Porcine Granulosa Cells Research Articles

Biphasic effects of leptin in porcine granulosa cells.

The direct effects of recombinant porcine leptin on porcine granulosa cells were studied to test the hypothesis that leptin, acting through the nuclear transcription factor signal transducer and activator of transcription 3 (STAT-3), modulates sterol regulatory element-binding protein 1 (SREBP1) thereby increasing steroidogenesis. In porcine granulosa cells in culture over 48 h, leptin at 10 ng/ml increased progesterone accumulation 3-fold while it was reduced by leptin at 1000 ng/ml. Leptin had no effect on progression of granulosa cells through the cell cycle nor on the frequency of cell death. Leptin treatment at 24 or 48 h of culture resulted in dose-dependent 2- to 4-fold increases in tyrosine phosphorylation of STAT-3. Leptin had a biphasic effect on the abundance of membrane-bound and transcriptionally active forms of SREBP1. In transient transfection of primary porcine granulosa cells, the plasmid expressing the transcriptionally active form of SREPB-1 induced transcription of the key regulator of steroidogenesis, the steroidogenic acute regulatory protein (StAR). StAR transcription was also increased by the low dose of leptin and was further upregulated in the presence of the SREBP plasmid. Leptin at 1000 ng/ml inhibited SREBP1-induced StAR expression. Thus, leptin, acting through STAT-3, modulates steroidogenesis in a biphasic and dose-dependent manner, and SREBP1 induction of StAR expression may be in the cascade of regulatory events.

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5 mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3 H -thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24 h resulted in proliferation arrest in vitro. Treatment with 0.5 mM mimosine significantly ( P<0.05) inhibited 3 H -thymidine incorporation after 24 h of culture (4.6% ± 0.1) and after 24 h of culture in serum deprived medium (41.3% ± 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24 h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.

Expression and distribution of AP-1 transcription factors in the porcine ovary.

The activator protein-1 (AP-1) transcription factors are important regulators of cell proliferation and differentiation. The developmental distribution of AP-1 family members in porcine ovary has not been previously investigated. We examined the expression of AP-1 factors in porcine ovarian follicles, granulosa cells, and corpora lutea at different stages of development. Immunoblot analyses confirmed that c-Jun, JunD, JunB, c-Fos, Fra-1, Fra-2, and FosB immunoreactive proteins were present in whole-cell extracts (WCE) of all antral follicles and midluteal phase corpora lutea (CL) as well as granulosa cells (GC) isolated from different-sized antral follicles. The intensities of c-Jun and c-Fos protein bands were decreased in CL WCE compared to antral follicles. In granulosa cells from preovulatory follicles (8-10 mm), Fra-2 exhibited a shift from 43 kDa to 46 kDa when compared to granulosa cells from smaller antral follicles. Separation of cytoplasmic and nuclear extracts was performed to determine if developmental differences between these fractions existed. Most AP-1 factors predominated in the nuclear fraction with notable exceptions. c-Fos predominated in the nucleus in GC and follicles but predominated in the cytoplasmic fraction of CL. With the exception of GC from 1-2-mm follicles, in which expression was similar between fractions, Fos-B was found predominantly in the cytoplasmic fraction. Fra-1 exhibited similar expression between cytoplasmic and nuclear fractions for all tissues. Immunohistochemical (IHC) analyses of porcine ovary sections were performed to determine the cellular distribution of these factors at different follicular stages, and immunopositive nuclei were evaluated. In primordial and primary unilaminar follicles, all AP-1 factors studied except for FosB were detected in granulosa nuclei. Granulosa cell nuclei of multilaminar preantral follicles were immunopositive for all factors, with lower expression of FosB. Antral follicles exhibited GC and thecal cell nuclear staining for all factors with the exception of FosB in theca. Luteal cells exhibited the most intense nuclear staining for JunD and Fra-2, whereas all other factors were present in luteal cell nuclei although to a lesser extent. IHC with FosB antibodies yielded mostly cytoplasmic staining but only weak luteal nuclear staining. In corpora albicantia, low levels of staining were seen for all AP-1 factors. The DNA-binding abilities of these factors in granulosa cells and CL were evaluated by EMSA. Nuclear extracts from granulosa cells from 1-2-mm or 8-10-mm antral follicles bound an AP-1 DNA consensus sequence and complexes consisted predominantly of c-Jun, JunD, JunB, c-Fos, and Fra-2. In CL, c-Jun, JunD, JunB, and Fra-2 were present in DNA-binding complexes, and c-Fos binding was not detected. In conclusion, our results suggest that expression and DNA-binding activity of AP-1 factors in follicular structures changes with luteinization. Differentiation to the luteal phenotype involves a reduction in nuclear c-Jun and c-Fos and a predominance of JunD and Fra-2.

