Leptin affects proliferation-, apoptosis- and protein kinase A-related peptides in human ovarian granulosa cells.

The aim of our in vitro studies was to understand the role of leptin in controlling proliferation, apoptosis, and protein kinase A (PKA) in human ovarian cells. We analyzed the in vitro effects of leptin (0, 1, 10 or 100 ng/ml) on the accumulation of proliferation-related peptides (PCNA, cyclin B1), apoptosis-associated peptide (Bax) and the intracellular signaling molecule PKA in cultured human granulosa cells using immunocytochemistry and Western immunoblotting. It was observed that leptin stimulated in a dose-dependent manner the accumulation of PCNA (at doses 1-100 ng/ml), cyclin B1 (at doses 10 or 100 ng/ml), Bax (at doses 10 or 100 ng/ml) and PKA (at doses 1-100 ng/ml) in cultured human ovarian cells. These observations suggest the ability of leptin to control directly human ovarian cell functions: proliferation, apoptosis, and intracellular messenger PKA.

Leptin directly controls secretory activity of human ovarian granulosa cells: possible inter-relationship with the IGF/IGFBP system.

AIMS: The aim of our in vitro studies was to understand the role of leptin and the insulin-like growth factor I/insulin-like growth factor protein (IGF/IGFBP) system in controlling human ovarian function. METHODS: We studied the action of leptin (0, 1, 10, or 100 ng/ml) and immunoneutralization of IGF-I using specific antiserum (0.1%) on the release of progesterone (P), estradiol (E), oxytocin (OT), IGF-I, IGFBP-3, and prostaglandins F (PGF) by these cells using radioimmunoassay/immunoradiometric assay. RESULTS: It was found that leptin stimulated the secretion of OT, IGFBP-3, and PGF. It suppressed the secretion of E and IGF-I, but not P, into the medium. The addition of antiserum against IGF-I decreased IGF-I output, increased P, OT, IGFBP-3, and PGF secretion, and had no effect on E release. Immunoneutralization of IGF-I also prevented or reversed the effects of leptin on P, E, IGF-I, IGFBP-3, PGF, but not on OT. CONCLUSIONS: These observations (1) demonstrate that leptin directly controls the secretory activity of human ovarian cells, (2) confirm the involvement of IGF-I in the regulation of ovarian cells, and (3) suggest an inter-relationship between leptin and the IGF/IGFBP system in the control of these functions and the involvement of IGF/IGFBP system in mediating leptin action on the ovary.

Thrombopoietin regulates proliferation, apoptosis, secretory activity and intracellular messengers in porcine ovarian follicular cells: involvement of protein kinase A.

Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their interrelationships within ovarian cells. We investigated the effect of TPO treatment (0, 1, 10 or 100 ng/ml) on the following characteristics of cultured porcine ovarian follicles, determined using SDS-PAGE and Western blotting, immunocytochemistry, RIA and ELISA: the expression of intracellular peptides associated with proliferation (PCNA), apoptosis (Bax), tyrosine kinase (TK, phosphotyrosine), Cdc2/p34 kinase, PKA and the transcription factor CREB-1, and the secretion of progesterone, androstenedione, estradiol-17beta, oxytocin, inhibin A, inhibin B, IGF-I, transforming growth factor-2beta (TGF-2beta) and IGF-binding protein 3 (IGFBP-3). The involvement of PKA-dependent pathways was examined by evaluating the effect of a PKA blocker (KT5720, 1 microg/ml), either alone or in combination with TPO, on the parameters listed above. A TPO-induced increase in expression of PCNA, Bax, PKA, TK, Cdc2/p34 and CREB was observed. Furthermore, TPO was able to inhibit androstenedione, estradiol, TGF-2beta and IGFBP-3 secretion, and to stimulate oxytocin, inhibin A, inhibin B and IGF-I secretion. Progesterone secretion was not stimulated. The PKA blocker KT5720, when given alone, reduced the expression of Bax and TGF-2beta, augmented the expression of PKA, CREB and oxytocin, but did not influence the secretion of progesterone, androstenedione, estradiol, IGFBP-3, inhibins A and B or IGF-I. When given together with TPO, the PKA blocker prevented or reversed the action of TPO on PKA, CREB, androstenedione, estradiol, IGFBP-3, oxytocin, but not its effect on Bax, TGF-2beta or inhibin B. On the other hand, treatment with KT5720 augmented the effect of TPO on progesterone, inhibin A and IGF-I. These results provide the first evidence that TPO may be a potent regulator of ovarian function (e.g. proliferation, apoptosis and the secretion of peptide hormones, steroids, growth factors and growth factor-binding protein, as well as of the expression of some intracellular messengers). Furthermore, they demonstrated the importance of PKA in controlling these functions and in mediating the effects of TPO on ovarian cells. It remains possible that other (TK- and Cdc2/p34-dependent) intracellular mechanisms are also involved in mediating TPO action on the ovary.

