Haem oxygenases produce carbon monoxide, which, like nitric oxide, is a gaseous messenger molecule that is one of several important survival factors in ovarian follicles. However, little is known about the expression and possible functions of these enzymes in granulosa cells. The purpose of this study was to investigate the expression and possible role of haem oxygenases in porcine granulosa cells (PGCs). We obtained frozen sections of porcine ovaries and PGCs from ovarian follicles of various sizes by needle aspiration, and examined the expression of haem oxygenase-1 (HO-1; inducible type) and HO-2 (constitutive type) in PGCs by immunohistochemistry, RT-PCR, western blotting and flow cytometry. Both types of haem oxygenase were identified in PGCs throughout follicular development, but HO-1 was expressed primarily in granulosa cells in atretic follicles. We also investigated the effect of haem oxygenases on apoptosis of granulosa cells (flow cytometry to detect subdiploid DNA fluorescence) and on expression of Fas ligand (quantitative analysis of western blotting and flow cytometry). In tightly bound PGCs, the mean proportion of apoptotic cells treated with 1 microM haemin (a haem oxygenase substrate) was approximately 1.7-fold greater than that in untreated controls, and zinc protoporphyrin IX (ZnPP IX; a haem oxygenase inhibitor) completely inhibited the increase in apoptosis induced by haemin in 24-h culture. Conversely, in weakly associated PGCs, the proportion of apoptotic cells was not altered by haemin. The quantity of Fas ligand protein was increased in a dose-dependent manner in tightly bound PGCs treated with haemin compared with controls, and the haemin-induced increase in Fas ligand protein was inhibited by ZnPP IX. Thus we identified inducible HO-1 and constitutive HO-2 in PGCs throughout follicular development, and we conclude that products of reactions catalysed by haem oxygenases are likely to be important autocrine/paracrine factors that regulate apoptosis in PGCs.

ABSTRACT

Epidermal growth factor (EGF) was present at a mean concentration of 266±18 (s.e.m.) pmol/mg wet tissue in the submandibular gland of 3-month-old male mice; it was also present in plasma at a concentration of 364±149 pmol/l. Sialoadenectomy (removal of the submandibular glands) decreased the plasma EGF content to undetectable levels (< 16·5 pmol/l), lowered the concentration of EGF in the skin from 1·22±0·11 to 0·47±0·08 fmol/mg wet tissue and reduced the thickness of the epidermis from 28·9±2·7 to 11·0±0·8 μm in 3 weeks (P < 0·001). Epidermal growth factor antiserum given to sialoadenectomized mice further decreased the thickness of the epidermis to 8·3 ±0·6 μm. No appreciable change was observed in the dermis and subcutaneous tissue. In sialoadenectomized mice, replacement of EGF prevented the decrease in thickness of the epidermis in a dose-dependent manner when started immediately after the operation. Treatment with EGF also effectively restored the normal morphology of the epidermis when its thickness had declined to its lowest level. These results suggest that EGF plays a physiological role in the maintenance of the epidermis.

J. Endocr. (1987) 113, 193–197

ABSTRACT

The present study was undertaken to investigate the effects of protein kinase C (PKC) activation and calcium mobilization on the release of prolactin from human decidual cells in early pregnancy. Decidua obtained from patients in early pregnancy was enzymatically dispersed and cultured with phorbol myristate acetate (PMA) and calcium ionophore A23187 in a cell culture system. Prolactin in the medium was measured by enzyme-immunoassay.

PMA, a PKC activator, dose-dependently attenuated the release of prolactin from cultured decidual cells, while a PKC inhibitor, H7, significantly (P < 0·001) diminished the effect of PMA on prolactin release. PMA had no effect on cell numbers or DNA synthesis in the decidual cells during culture. It did not significantly increase the generation of inositol phosphate in decidual cells prelabelled with myo[³H]inositol and it had no effect on intracellular calcium concentration ([Ca^{2 +} ]_i). Calcium ionophore A23187, a Ca^{2 +}-mobilizing agent, also significantly (P<0·001) attenuated the release of prolactin and potentiated the PMA-induced suppression of prolactin release from decidual cells.

These findings suggest that activation of PKC and mobilization of Ca²⁺ may be involved in regulating prolactin release from human decidual cells. The PMA-induced suppression of prolactin release is not triggered by phosphoinositide hydrolysis nor by the increase in [Ca^{2 +} ]_i in decidual cells.