Synergistic action of growth hormone and insulin-like growth factor I (IGF-I) on proliferation and estradiol secretion in porcine granulosa and theca cells cultured alone or in coculture

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [ 3 H ]thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [ 3 H ]thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [ 3 H ]thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.

Correlation of mitogen-activated protein kinase activities with cell survival and apoptosis in porcine granulosa cells.

The regulation of granulosa cell survival and death is critical for determining the fate of ovarian follicles. Mitogen-activated protein kinases (MAPKs) play central roles in various cellular responses, but the relationship between MAPK activities and granulosa cell survival as well as death is poorly understood. The present study examines the roles of the extracellular signal-regulated kinase (ERK) and p38 MAPK activities in porcine granulosa cells in response to survival factors and oxidative stress. Cell survival and apoptosis were evaluated by Trypan blue staining, DNA fragmentation, and chromatin staining with Hoechst 33342. Cell survival induced by serum or by follicle-stimulating hormone (FSH) was inhibited when ERK activity was attenuated with PD98059, which led to the induction of apoptosis. The p38 inhibitor SB203580 significantly decreased the cell survival evoked by FSH, but not by serum. Even in the presence of 10% serum, H(2)O(2) caused apoptosis, indicating that H(2)O (2) may be an atretogenic factor or its mediator. Interestingly, this induction of apoptosis was also prevented by SB203580, suggesting that p38 is involved in an apoptotic pathway induced by H(2)O (2) as well as in a survival pathway evoked by FSH in granulosa cells. These results indicate that whereas ERK activity is critical to the survival of granulosa cells, p38 activity contributes to their survival or apoptosis depending on the stimulus.

Involvement of MAP kinase in the mediation of GH action on ovarian granulosa cells

Growth hormone (GH), prostaglandins F (PGF) and prostaglandins E (PGE) are important regulators of ovarian function. Therefore, interrelationships between GH and these substances and their intracellular mechanisms might be of physiological significance in the ovary. The aims of this study on cultured porcine ovarian granulosa cells were to determine the effect of GH on the secretion of oxytocin (OT), PGF and PGE and whether MAP kinase could be involved in the mediation of GH action. Experiments were carried out with cultured porcine granulosa cells to investigate the effects of exogenous pGH (1–100 ng/ml) on the expression of MAP kinase (ERK-1, -2) and of PGH (1–100 ng/ml) and the MAP kinase blocker PD 98059 (1 μg/ml) on the secretion of PGF, PGE and OT. The cellular content of ERK-1 and -2 was analyzed by Western immunoblotting and immunocytochemistry, whilst PGF, PGE and OT accumulation in the medium was measured by RIA. Addition of GH to culture medium significantly altered the pattern of ovarian ERK MAP kinase on SDS-PA gels: the 44 and 42 kDa bands were reduced and additional 50 and 48 kDa bands appeared. Moreover, there was an increase in the percentage of cells containing ERK MAP kinase. GH stimulated the secretion of PGF (at a concentration of 1 ng GH per ml medium) and OT (100 ng GH per ml), but not PGE. The MAP kinase blocker alone did not affect PGF, PGE and OT secretion but did prevent the stimulatory effects of GH on PGF and induced stimulatory action of GH (10 ng/ml) on PGE. GH-stimulated OT secretion was unaffected. These observations confirm the role of GH in regulating porcine ovarian PGF, PGE and OT secretion and the presence of ERK MAP kinase in porcine granulosa cells. Furthermore, our studies demonstrate that MAP kinase-dependent intracellular mechanisms are dependent on GH, and that these mechanisms are involved in the mediation of GH action on ovarian PGF and PGE but not OT secretion.

4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

BackgroundIon channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.MethodsGranulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size.Results4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production.Conclusion4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Involvement of prostanoids in the inhibitory effect of endothelin-1 on progesterone production of ovarian granulosa cells.

