Interrelationships between sirtuin 1 and transcription factors p53 and NF-κB (p50/p65) in the control of ovarian cell apoptosis and proliferation.

The roles of the mTOR system enzyme sirtuin 1 (SIRT1), the transcription factor p53 and the nuclear factor kappaB (NF-κB) and their interrelationships in the control of ovarian function have not been well studied. We examine, in vitro, the involvement of SIRT1, p53 and the p65 and p50 subunits of NFκB and their interrelationships in the control of the apoptosis and proliferation of porcine ovarian granulosa cells. Monolayers of primary granulosa cells were transfected with cDNA constructs encoding SIRT1, p53, p65 or p50 alone or were co-transfected with gene constructs for SIRT1 together with p53, p65 or p50. The accumulation of SIRT1, markers of proliferation (mitogen-activated protein kinase or extracellular-signal-regulated kinases 1,2) and a marker of apoptosis (caspase 3) was detected by immunocytochemistry. Transfection of cells with a SIRT1 gene construct alone promoted the accumulation of SIRT1 and decreased the accumulation of proliferation markers but did not affect the marker of apoptosis. Transfection of cells with gene constructs encoding p53, p50 or p65 decreased the expression of proliferation markers but not the apoptosis marker. Co-transfection of cells with SIRT1 cDNA changed the action of p65 on cell proliferation from inhibitory to stimulatory. SIRT1 overexpression induced the pro-apoptotic action of p53 and p50 but not of p65 constructs. Thus, SIRT1, p53 and NF-κB are involved in the control of both the proliferation and the apoptosis of ovarian cells. These novel data on the cross-talk between the mTOR/SIRT1 system and the transcription factors p53 and NF-κB show both the inhibitory (proliferation) and stimulatory (apoptosis) influences of SIRT1 on transcription factor action in ovarian cells.

The involvement of SIRT1 and transcription factor NF-κB (p50/p65) in regulation of porcine ovarian cell function.

The role of either mTOR system/enzyme sirtuin1 (SIRT1) or transcription factor NF-κB in the direct control of ovarian function has not been estabished. The aim of our in vitro experiments was to examine the involvement of SIRT1 and the p65 and p50 subunits of NFκB in control of porcine ovarian granulosa cell functions and the interrelationships between SIRT1, NFκB (p65, p50) 30 and FSH in the ovary. Monolayers of primary granulosa cells were transfected with gene constructs encoding either SIRT1 or p65 and p50, and thereafter cultured with, or without, addition of FSH. The accumulation of markers of proliferation (cyclin B1 and cyclin-dependent protein kinase Cdc2/p34) and proteins p50, p65 and SIRT1 in the cells was detected by using SDS-PAGE/Western immunoblotting and immunocytochemistry. The secretion of progesterone (P4) and insulin-like growth factor I (IGF-I) was measured by using radioimmunoassay. It was observed that transfection of cells with a SIRT1 gene construct promoted accumulation of proliferation markers, Cdc2/p34, cyclin B1, decreased accumulation of p50 and p65 and stimulated release of P4 and IGF-I. Co-transfection of cells with cDNA p50 and cDNA p65 enhanced the accumulation of SIRT1 and the release of P4 but did not influence the release of IGF-I. Adding FSH to the culture medium stimulated accumulation of both subunits of NF-κB, as well as accumulation of Cdc2/p34, cyclin B1 and release of both P4 and IGF-I. The ability of FSH to promote NF-κB accumulation, the similarity of the main effects of FSH, SIRT1 and NF-κB, as well as the inability of NF-κB to substantially modify the the majority of FSH effects suggest that SIRT1/NF-κB system could be a mediator of FSH action on ovarian cell functions. On the other hand, SIRT1 was able to inhibit NF-κB and to change stimulatory the effect of FSH on NF-κB from stimulatory to inhibitory. This could suggest the existence of negative feedback control of FSH/NF-κB system by high amounts of SIRT1. Our observations (1) confirm the previous data on proliferation, P4 and IGF-I release in ovarian cells and their up-regulation by FSH, (2) demonstrate the presence of SIRT1, NF-κB/p50 and NF-κB/p65 in these cells, (3) show for the first time the involvement of SIRT1 and NF-κB in direct control of proliferation and secretory activity of ovarian cells, (4) represent the first data on interrelationships between FSH, SIRT1 and NF-κB within the ovary.