Aneuploidy in the transgenic rabbit.

The aim of this study was to determine whether there are differences in the karyotypes between transgenic and non-transgenic or control rabbits. New Zealand White transgenic rabbits (F1 generation) were obtained after breeding of transgenic founder rabbits that were derived from single--SM--or double microinjection--DM--with a WAP-hFVIII transgene. C-metaphase plates were obtained from short-time culture of peripheral blood lymphocytes synchronized by the addition of colcemide. A significantly higher rate of aneuploidy was observed in c-metaphase spreads of transgenic (56-66%) rabbits, as compared to non-transgenic ones (28-38%) (P < 0.05; P < 0.01). The patterns of chromosome banding were identical in both groups of rabbits. No structural aberrations were revealed in either group. These findings demonstrate that transgenic rabbits have a higher frequency of numerical chromosomal aberrations in their peripheral blood lymphocytes than normal rabbits, but without apparent deleterious effects on health or reproduction.

Do GH, IGF-I and oxytocin interact by regulating the secretory activity of porcine ovarian cells?

The aims of this study on porcine ovarian granulosa cells were to examine the effect of GH on oxytocin (OT), IGF-I and IGF-I receptors, IGF-binding protein-3 (IGFBP-3), progesterone and prostaglandin E (PGE), as well as to determine whether IGF-I and/or OT may be mediators of GH action. The cells were cultured either with porcine GH (pGH) (1 ng/ml to 10 microg/ml or 100 ng/ml only), antiserum against IGF-I (0.1%), antiserum against OT (0.1%) or a combination of GH (10 ng/ml) with antiserum against IGF-I or antiserum against OT (0.1%). The secretion of IGF-I, OT, IGFBP-3, progesterone and PGE was determined using RIA/IRMA, whilst the IGF-I binding sites were measured using a radioreceptor assay. It was observed that pGH increased the secretion of IGF-I and the abundance of IGF-I binding sites in granulosa cells. Furthermore, GH inhibited OT release, stimulated progesterone and PGE output, but had no significant effect on IGFBP-3 secretion. Immunoneutralization of IGF-I by antiserum against IGF-I inhibited PGE secretion, but it did not influence progesterone or IGFBP-3 secretion. Binding of OT by antiserum suppressed IGFBP-3, PGE, but not progesterone secretion. Neither immunoneutralization of IGF-I nor OT substantially prevented the effects of GH on progesterone, IGFBP and PGE. These observations demonstrate the involvement of GH, IGF-I and OT in the control of porcine ovarian secretory activity and the ability of GH to regulate IGF-I and OT production and IGF-I reception. Nevertheless, lack of correlation between the effects of GH, antiserum against IGF-I and antiserum against OT, as well as the inability of blockade of IGF-I or OT to prevent the effects of GH, suggests that IGF-I and OT, despite their dependence on GH, do not mediate GH action on ovarian cells.

Secretory activity of bovine ovarian granulosa cells transfected with sense and antisense insulin-like growth factor (IGF) binding protein-3 and the response to IGF-I, GH, LH, oxytocin and oestradiol.

The aim of our in vitro experiments was to examine if IGF binding protein (IGFBP)-3 is involved in control of bovine ovarian secretory activity. For this purpose we performed the transfection of bovine granulosa cells with cDNA sense and antisense constructs increasing or inhibiting IGFBP-3 synthesis. The release of IGFBP-3, progesterone, oxytocin, IGF-I and prostaglandins F (PGF) and E (PGE) by control and transfected cells was compared. The transfected ovarian cells were cultured with and without bLH (100 ng/ml), bGH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and oestradiol-17beta (100 ng/ml). The concentration of IGFBP-3 produced was assessed using ligand and western blotting and secretion of progesterone, oxytocin, IGF-I, PGF and PGE was evaluated using RIA/IRMA techniques. Transfection of cells with the sense IGFBP-3 cDNA construct resulted in the expected increase in IGFBP-3 release, whereas the antisense IGFBP-3 construct induced the expected reduction in IGFBP-3 output. The granulosa cells transfected to overexpress IGFBP-3 had an increase in IGF-I, PGF and PGE release, and a decrease in basal and hormone- or growth factor-induced accumulation of progesterone and oxytocin. The granulosa cells transfected to have reduced IGFBP-3 expression gave primarily significant opposite findings. The present results suggest the involvement of IGFBP-3 in control of bovine ovarian steroid, peptide hormone, growth factor and prostaglandin release. IGFBP-3 is a physiological stimulator of IGF-I and prostaglandin release and an inhibitor of steroid and peptide hormone output.

The role of IGF-I, cAMP/protein kinase A and MAP-kinase in the control of steroid secretion, cyclic nucleotide production, granulosa cell proliferation and preimplantation embryo development in rabbits.