The present study was undertaken to investigate the possible involvement of prostanoids in the inhibitory effect of endothelin-1 (ET-1) on progesterone production of ovarian granulosa cells. ET-1 (1 x 10(-7) M) decreased the basal and follicle-stimulating hormone- (FSH) stimulated progesterone production from both human and porcine granulosa cells. Indomethacin dose-dependently inhibited progesterone release, but did not alter the inhibitory effect of ET-1 (1 x 10(-7) M) on progesterone production of cultured ovarian granulosa cells. This study shows that ET-1 suppresses basal and FSH-stimulated progesterone production by ovarian granulosa cells, but this effect is not mediated by prostanoids.

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization.

Effects of VEGF and bFGF on proliferation and production of steroids and nitric oxide in porcine granulosa cells.

Ovarian angiogenesis, which is currently considered to be of crucial importance in controlling the growth of developing follicles, is a physiological process driven by a variety of angiogenic factors. Among these, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been recognized as key players in promoting cell growth and differentiation. Porcine granulosa cells from small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles were seeded at different densities in DMEM:Ham's F12 (1:1) with or without different concentrations of VEGF or bFGF. After 48 h of culture, media were assayed for oestradiol (E2) 17beta, progesterone (P4), nitric oxide (NO) and VEGF levels; in addition, cell proliferation was evaluated by 3H-thymidine incorporation assay. Both bFGF and VEGF effects on E2 and P4 production by cultured granulosa cells resulted to be dependent on follicle size. The bFGF was always ineffective in modulating cell proliferation, while VEGF exerted an inhibitory effect on the proliferation in the small follicle group and a stimulatory one in the medium and large follicle groups. The bFGF consistently reduced NO levels in culture media. The VEGF appeared to be ineffective in modifying NO production in the small follicle group, while it was stimulatory in the medium follicle group and inhibitory in the large follicle group. Basal VEGF production was higher in cells from the large follicle as compared with the small and medium follicle groups, and it was unaffected by bFGF. These results suggest that VEGF plays a modulatory role in granulosa cell functional activity and it is possibly involved in the regulation of follicle growth; on the contrary, bFGF does not appear to represent a significant regulatory factor in our cellular model, except for an inhibitory action on the production of NO, whose anti-angiogenic properties need to be further substantiated.

Characterization of a recombinant porcine follistatin in a heat shock expression system

The role of follistatin in ovarian function has yet to be fully elucidated; it is present in low concentration in vivo and it is difficult to obtain suitable quantities of follistatin for characterization. We have cloned porcine follistatin cDNA in an expression system that uses the heat shock protein promoter BoHSP70. Recombinant follistatin with apparent molecular masses of 39, 46, 48, 50 kDa was expressed and secreted into culture medium at concentrations of 400–500 μg × 20 mL-1 × 150 cm-2 flask (4 × 107 cells). In ligand blots, the recombinant follistatin was shown to be immunologically similar to native follistatin and to bind to recombinant activin A. Porcine granulosa cells dissected from 1–3 mm follicles from immature gilts were cultured with recombinant follistatin or with a follistatin monoclonal antibody to examine the activity of the recombinant follistatin. At a physiologically relevant concentration of 1 μg mL-1 the recombinant porcine follistatin suppressed the accumulation of estradiol-17β (P &lt; 0.05). There was a trend for estradiol-17β accumulation in the presence of 10 μg mL-1 of monoclonal anti-follistatin antibody (P = 0.1). This expression system consistently produced large quantities of recombinant porcine follistatin, which is immunologically and biologically similar to follistatin, and is capable of independently inhibiting estratiol-17βproduction in vitro. Key words: Porcine, follistatin, heat shock promoter, glycoprotein, ovary, estradiol

Potassium channel antagonists influence porcine granulosa cell proliferation, differentiation, and apoptosis.

This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

Effects of progestins on progesterone synthesis in a stable porcine granulosa cell line: control of transcriptional activity of the cytochrome p450 side-chain cleavage gene.

