GnRH as a cell proliferation regulator: mechanism of action and evolutionary implications

Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Studies on GnRH have demonstrated that GnRH has both stimulatory and inhibitory effects on cell proliferation depending on the cell type; however, the mechanisms of these effects remain to be elucidated. Against this background we used four human cell lines, TSU-Pr1, Jurkat, HHUA and DU145, and newly found that GnRH increased or decreased the colony-formation depending on the cell line. Moreover, we demonstrated that the stimulatory and inhibitory effects of GnRH exhibit distinct ligand selectivities. In order to investigate the molecular basis of these phenomena, analyses of the expression of human GnRH receptors were performed and, moreover, the effects of GnRH were analyzed under conditions in which human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor, which had been suspected of being nonfunctional because of alterations in its sequence, is involved in the effects of GnRH on cell proliferation. In this article, the influence of the autocrine activities of the cells is also reviewed, focusing on the characteristics of substances secreted from the four cell lines. Based on recent studies of GnRH and its receptors and our up-to-date findings, the evolutionary implications of GnRH action are discussed.     

Human type II GnRH receptor mediates effects of GnRH on cell proliferation

GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Progress in studies on GnRH demonstrated that GnRH has both inhibitory and stimulatory effects on cell proliferation depending on the cell type, and the mechanisms of these effects have been intensively studied. However, even human GnRH receptors which mediate GnRH stimulation have not been completely identified. In the present study, we showed that the inhibitory and stimulatory effects of GnRH on colony-formation using four cell lines and have demonstrated that the inhibitory and stimulatory effects of GnRH exhibit distinctly different patterns of ligand sensitivity. This result strongly suggests that the two opposite effects of GnRH occur via different types of GnRH receptors, however expressional analyses of human GnRH receptors did not exhibit the significantly different pattern between negatively and positively responding cell lines. Then, in order to identify the GnRH receptors involved in the two opposite effects, effects of GnRH were analysed under the conditions that human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor mediates GnRH stimulation and its splice variant determines the direction of the response to GnRH. These results are the first clear evidence for the functionality of human type II GnRH receptor. Therefore our novel findings are quite noticeable and will greatly contribute to the studies on the mechanisms of the effects of GnRH on cell proliferation in the future.     

Influence of serum supplements in culture medium on gonadotropin-releasing hormone effects on colony formation

A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcinomas of breast, ovary, endometrium, prostate, pancreas, and liver origin. In contrast, GnRH increases the proliferative activity of lymphoid tissues and cells, which suggests that GnRH is also an important immunomodulator. In a previous study, we demonstrated that the colony-forming efficiencies of HHUA (derived from human endometrial carcinoma) and Jurkat (derived from human mature leukemia) cells are affected by the GnRH agonist Buserelin, and that the conditioned media of HHUA and Jurkat cells severely affect the Buserelin activity. The latter finding suggests that substances in the culture medium have some relation to the GnRH activity. Therefore, in the present study, to evaluate the effect of serum supplements on the colony-forming efficiency assay, the assay was performed using 3 lots of fetal bovine serum (FBS) and 2 lots of Nu-Serum I, a semi-synthetic serum supplement. The results showed that the colony-forming efficiencies of HHUA and Jurkat cells fluctuated greatly depending on the lot of FBS. In contrast, Buserelin significantly affected the colony-forming efficiency to similar extents in the media containing both the lots of Nu-Serum I. These results strongly suggest that the constituents of the serum supplements also influence the effect of GnRH on cell proliferation. For further studies about the relationship between substances in the culture medium and the GnRH effects on cell proliferation, it will be necessary to use a completely defined medium, and that a semi-synthetic serum supplement such as Nu-Serum I will also be useful.     

Direct effect of a gonadotropin-releasing hormone agonist on the growth of canine mammary tumour cells

Gonadotropin-releasing hormone (GnRH) agonist exert "in vivo" an inhibitory action on the growth of hormone-dependent canine mammary tumours (Lombardi et al. [1999] J. Vet. Pharmacol Ther. 22(1):56-61). The present experiments have been performed "in vitro" in order to investigate the mechanisms involved in this direct antiproliferative action of GnRH agonists. In particular, the aim was to study whether these compounds might exert their antiproliferative effect by interfering with the stimulatory action of epidermal growth factor (EGF). To this purpose, the effects of GnRH agonist, Goserelin (GnRH-A), on the mitogenic action of EGF, on EGF-activated intracellular signaling mechanisms (intracellular calcium and nitric oxide production) as well as on ATP induced cell proliferation and signalling, and on the binding of EGF receptors have been evaluated in primary culture of canine mammary tumour cells. The results of these "in vitro" studies show that GnRH-A counteracts the mitogenic action of EGF and ATP, decreases the EGF/ATP-induced calcium signalling and reduces EGF binding, probably by means of NO-induced [Ca2+]i downregulation. These data suggest that GnRH agonists may inhibit the proliferation of the tumour cells by interfering with the stimulatory action of EGF.     