The aim of this study was to investigate the actions of insulin-like growth factor I (IGF-I) on the secretory and proliferative functions of rabbit ovarian cells and on early embryogenesis. It was found that addition of IGF-I at a lower concentration (1 ng/ml) stimulated progesterone secretion by cultured rabbit granulosa cells, whilst higher concentrations of IGF-I (10, 100 ng/ml) were inhibitory. IGF-I had no effect on estradiol secretion. Cyclic AMP secretion was slightly increased after addition of IGF-I at 10 ng/ml, but not by higher concentrations. Cyclic GMP secretion was stimulated by IGF-I at 100 ng/ml only. A blocker of protein kinase A, Rp-cAMPS, did not alter progesterone and estradiol secretion but did prevent the action of IGF-I on progesterone secretion. An immunocytochemical study demonstrated that IGF-I significantly increased the proportion of proliferating cell nuclear antigen-positive (PCNA-positive) cells. Rp-cAMP did not change cell proliferation but partially prevented the proliferation-stimulating effect of IGF-I. IGF-I (100 ng/ml) significantly increased the proportion of divided zygotes and the number of embryos reaching the morula/blastocyst stage. Blockers of PKA, Rp-cAMPS and KT5720, reversed the effects of IGF-I on zygote cleavage and embryo development. Addition of IGF-I (100 ng/ml) significantly increased MAPK within the cells (proportion showing immunoreactivity to ERK-1 and ERK-3 antibodies and intensity of a 42 kDa band related to ERK-2). Rp-cAMPS suppressed the basal ERK-2 immunoreactivity but not that of ERK-1 or ERK-3. It completely inhibited the IGF-I-induced activation of ERK-3 but not that of ERK-1 or ERK-2. This in vitro study demonstrates that IGF-I is a potent stimulator of ovarian secretion, proliferation and embryogenesis in rabbit. Its effects are mediated by cAMP/PKA- and, probably by, MAPK-dependent intracellular mechanisms.

Effect of four cGMP analogues with different mechanisms of action on hormone release by porcine ovarian granulosa cells in vitro.

The aim of our in-vitro experiments was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian hormone secretion, as well as to understand, whether cGMP effect on the ovary may be mediated by either protein kinase G (PKG), cGMP-gated ion channels (CGI) or cGMP-specific phosphodiesterases (PDE). We compared the effects of the cGMP analogues 8-pCPT-cGMP, an activator of PKG 1-alpha, 1-beta and type II and of CGI, but not of PDE: Rp-8-pCPT-cGMPS and Rp-8-Br-cGMPS, inhibitors of PKG, stimulators of CGI with no effect of PDE, and Rp-8-Br-PET-cGMPS, an inhibitor of both, PKG and CGI and stimulator of PDE (all at 0.01, 0.1, 1, 10 or 100 nM), on the release of oxytocin (OT) and progesterone (P) by cultured porcine granulosa cells. It was observed, that Rp-8-pCPT-cGMPS significantly (p<0.05) suppressed OT release when given at 1 or 10 nM. Rp-8-Br-cGMPS increased OT output, when given at 1-10 nM too, but decreased it at 100 nM. Rp-8-Br-PET-cGMPS inhibited OT release at 1 nM. No influence of 8-pCPT-cGMP on OT output was found. 8-pCPT-cGMP stimulated P release at 0.1, 10 or 100 nM. All other cGMP analogues studied suppressed P release at all doses used. The present observations suggest the involvement of cGMP-dependent intracellular mechanisms in control of ovarian steroid and nonapeptide hormone release. The lack of association between patterns of influence of cGMP analogues on CGI and PDE, and the coincidence of the majority of effects of cGMP analogues on P, OT and PKG may indirectly indicate that cGMP action on release of ovarian hormones is mediated mainly by PKG, but not by CGI or PDE.

Effect of growth hormone and inhibitors of protein kinase A on IGF-I, oxytocin and progesterone release by cultured bovine granulosa cells.

In our experiments we studied the action of GH on the release of nonapeptide, steroid hormones and growth factor, in bovine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these GH effects. For this purpose, the effects of exogenous bGH (0.001-10 mg/ml), PKA blockers KT5720 (100 ng/ml) and Rp-cAMPS (1 mmol), alone and in combination, on IGF-I, oxytocin and progesterone secretion were investigated. It was found that GH addition to culture medium strongly (p<0.05) stimulated IGF-I (at a concentration of 0.01-0.1 mgGH/ml medium), oxytocin (0.01-10 mgGH/ml) and progesterone (0.01-1 mgGH/ml medium) secretion into the culture medium. PKA blockers KT5720 and Rp-cAMPS given alone did not affect release of these substances. Rp-cAMPS partially prevented GH effect on IGF-I release, but enhanced GH action on progesterone output. KT5720 did not modify action of GH on oxytocin release. These observations confirm the involvement of GH in the control of IGF-I, oxytocin and progesterone release by bovine ovarian granulosa cells. Effects of PKA blocker on several GH-induced effects suggest that GH effects on IGF-I and progesterone, but not on oxytocin release may be partially mediated by the cAMP/PKA-dependent intracellular mechanisms.