The purpose of the present study was to examine the effects of progestins on progesterone synthesis and expression of the cytochrome P450 cholesterol side-chain cleavage gene (P450(scc)) in a stable porcine granulosa cell line, the JC-410. Cells were incubated for 48 h with the synthetic progestogen-levornorgestrel with or without RU486 (progesterone and glucocorticoid receptor antagonist) or RWJ26819 (progesterone agonist without affinity to glucocorticoid receptors). Both levonorgestrel and RU486 enhanced progesterone accumulation in a dose-dependent manner. RU486 did not antagonize the effects of levonorgestrel, and RWJ26819 had no effect on progesterone production in cultured JC-410 cells. Progesterone and levonorgestrel increased steady state P450(scc) mRNA levels after 3-6 h of treatment. Progesterone and RU486 at 0.1, 1, and 10 microM increased the transcription rate of P450(scc) transiently expressed in JC-410 cells after 18 h of incubation; 30 microM had no effect, and 100 microM suppressed transcription. Levonorgestrel did not affect transcription of the P450(scc) gene, and RWJ26819 reduced its transcription. Progesterone and RU486 significantly decreased the number of cells and total protein content after 72 and 24 h of incubation, respectively. Levonorgestrel had no effect, whereas RWJ26819 increased (24 h) but subsequently reduced (72 h) cell number and protein content. The present results indicate that progestins are capable of directly modulating progesterone biosynthesis in porcine JC-410 granulosa cells. These effects may be exerted in part through the regulation of P450(scc) gene expression. Ostensible differences exist between progesterone and its synthetic analogues in the control of progesterone secretion in the stable porcine granulosa cell line in vitro.

Molecular basis of voltage-dependent potassium currents in porcine granulosa cells.

The major objective of this study was to elucidate the molecular bases for K(+) current diversity in porcine granulosa cells (GC). Two delayed rectifier K(+) currents with distinct electrophysiological and pharmacological properties were recorded from porcine GC by using whole-cell patch clamp: 1) a slowly activating, noninactivating current (I(Ks)) antagonized by clofilium, 293B, L-735,821, and L-768,673; and 2) an ultrarapidly activating, slowly inactivating current (I(Kur)) antagonized completely by clofilium and 4-aminopyridine and partially by tetraethylammonium, charybdotoxin, dendrotoxin, and kaliotoxin. The molecular identity of the K(+) channel genes underlying I(Ks) and I(Kur) was examined using reverse transcription-polymerase chain reaction and immunoblotting to detect K(+) channel transcripts and proteins. We found that GC could express multiple voltage-dependent K(+) (Kv) channel subunits, including KCNQ1, KCNE1, Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kvbeta1.3, and Kvbeta2. Coimmunoprecipitation was used to establish the hetero-oligomeric nature of granulosa cell Kv channels. KCNE1 and KCNQ1 were coassociated in GC, and their expression coincided with the expression of I(Ks). Extensive coassociation of the various Kv alpha- and beta-subunits was also documented, suggesting that the diverse electrophysiological and pharmacological properties of I(Kur) currents may reflect variation in the composition and stoichiometry of the channel assemblies, as well as differences in post-translational modification of contributing Kv channel subunits. Our findings provide an essential background for experimental definition of granulosa K(+) channel function(s). It will be critical to define the functional roles of specific GC K(+) channels, because these proteins may represent either novel targets for assisted reproduction or potential sites of drug toxicity.

Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ovine, Bovine, Porcine, and Equine Ovarian Follicles: Involvement in IGF Binding Protein-4 Proteolytic Degradation and mRNA Expression During Follicular Development

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ovine, Bovine, Porcine, and Equine Ovarian Follicles: Involvement in IGF Binding Protein-4 Proteolytic Degradation and mRNA Expression During Follicular Development

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29–1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

The diverse mechanism of action of dichlorodiphenyldichloroethylene (DDE) and methoxychlor in ovarian cells in vitro