The regulation of haemopoiesis in long-term bone marrow cultures: I. Role of L-cell CSF

The effects of L-cell conditioned medium which contains granulocyte/macrophage colony stimulating factor (CSF); of highly purified L-cell CSF; and the antiserum directed against L-cell CSF, have been investigated in long-term murine bone marrow cultures. Treatment of cultures with CSF containing conditioned medium led to a rapid decline in haemopoiesis. However, this inhibition of in vitro haemopoiesis is probably caused by materials other than CSF, since the addition of highly purified L-cell CSF had no appreciable effect upon long-term haemopoietic cell proliferation or differentiation. Furthermore, the inhibitory activity of L-cell conditioned medium was not abrogated following neutralization of the CSF activity by CSF antiserum. The direct addition of CSF antiserum did not inhibit granulocyte or macrophage formation. These results suggest that long-term cultures of murine marrow cells may show extensive interactions with stromal cells which are not influenced by exogenous stimulatory or inhibitory factors.     

IGF-I and MAP kinase involvement in the stimulatory effects of LNCaP prostate cancer cell conditioned media on cell proliferation and protein synthesis in MC3T3-E1 osteoblastic cells

Bone metastases from prostate cancer cause abnormal new bone formation, however, the factors involved and the pathways leading to the response are incompletely defined. We investigated the mechanisms of osteoblast stimulatory effects of LNCaP prostate carcinoma cell conditioned media (CM). MC3T3-E1 osteoblastic cells were cultured with CM from confluent LNCaP cells. LNCaP CM stimulated MAP kinase, cell proliferation (3H-thymidine incorporation), and protein synthesis (14C-proline incorporation) in the MC3T3-E1 cells. The increases in cell proliferation and protein synthesis were prevented by inhibition of the MAP kinase pathway. IGF-I mimicked the effects of the CM on the MC3T3-E1 cells and inhibition of IGF-I action decreased the LNCaP CM stimulation of 3H-thymidine and 14C-proline incorporation and MAP kinase activity. The findings indicate that IGF-I is an important factor for the stimulatory effects of LNCaP cell CM on cell proliferation and protein synthesis in osteoblastic cells, and that MAP kinase is a component of the signaling pathway for these effects.     

Growth of the androgen-dependent tumor of the prostate: Role of androgens and of locally expressed growth modulatory factors

The crucial role played by androgens in the growth of prostatic carcinoma is now well established. However, the mechanisms of this proliferative action are still poorly understood. Experiments have been performed to clarify: (1) the metabolism of androgens in prostatic tumor cells; and (2) the role played by locally produced growth factors in the autocrine regulation of prostatic tumor cell proliferation and the possible regulation exerted by testosterone (T) on the activity of these factors. These studies have been performed by utilizing the human androgen-responsive prostatic cancer LNCaP cell line. (1) By incubating LNCaP cells with different ¹⁴C-labeled androgenic precursors, it has been shown that all the major key enzymes involved in the metabolism of androgens (5-reductase, 17β-hydroxysteroid-oxidoreductase, 3- and 3β-hydroxysteroid-oxidoreductases) are present and active in these cells. In particular, the 5-reductase, which converts T and Δ₄ to DHT and 5-A respectively, seems to be more active when Δ₄ is the substrate, suggesting a preference for this precursor. (2) The hypothesis that LNCaP cells might produce LHRH (or a LHRH-like peptide) has been verified by RT-PCR, performed in the presence of a pair of specific oligonucleotide primers. A cDNA band of the expected size (228 bp), which specifically hybridized with a ³²P-labeled LHRH oligonucleotide probe, has been obtained in LNCaP cells. To clarify the possible role played by this factor in the regulation of tumor growth, LNCaP cells, cultured in steroid-free conditions, have been treated with a LHRH antagonist; the treatment resulted in a significant increase of cell proliferation. Taken together, these data indicate that a LHRH (or LHRH-like) growth modulatory system is expressed in LNCaP cells and plays an inhibitory role in the regulation of tumor cell proliferation. This system seems to be regulated in a negative way by steroids. Growth factors endowed with stimulatory activity, such as EGF and TGF, have also been shown to be produced by LNCaP cells. The present studies show that the immunoprecipitation of the EGF receptor with a specific monoclonal antibody (Ab225) reveals a protein band of the expected size (170 kDa) which is phosphorylated even in basal conditions. Moreover, the treatment of LNCaP cells, cultured in serum-free conditions, either with a monoclonal antibody against the EGF receptor, or with immunoneutralizing antibodies against EGF and TGF, results in a significant decrease of cell proliferation. These observations clearly confirm the expression, in prostatic tumor cells, of an EGF/TGF loop which exerts a stimulatory action on cell proliferation. T seems to exert a positive regulation on this loop, at least in terms of EGF receptor concentration.     

Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production

Programmed death-1 ligand (PD-L)1 and PD-L2 are ligands for programmed death-1 (PD-1), a member of the CD28/CTLA4 family expressed on activated lymphoid cells. PD-1 contains an immunoreceptor tyrosine-based inhibitory motif and mice deficient in PD-1 develop autoimmune disorders suggesting a defect in peripheral tolerance. Human PD-L1 and PD-L2 are expressed on immature dendritic cells (iDC) and mature dendritic cells (mDC), IFN-gamma-treated monocytes, and follicular dendritic cells. Using mAbs, we show that blockade of PD-L2 on dendritic cells results in enhanced T cell proliferation and cytokine production, including that of IFN-gamma and IL-10, while blockade of PD-L1 results in similar, more modest, effects. Blockade of both PD-L1 and PD-L2 showed an additive effect. Both whole mAb and Fab enhanced T cell activation, showing that PD-L1 and PD-L2 function to inhibit T cell activation. Enhancement of T cell activation was most pronounced with weak APC, such as iDCs and IL-10-pretreated mDCs, and less pronounced with strong APC such as mDCs. These data are consistent with the hypothesis that iDC have a balance of stimulatory vs inhibitory molecules that favors inhibition, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of iDC. PD-L1 expression differs from PD-L2 in that PD-L1 is expressed on activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells. In contrast, PD-L2 is expressed on placental endothelium and medullary thymic epithelial cells. PD-L1 is also highly expressed on most carcinomas but minimally expressed on adjacent normal tissue suggesting a role in attenuating antitumor immune responses.     

Effects of lymphocytes from Marek's disease-infected chickens on mitogen responses of syngeneic normal chicken spleen cells.

PHA responses have been measured in lymphoid cell cultures prepared by mixing normal chicken spleen cells with spleen or thymus cells from syngeneic chickens infected with the oncogenic herpesvirus MDV. Results of these studies may be summarized as follows: 1) spleen cells from MDV-infected chickens with visceral lymphomas inhibit the PHA response of normal spleen cells possibly by release of soluble inhibitory factors in response to the mitogen; 2) lymphoid cells from asymptomatic MDV-infected chickens, although hyporeactive themselves to PHA, can have a stimulatory effect on PHA responses of normal spleen cells in mixed cultures; 3) spleen cells from MDV-infected chickens, effectively protected from viral oncogenesis by HVT vaccination, show normal reactivity to PHA in spearate cultures and may react in mixed cultures like normal lymphocytes, with neither a pronounced stimulatory nor inhibitory effect on the PHA response of normal spleen cells.     