Isolated porcine ovarian follicles as a model for the study of hormone and growth factor action on ovarian secretory activity.

The aim of our in vitro experiments with isolated porcine ovarian follicles was to study the effects of gonadotropins, GH, IGF-I and oxytocin (OT) on release of ovarian steroid, OT, IGF-I, insulin-like growth factor-binding protein-3 (IGFBP-3), prostaglandin F (PGF), prostaglandin E (PGE) and cAMP. It was found that quarters of ovarian follicles cultured for 8 days produced significant amounts of progesterone, estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation from the 3rd to the 8th day of culture. Addition of serum promoted progesterone, estradiol and OT release, whilst accumulation of IGFBP-3 was maintained to a greater extent in serum-free medium. GH (10 ng/ml or above) was able to inhibit androstenedione, OT, PGF and IGFBP-3, to stimulate IGF-I and cAMP, and to alter testosterone and PGE release by follicles cultured in serum-supplemented and/or serum-free medium. IGF-I (10 ng/ml or more) inhibited androstenedione and PGF secretion, stimulated testosterone, estradiol, OT and cAMP production, but did not influence progesterone, IGFBP-3 or PGE output in these conditions. OT (100 ng/ml) was able to inhibit androstenedione and to stimulate testosterone, IGF-I, PGF and PGE, but not estradiol or IGFBP-3 release. A stimulatory effect of LH on progesterone and OT and an inhibitory influence of LH on estradiol secretion in the serum-supplemented medium were observed. FSH in these conditions stimulated OT, but not progesterone or estradiol secretion. The use of this experimental model suggests the involvement of gonadotropins, OT, GH and IGF-I in the control of ovarian steroid and nonapeptide hormone, growth factor, growth factor-binding protein, prostaglandin and cyclic nucleotide production. The stimulatory effect of GH on IGF-I, and the stimulatory influence of IGF-I on OT, as well as coincidence of the majority of effects of IGF-I and OT, suggest the existence of a GH-IGF-I-OT axis. On the other hand, the different patterns of action of GH and IGF-I on OT, estrogen and IGFBP-3 suggest that part of the GH effect on ovarian cells is IGF-I independent.

The ability of steroid hormones to control cAMP and cGMP production by human granulosa cells in culture.

The aim of our in vitro experiments was to discover the effects of steroid hormones (progesterone, androstenedione and estradiol-17 beta, 10, 100, 1000 or 10,000 pg/ml medium) on the output of cyclic nucleotides adenosine 2',3'-cyclic monophosphate (cAMP) and guanosine 2',3'-cyclic monophosphate (cGMP) by granulosa cells isolated from human ovaries. The dramatic increase of cAMP release after progesterone treatment was observed. Low doses of androstenedione (10 or 100 pg/ml) significantly decreased while higher doses (1000 or 10,000 pg/ml) tended to increase cAMP output. All doses of estradiol significantly inhibited cAMP release. Low doses (10 or 100 pg/ml) of all investigated steroid inhibited while higher doses (1000 or 10,000 pg/ml) stimulated cGMP output. cGMP-stimulating effects of progesterone and androstenedione were expressed significantly more than that of estradiol. The influence of progestagen, androgen and estrogen on cyclic nucleotide release by granulosa cells may suggest the involvement of cAMP- and cGMP-dependent intracellular mechanisms in the realization of steroid hormone effects within human ovaries.

Prevention of postoperative thromboembolisms in general surgery by the combination of heparin and dihydroergotamine.

The effect and the degree of safety of administering a fixed combination of 5000 IU of heparin + 0.5 mg dihydroergotamine (HDHE s.c. per every 12 hours) as opposed to 5000 IU of heparin (LDH s.c. every 8 hours) was assessed in a prospective randomized study on 86 patients having undergone major abdominal operation. Postoperatively a deep vein thrombosis was detected by the radiofibrinogen test in 10% of the 40 patients of the HDHE group and in 13% of 46 of the LDH group. Four patients died. At autopsy neither fatal nor a contributing pulmonary embolism was found. 'Non-lethal' pulmonary embolism diagnosed by lung perfusion scintigraphy and by chest X-rays, developed in 2 patients treated with LDH and in one treated with HDHE. Two-thirds of the dose of heparin were identically effective in prevention of venous thromboembolisms than the whole dose if heparin was combined with DHE. The decrease of the heparin dose significantly reduced the number of wound haematomas and of suffusion due to injection.