL concentrations and potential age-related changes in the sera of healthy young and elderly individuals (n = 75) selected according to the SENIEUR-protocol were investigated by a highly sensitive (detection limit: 2 pg/ml) and specific (cross-reactivity with prolactin and human growth hormone (hGH) of less than 0.001% and 0.0001%, respectively) monoclonal antibody-based time-resolved fluoroimmunoassay (IFMA) established in our laboratory. All individuals, even the aged probands (mean age: 72 +/- 3a), had hPL-levels below 20 pg/ml, in contrast to glycoprotein hormones, such as luteinizing hormone or human chorionic gonadotropin (hCG). To determine the significance of hPL as a tumour marker, serum samples of 12 testicular cancer patients with highly elevated levels of holo-hCG (mean: 42.490 ng/ml) at diagnosis were followed over 6-12 months and analysed with the hPL-IFMA. Elevation of hPL was seen in 10 patients, but the respective levels were 2-3 orders of magnitude smaller than those of holo-hCG and returned earlier to undetectable values. These in vivo data were compared to the hPL secretion pattern of the choriocarcinoma cell lines JAR and BeWo in vitro. In tissue culture supernatants of the two cell lines hPL was detected only in JAR cells, whereas both cell lines secreted holo-hCG. In conclusion, the fact that hPL is not physiologically present in peripheral blood but is produced ectopically in the human testis and ovary suggest auto/paracrine functions of this molecule. The significance of hPL as a tumour marker for patients with testicular cancer is limited as it provides no additional information to holo-hCG.

Selective growth hormone/placental lactogen gene transcription and hormone production in pre- and postmenopausal human ovaries.

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.