GnRH in non-hypothalamic reproductive tissues

Gonadotropin releasing hormone (GnRH) is a hypothalamic neuronal secretory decapeptide that plays a pivotal role in mammalian reproduction. GnRH and its analogues are used extensively in the treatment of hormone dependent diseases and assisted reproductive technology. Fourteen structural variants and three different forms of GnRH, named as hypothalamic GnRH or GnRH-I, mid brain GnRH or GnRH-II and GnRH-III across various species of protochordates and vertebrates have been recognised. The hormone acts by binding to cell surface transmembrane G protein coupled receptors (GPCRs) and activates Gq/11 subfamily of G proteins. Although hypothalamus and pituitary are the principal source and target sites for GnRH, several reports have recently suggested extra-hypothalamic GnRH and GnRH receptors in various reproductive tissues such as ovaries, placenta, endometrium, oviducts, testes, prostrate, and mammary glands. GnRH-II appears to be predominantly expressed in extra pituitary reproductive tissues where it produces its effect by PLC, PKA2, PLD, and AC cell signalling pathways. In these tissues, GnRH is considered to act by autocrine or paracrine manner and regulate ovarian steroidogenesis by having stimulatory as well as inhibitory effect on the production of steroid hormones and apoptosis in ovarian follicle and corpus luteum. In male gonads, GnRH has been shown to cause a direct stimulatory effect on basal steroidogenesis and an inhibitory effect on gonadotropin-stimulated androgen biosynthesis. Recent studies have shown that GnRH is more abundantly present in ovarian, endometrial and prostrate carcinomas. The presence of type-II GnRH receptors in reproductive tissues (e.g. gonads, prostrate, endometrium, oviduct, placenta, and mammary glands) suggests existence of distinct role(s) for type-II GnRH molecule in these tissues. The existence of different GnRH forms indicates the presence of distinctive cognate receptors types in vertebrates and is a productive area of research and may contribute to the development of new generation of GnRH analogues with highly selective and controlled action on different reproductive tissues and the target-specific GnRH analogues could be developed.     

The partial purification and characterization of GnRH-like activity from prostatic biopsy specimens and prostatic cancer cell lines

We have investigated the possibility of the secretion of gonadotrophin-releasing-hormone (GnRH)-like peptides by prostatic cancer cells in culture and their presence in cytosolic preparations from human prostatic biopsy specimens. A GnRH-specific radioimmunoassay showed GnRH-like activity in concentrated cytosolic preparations and conditioned media from DU 145, an androgen-insensitive human prostatic cell line and from LNCaP, an androgen-responsive prostatic cancer cell line. GnRH immunoreactivity in culture media correlated directly with cell numbers. HPLC demonstrated that this GnRH-like material co-migrated with synthetic GnRH. This homology between synthetic GnRH and partially purified prostatic GnRH was confirmed following V8 protease and trypsin digestion which resulted in similar alterations in HPLC characteristics. The mean content of GnRH-like activity/g specimen tissue was significantly more in malignant tissue (88.5 ± 80.5 fmol) than in benign (29.6 ± 22 fmol), though more specimens of benign tissue were positive (37/54) than malignant tissue (6/22). This observation, taken with an earlier finding of GnRH-specific receptors in a hormone-sensitive cell line and human cancer specimens provides supportive evidence for the autocrine hypothesis of cell regulation.     

Mechanisms of haemopoietic stem cell proliferation control

The control of stem cell (CFU-S) proliferation is mediated by short-range acting factors which can be detected by the proliferation modifying activities present in media conditioned by haemopoietic cells. A specific inhibitor of stem cell proliferation is obtained from haemopoietic tissue containing minimally proliferating CFU-S, whilst stimulatory material is obtained from cell suspensions containing rapidly proliferating CFU-S. Used competitively, these factors, which are detected in different molecular weight range fractions, manipulate the rate of CFU-S proliferation in a manner compatible with a physiological control mechanism. In addition, a long-term bone marrow culture system has been shown to provide an in vitro model of stem cell control. Fractionation of cell populations from haemopoietic tissues reveals marked concentration differences of the CFU-S proliferation modifying activities depending on the proliferative state of the CFU-S. However, irrespective of whether the tissue contains stem cells that are actively or minimally proliferating, both stimulatory and inhibitory activities are detected. From dose-response studies it is concluded that stem cell proliferation is controlled by an appropriate balance of stimulatory and inhibitory factors which, however, are not produced by the stem cells themselves.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.     

Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.     

Pulsed ultrasound promotes melanoblast migration through upregulation of macrophage colony‐stimulating factor/focal adhesion kinase autocrine signaling and paracrine mechanisms

Repigmentation of vitiliginous lesions relies on the proliferation and migration of melanoblasts from hair follicles to the epidermis. Pulsed ultrasound has been demonstrated to have stimulatory effects on cell proliferation and migration and has been applied clinically to enhance tissue repair. To clarify the biologic effects and signaling mechanisms of pulsed ultrasound on melanoblast proliferation and migration, two melanoblast cell lines, the undifferentiated NCCmelb4 cells and the differentiated NCCmelan5 cells, were examined. We demonstrated that pulsed ultrasound increased cell migration in a dose‐dependent manner without altering cell proliferation. Pulsed ultrasound enhanced autocrine secretion of macrophage colony‐stimulating factor (M‐CSF), which subsequently activated the focal adhesion kinase (FAK) pathway to promote melanoblast migration. Furthermore, conditioned medium from mouse embryonic fibroblasts NIH 3T3 and primary human keratinocytes treated with pulsed ultrasound could stimulate melanoblast migration through a paracrine effect. Our results provide a novel mechanism to promote migration of melanoblasts by pulsed ultrasound stimulation.     