Journal of Endocrinology (1993) 137, 335–340

It has been recognized that tissue-specific growth factors and angiogenic factors play important roles in the growth of tumors and in the tissue-repair system. In uterine myometrial smooth muscle cells, it has also been reported that the platelet-derived growth factor (PDGF) binds to PDGF receptors and stimulates proliferation. In this paper, we examine whether or not PDGF is able to stimulate production of vascular endothelial growth factor (VEGF) in cultured human myometrial smooth muscle cells. PDGF treatment enhanced immunoreactive VEGF production as well as cell proliferation. Production of VEGF121 and VEGF165 in the cells was detected by reverse transcription-polymerase chain reaction analysis, but the PDGF treatment did not change the ratio of VEGF165 to VEGF121. The effect of PDGF on cell proliferation leveled off at 10 ng/ml, whereas its effect on VEGF production continued to increase linearly at concentrations above 10 ng/ml. Upon treatment of the cells with antibody against VEGF, the cell proliferation increased linearly even at PDGF concentrations above 10 ng/ml. The enhanced [3H]thymidine incorporation by PDGF was abolished by either mitogen-activated protein kinase kinase (MAPKK) inhibitor or protein kinase C (PKC) inhibitor. In contrast, VEGF production was abolished by MAPKK inhibitor, but not by PKC inhibitor. These results indicate that PDGF stimulates both cell proliferation and VEGF production in partly different signal pathways, and thus PDGF might play a role in the physiology and pathology of the myometrium.

ABSTRACT

It is well known that lactotrophs are in close proximity to gonadotrophs in the lateral region of the pituitary gland, and thus there is interest in interactions between these two types of cell. The present study was undertaken to investigate the role of angiotensin II (AII) in gonadotrophin-releasing hormone (GnRH)-induced prolactin release, and to examine the effect of oestradiol on the paracrine interaction among anterior pituitary cells of young male rats. Over a 3-day period, one group of rats was injected twice with polyoestradiol phosphate (0·5 μg/g body weight; PEP-treated group), and a second with saline (control group). Their anterior pituitary glands were enzymatically dispersed and, subsequently, the cells were allowed to reaggregate for 48 h.

A 20-min perifusion with 100 nmol GnRH/1 increased (P<0·01) prolactin release from these anterior pituitary cell aggregates. The integrated value for prolactin release was 9·1 ±2·9 ng/10⁷ cells. In the PEP-treated group, basal release of prolactin was greater than that in the control group (P<0·01). However, during exposure to GnRH, the integrated amount of prolactin release by the PEP-treated group (12·5 ± 4·8 ng/10⁷ cells) was not significantly different from that of the control group, although in each individual experiment the GnRH-stimulated prolactin release from the PEP-treated cells was higher than that from the cells that had not been exposed to PEP. The release of angiotensin I (AI) from these perifused pituitary aggregates was significantly (P<0·01) increased by GnRH. In contrast, GnRH-stimulated release of prolactin was significantly (P<0·01) suppressed by 100 nmol saralasin/l, a specific AII antagonist, in both control and PEP-treated groups, whereas saralasin did not attenuate GnRH-induced LH release. GnRH-induced LH release was suppressed by PEP treatment during the first 2 min of perifusion, but enhanced throughout the remaining 18 min. In PEP-treated cell aggregates, the release of AI was increased during the later period. These data demonstrate that GnRH is capable of stimulating prolactin release through a mechanism that may involve the release of angiotensin.

Journal of Endocrinology (1990) 125, 225–232

University of Chicago, Pritzker School of Medicine, Chicago, Illinois 60637, U.S.A.

(Received 18 July 1975)

The biochemical basis of the genetically determined difference in sexual hair growth between oriental and caucasian men has not been established. Oriental men have plasma testosterone levels equal to those of Occidentals. However, the former group has low 17-oxosteroid excretion and beard growth can be induced by androgen, findings compatible with hypoandrogenism (Kobayashi, Lobotsky & Lloyd, 1966). Available data do not exclude differences in plasma testosterone binding or levels of other androgens. Therefore, the following studies were undertaken.

Six, 24 ± 0·81 (S.D.) -year-old normal graduate students of oriental descent (3 Chinese, 3 Japanese) who were unable to grow beard, moustache, or sideburns were compared with six, 24 ± 4·6-year-old, Caucasian graduate students who were fully virile. Hormone levels were measured in single, fasting, 08.00 h blood specimens.

Plasma testosterone, androstenedione, dihydrotestosterone, and dehydroepiandrosterone (DHA) were

ABSTRACT

Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using ¹²⁵I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0·59 nmol/l and the maximal binding capacity was 1·84 pmol/mg protein. Affinitylabelling of ¹²⁵I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1−100 nmol/l) induced rapid and transient increases in [Ca²⁺]_i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[³H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ET_B) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca²⁺]_i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells.