Dichlorodiphenyldichloroethylene (DDE), the most stable metabolite of the organochlorine insecticide dichlorodiphenyltrichloroethane (DDT), and the DDT analog methoxychlor can have adverse effects on reproduction. These chemicals have been identified as having estrogenic activity. The aim of the current study was to examine the effects of dichlorodiphenyldichloroethylene (DDE), methoxychlor, and estradiol-17β on steroidogenesis and FSH responsiveness in ovarian cells in vitro. Experiments were performed on a primary culture of porcine granulosa cells and a culture of Chinese hamster ovary (CHO) cells, the latter stably transfected with the FSH receptor (CHO-FSH-R). DDE (10 μM) and estradiol-17β (0.1 μM) but not methoxychlor (10 μM), increased proliferation of the granulosa cells. DDE (100 and 10 μM, respectively) decreased FSH-stimulated cAMP synthesis in the granulosa and CHO-FSH-R cells. DDE also decreased progesterone synthesis in the granulosa cells. Methoxychlor (10 μM) inhibited progesterone synthesis in the granulosa cells, but it did not affect the generation of cAMP in either type of cells studied. However, methoxychlor inhibited estradiol-17β-stimulated progesterone synthesis in the granulosa cells. We conclude that DDE primarily inhibited the generation of cAMP, while methoxychlor supressed progesterone synthesis through a mechanism distal to cAMP generation. The present results indicate that DDE and methoxychlor are not limited to a mimicking of the endocrine effects of estradiol-17β in cultured ovarian cells. Therefore, a non-estrogenic component of the endocrine disrupting activities of DDE and methoxychlor must be considered in evaluating their reproductive toxicity.

Follicle-stimulating hormone (FSH) induced internalization of porcine FSH receptor in cultured porcine granulosa cells and Chinese hamster ovary cells transfected with recombinant porcine FSH receptor cDNA.

In order to study the fate of human follicle-stimulating hormone (FSH) when hormone binds to its receptor, a quick biochemical method that can differentiate between the surface-bound and internalized hormone was used to determine the internalization induced by FSH in cultured both porcine granulosa cells and Chinese hamster ovary (CHO) cells expressing recombinant porcine FSH receptor. The results showed that FSH was slowly internalized, and the internalized radioactivity (acid resistant) reached a peak 10-12 h after addition of 125I-hFSH. It was suggested that FSHR do not get internalized rapidly under physiological circumstances precisely because the appropriate sequences are absent.

Role of tumor necrosis factor in preovulatory follicles of swine.

The effects of tumor necrosis factor (TNF) on cultured porcine granulosa cells that were obtained from preovulatory follicles were studied with regard to following parameters: 1) TNF receptor type I expression, 2) progesterone receptor and transforming growth factor beta receptor type II (TbetaR II) as markers of luteinization, 3) proliferation, and 4) apoptosis. For comparative purposes the effects of TNF were also studied on insulin/forskolin-treated cells, as this treatment is well established to induce luteinization. Cytochemical methods followed by semiquantitative analysis were used. Our data show that TNF treatment upregulates TNF receptor type I expression in granulosa cells. TNF downregulates the expression of TbetaR II of insulin/forskolin-stimulated and of unstimulated cells. The progesterone receptor is also downregulated by the cytokine after insulin/forskolin-induced luteinization. Supplementation of the medium with TNF leads to increased proliferation and at the same time it induces apoptosis. Our results indicate that TNF exerts an inhibitory influence on luteinization and that TNF influences the balance between follicular growth (proliferation) and atresia (apoptosis).

Porcine theca cells produce immunoreactive beta-endorphin and change steroidogenesis in response to opioid agonist.

In earlier in vitro experiments opioids affected steroidogenesis in porcine luteal and granulosa cells. The present studies were undertaken to examine the effects of FK 33-824 (opioid agonist) alone or in combination with LH, PRL or naloxone (NAL, opioid antagonist) on steroidogenesis in cultured porcine theca cells. Moreover, we have tested beta-endorphin-like immunoreactivity (beta-END-LI) concentrations in culture media under control conditions and following treatments of theca cells with LH, PRL, progesterone (P4), oestradiol (E2) or testosterone (T). FK 33-824 and NAL significantly increased P4 release by theca cells and inhibited stimulatory effect of LH on this steroid output. PRL-induced P4 secretion from the cells was blunted only by FK 33-824. Secretion of androstenedione (A4) and T was essentially elevated in the presence of FK 33-824 and this potentiation of both androgen release was completely abolished by PRL. NAL blocked stimulatory effect of the opioid agonist only in case of T. Secretion of oestradiol and oestrone was completely free from the influence of both the opioid agonist and antagonist. Pig theca cells were able to produce beta-END-LI but none of tested hormones (LH, PRL, P4, E2 and T alone or in combination) significantly affected this production. In conclusion, these data indicate that porcine theca cells may produce beta-END-LI and change their steroidogenesis in response to opioid peptides.