Direct opioid regulation of pituitary release of bovine luteinizing hormone

Although endogenous opioid peptides (EOP) are thought to alter pituitary release of luteinizing hormone (LH) by modifying the release of gonadotropin-releasing hormone (GnRH) from the brain, EOP may also directly affect the release of LH from pituitary cells. This hypothesis was tested using dispersed cells from the bovine anterior pituitary gland. Pituitaries were enzymatically dissociated, preincubated for 18 h and then cultured for either 2 or 24 h with GnRH, naloxone, methionine-enkephalin (Met-enk) or their combinations. Basal release of LH into media was 18.2 and 38.4 ng/100,000 cells after culture for 2 or 24 h, respectively. When cultured for 2 or 24 h with 10 nM GnRH, LH release was 296% and 131% of the basal release for each culture period. Cellular viability (75% vs 68%) and total (cells + medium) LH (128 vs 134 ng/100,000 cells) did not differ (P greater than .05) between cells cultured for 2 or 24 h. Naloxone (1 microM) increased (P less than .01) basal release of LH by 57% after 2 h of culture but not after 24 h of culture. Naloxone did not augment the amount of LH released in response to 10 nM GnRH. Addition of Met-enk (1 nM to 1 microM) suppressed (P less than .05) basal release of LH (23% to 62%) after 2 h of culture. Similar suppressive effects (8% to 49%) occurred in a dose-dependent manner (0.1 nM to 1 microM) after 24 h of culture. Met-enk (1 and 100 nM) antagonized (P less than .05) the stimulatory effect of naloxone and reduced (P less than .05) the amount of LH released in response to GnRH after 2 h of culture. In summary, the stimulatory effect of naloxone on the basal release of LH suggests that EOP may directly regulate pituitary cell function; the inhibitory effect of physiological concentrations of Met-enk on the basal in vitro release of LH suggests that EOP may directly affect the release of LH in vivo; the antagonism between the stimulatory effect of naloxone and the inhibitory effect of Met-enk is consistent with effects exerted through opioid receptors; and the stimulatory effect of GnRH may be partially reduced by Met-enk. These results are consistent with the hypothesis that opioids may directly modulate the release of LH at the pituitary level.     

Effects of lymphokines and immune complexes on murine placental cell growth in vitro

Isolated murine placental cells obtained at Day 16 of allogeneic gestation (C3H x DBA/2J) were cultured for 3 days alone or in coculture with irradiated mouse splenocytes at the end of which 3H-thymidine was added for an additional 18-h culture to assess cell proliferation. Placental cell proliferation was significantly enhanced at spleen cell:placental cell ratios of 10:1 and 25:1 above that observed in the absence of added spleen cells. The stimulatory effect of irradiated allogeneic (C3H plus Balb/cJ) spleen cell cultures was significantly greater (approximately 2-fold) than that of isogeneic spleen cells (C3H alone). Conditioned medium from murine spleen cells cultured with concanavalin A (ConA) to induce lymphokine production had dose-dependent inhibitory effects on proliferation when added to placental cell cultures over a range of concentrations from 10 to 40% (vol:vol). Addition of pseudo "immune complexes" in the form of heat-aggregated human gamma globulin (AHGG) to culture medium failed to alter placental cell proliferation over a range of concentrations from 2 to 200 micrograms/ml either in the absence or presence of ConA-conditioned medium. In contrast to late-gestational stage placental cells, cell suspensions obtained from Days 8-9 murine ectoplacental cone (EPC) outgrowths, or from earlier stage placentas (Days 12-14) responded to low concentrations of conditioned medium from ConA-stimulated splenocytes with increased proliferation. The effect was less impressive on placental cells at gestational ages later than 12 days than on earlier stage preparations. On all placental cell suspensions tested, as well as EPC cells, a clear-cut inhibition of growth was observed at high doses of conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)