Journal of Endocrinology (1992) 134, 59–66

ABSTRACT

Five synthetic analogues of human parathyroid hormone (hPTH), (Tyr³⁴)hPTH(3–34) amide, (5–34) amide, (7–34) amide, (8–34) amide and (9–34) amide, were tested for their ability to antagonize hPTH action specifically in intact cultured cells. Clonal rat osteogenic sarcoma cells were used (UMR 106–06 line) which respond to PTH with an increase in cyclic AMP (cAMP) formation. The most potent antagonists were (Tyr³⁴)hPTH(3–34) amide and (5–34) amide, which inhibited the effect of hPTH(2·4 nmol/l) with half maximally effective concentrations of 0·1 μmol/l. When conditioned medium was used from a human lung cancer cell line producing osteoblast adenylate cyclase-stimulating activity, these two analogues were capable of inhibiting the increase in cAMP production. The specificity of the antagonism was indicated by the inability of the analogues to influence the effects of prostaglandin E₂ or of calcitonin, which are alternative stimulators of cAMP production in the osteogenic sarcoma cells. Only (Tyr³⁴)hPTH(3–34) amide showed some PTH-like agonist activity at high concentrations. These analogues should prove valuable in the investigation of PTH actions on target cells and of tumour products which appear to act through the PTH receptor.

J. Endocr. (1986) 108, 261–265

ABSTRACT

Parathyroid hormone (PTH)-like bioactivity, assayed as adenylate cyclase response in UMR 106-01 osteogenic sarcoma cells, was present in extracts of sheep fetal and maternal parathyroid glands and placenta. Preincubation of extracts with PTH(1–34) antiserum inhibited approximately 40% of the bioactivity in fetal parathyroid extracts, 50% in maternal parathyroid extracts, but only 10% of the bioactivity in the placental extract. Partial purification of placental extracts by chromatography yielded fractions containing PTH-like bioactivity which were similar in behaviour to that of PTH-related protein (PTHrP) from a human lung cancer cell line (BEN). An antiserum against synthetic PTHrP(1–16) partially inhibited the bioactivity of the placental extract and synthetic PTHrP(1–34), but had no effect on the bioactivity of bovine PTH(1–34) or bovine PTH(1– 84). The placental PTH-like bioactivity was higher in mid- than in late gestation. Fetal parathyroid glands contained the highest PTH-like bioactivity.

Thyroparathyroidectomy of one fetal twin lamb in each of 16 ewes between 110 and 125 days of gestation resulted in decreases of the plasma calcium concentration and reversal of the placental calcium gradient that existed between the ewe and the intact fetus. Perfusion of the placenta of each twin in anaesthetized ewes was carried out sequentially with autologous fetal blood in the absence of the exsanguinated fetus. The plasma calcium concentration in the blood perfusing the placenta of each twin increased, but reached a plateau at a lower concentration in the perfusing blood of thyroparathyroidectomized fetuses than in that of the intact fetuses. Addition of extracts of fetal parathyroid glands or of partially purified PTHrP resulted in further increases in plasma calcium in the autologous blood perfusing the placentae of thyroparathyroidectomized fetuses, but addition of bovine PTH(1–84) or rat PTH(1–34) had no effect. The presence of this PTH-like protein in the fetal parathyroid gland and placenta may contribute to the relative hypercalcaemia of the fetal lamb. This protein, which is similar to PTHrP associated with humoral hypercalcaemia of malignancy, stimulates the placental calcium pump responsible for maintaining a relative fetal hypercalcaemia during gestation.

J. Endocr. (1988) 117, 261–271

This study explores the effects of chronic administration of vitamin D(3) compounds on several biological functions in mice. Knowledge of long-term tolerability of vitamin D(3) analogs may be of interest in view of their potential clinical utility in the management of various pathologies such as malignancies, immunological disorders and bone diseases. Four unique vitamin D(3) analogs (code names, compounds V, EO, LH and LA) and 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) were administered i.p. for 55 weeks to Balb/c mice. Each analog had previously been shown to have potent in vitro activities. After 55 weeks of administration, the mice had a profound decrease in their serum levels of interleukin-2 (IL-2). Likewise, several analogs depressed serum immunoglobulin G concentrations (compounds LH and LA), but levels of blood lymphocytes and splenic lymphocyte subsets (CD4, CD8 and CD19) were not remarkably depressed. The percent of committed myeloid hematopoietic stem cells was 4- to 5-fold elevated in the bone marrow of the mice that received analogs LH and V; nevertheless, their peripheral blood white and red cell counts and platelets were not significantly different in any of the groups. The mice that received 1,25(OH)(2)D(3) had a decrease in bone quantity and quality with a decrease in cross-sectional area and cortical thickness, and a 50% reduction in both stiffness and failure load compared with the control group. In contrast, the cohort that received a fluorinated analog (compound EO) developed bones with significantly larger cross-sectional area and cortical thickness as well as stronger mechanical properties compared with the control group. At the conclusion of the study, body weights were significantly decreased in all experimental mice. Their blood chemistries were normal. Extensive gross and microscopic autopsy analyses of the mice at the conclusion of the study were normal, including those of their kidneys. In conclusion, the vitamin D(3) analogs were fairly well tolerated. They did suppress immunity as measured by serum IL-2 and may provide a means to depress the immune response after organ transplantation and for autoimmune diseases. Use of these analogs prevented the detrimental effects of vitamin D(3) administration on mechanical and geometric properties of bone, while one analog (compound EO) actually enhanced bone properties. These results suggest that long-term clinical trials with the analogs are feasible.