Transcription factor NF-κB (p50/p50, p65/p65) controls porcine ovarian cells functions.

The aim of these in vitro studies was to examine the involvement of transcription factor NF-κB (p50/p50, p65/p65) and FSH in control of porcine ovarian granulosa cells functions and the possible role of dimers p50/p50, p65/p65 in mediating FSH actions on these cells. Monolayer of primary granulosa cells was transfected with plasmids encoding human p50 cDNA and p65 cDNA, and cultured with or without addition of FSH (0, 1, 10 or 100 ng/ml). The accumulation of proteins p50 and p65, as well as of proliferation markers (PCNA and MAPK/ERK1,2) and marker of apoptosis (Bax) in cells was detected by using SDS-PAGE-Western immunoblotting and immunocytochemistry. DNA fragmentation was evaluated by TUNEL assay. Release of hormones insulin-like growth factor I (IGF-I), progesterone (P(4)), oxytocin (OT), prostaglandins E(2) (PGE(2)) and F(2α) (PGF(2α)) was measured by using RIA. We observed, that p50/p50 promoted the accumulation of PCNA, MAPK/ERK1,2, the release of OT, PGF(2α); inhibited the occurrence of TdT-positive cells, the release of IGF-I and P(4), and did not influence the accumulation of Bax and the release of PGE(2). p65/p65 enhanced the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, PGE(2) and PGF(2α); decreased the percentage of cell containing TdT and did not affect the release of P(4). FSH stimulated the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, P(4), PGE(2); but reduced the proportion of TdT-positive cells and the release of PGF(2α). These observations suggest (1) the involvement of NF-κB (p50/p50) in stimulation of proliferation, inhibition of apoptosis and in either stimulation (OT, PGE(2)) or inhibition (IGF-I, P(4), but not PGF(2)) of hormones release by porcine ovarian granulosa cells; (2) the involvement of NF-κB (p65/p65) in stimulation of proliferation and mitochondrial/Bax-related apoptosis, inhibition of nuclear/TdT-related apoptosis and in stimulation of ovarian hormones (IGF-I, OT, PGE(2), PGF(2α), but not P(4)) release; (3) the role of FSH in up-regulation of both ovarian cell proliferation and mitochondrial/Bax-related apoptosis, in inhibition of nuclear/TdT-related apoptosis, in promotion of IGF-I, P(4), OT, PGE(2) and suppression of PGF(2α) release by porcine ovarian cells. The majority of results demonstrates the involvement of NF-κB (p50/p50 and p65/p65) and FSH in control of basic ovarian functions (proliferation, apoptosis, and secretory activity), but not the functional interrelationships between these regulators.

Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos.

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

A role for IL-15 in driving the onset of spontaneous autoimmune thyroiditis?

The obese strain (OS) of chickens, which suffers from spontaneous autoimmune thyroiditis, is an excellent animal model for Hashimoto's thyroiditis and provides a unique opportunity to investigate the mechanisms underlying and driving the onset of the disease. Following recent advances in cloning chicken cytokines, we can now begin to investigate the role of cytokines in driving the lymphoid infiltration of the thyroid seen in these birds from day 7 posthatch. Using real-time quantitative RT-PCR, we characterized the expression of IFN-gamma, IL-1beta, IL-2, IL-6, IL-8, IL-15, and IL-18 in thyroids from OS birds and control CB line birds, both in the embryo just before hatch (embryonic day 20) and at 3 and 5 days posthatch. All of these cytokines were up-regulated compared with levels in thyroids from CB birds, at least at some time points, with some evidence for coordination of regulation, e.g., for the proinflammatory cytokines IL-1beta and IL-8. Only IL-15 was up-regulated at all time points. IL-15 was also shown to be up-regulated in spleens of OS birds at embryonic day 20 and 5 days posthatch, suggesting that IL-15 is constitutively up-regulated in this line of birds. This could explain the general immune system hyperreactivity exhibited by OS chickens and may be a factor driving the lymphoid infiltration of the thyroid.