Cultured Porcine Granulosa Cells Research Articles

New Gene Markers of Exosomal Regulation Are Involved in Porcine Granulosa Cell Adhesion, Migration, and Proliferation.

Exosomal regulation is intimately involved in key cellular processes, such as migration, proliferation, and adhesion. By participating in the regulation of basic mechanisms, extracellular vesicles are important in intercellular signaling and the functioning of the mammalian reproductive system. The complexity of intercellular interactions in the ovarian follicle is also based on multilevel intercellular signaling, including the mechanisms involving cadherins, integrins, and the extracellular matrix. The processes in the ovary leading to the formation of a fertilization-ready oocyte are extremely complex at the molecular level and depend on the oocyte's ongoing relationship with granulosa cells. An analysis of gene expression from material obtained from a primary in vitro culture of porcine granulosa cells was employed using microarray technology. Genes with the highest expression (LIPG, HSD3B1, CLIP4, LOX, ANKRD1, FMOD, SHAS2, TAGLN, ITGA8, MXRA5, and NEXN) and the lowest expression levels (DAPL1, HSD17B1, SNX31, FST, NEBL, CXCL10, RGS2, MAL2, IHH, and TRIB2) were selected for further analysis. The gene expression results obtained from the microarrays were validated using quantitative RT-qPCR. Exosomes may play important roles regarding intercellular signaling between granulosa cells. Therefore, exosomes may have significant applications in regenerative medicine, targeted therapy, and assisted reproduction technologies.

The Mechanisms of BDNF Promoting the Proliferation of Porcine Follicular Granulosa Cells: Role of miR-127 and Involvement of the MAPK-ERK1/2 Pathway.

As a member of the neurotrophic family, brain-derived neurotrophic factor (BDNF) provides a key link in the physiological process of mammalian ovarian follicle development, in addition to its functions in the nervous system. The emphasis of this study lay in the impact of BDNF on the proliferation of porcine follicular granulosa cells (GCs) in vitro. BDNF and tyrosine kinase B (TrkB, receptor of BDNF) were detected in porcine follicular GCs. Additionally, cell viability significantly increased during the culture of porcine GCs with BDNF (100 ng/mL) in vitro. However, BDNF knockdown in GCs decreased cell viability and S-phase cells proportion-and BDNF simultaneously regulated the expression of genes linked with cell proliferation (CCND1, p21 and Bcl2) and apoptosis (Bax). Then, the results of the receptor blocking experiment showed that BDNF promoted GC proliferation via TrkB. The high-throughput sequencing showed that BDNF also regulated the expression profiles of miRNAs in GCs. The differential expression profiles were obtained by miRNA sequencing after BDNF (100 ng/mL) treatment with GCs. The sequencing results showed that, after BDNF treatment, 72 significant differentially-expressed miRNAs were detected-5 of which were related to cell process and proliferation signaling pathways confirmed by RT-PCR. Furthermore, studies showed that BDNF promoted GCs' proliferation by increasing the expression of CCND1, downregulating miR-127 and activating the ERK1/2 signal pathway. Moreover, BDNF indirectly activated the ERK1/2 signal pathway by downregulating miR-127. In conclusion, BDNF promoted porcine GC proliferation by increasing CCND1 expression, downregulating miR-127 and stimulating the MAPK-ERK1/2 signaling cascade.

The toxic influence of silver and titanium dioxide nanoparticles on cultured ovarian granulosa cells

The application of metal nanoparticles in modern society is growing, but there is insufficient data concerning their influence on reproductive processes and comparison of their biological activity. The present experiments aimed to compare the effects of silver and titanium dioxide nanoparticles (AgNPs and TiO2NPs) on ovarian granulosa cell functions. AgNPs and TiO2NPs were added to culture of porcine granulosa cells at doses 0, 0.01, 0.1, 1 or 10 μg/mL. The mRNAs for proliferating cell nuclear antigen (PCNA), cyclin B1, bax and caspase 3 were quantified by RT-PCR; release of progesterone was analyzed by ELISA. It was shown that both AgNPs and TiO2NPs significantly reduced all the measured parameters. ED50 of the inhibitory influence of AgNPs on the main ovarian cell parameters was higher than ED50 of TiO2NPs. The ability of AgNPs and TiO2NPs to suppress ovarian granulosa cell functions should be taken into account by their application.

Interrelationships between kisspeptin and FSH in control of porcine ovarian cell functions

The existing knowledge of the direct action of kisspeptin on the ovary needs to be expanded. In our study, the direct effects of kisspeptin on basic ovarian cell functions and their response to FSH were examined. We studied the effect of kisspeptin alone (0, 1, 10, and 100 ng/mL) and of kisspeptin (1, 10, and 100 ng/mL) in combination with FSH (10 ng/mL) on cultured porcine granulosa cells. Markers of viability, proliferation (accumulation of proliferating cell nuclear antigen [PCNA] and cyclin B1), and apoptosis (accumulation of bax and caspase 3), as well as the release of steroid hormones and IGF-I were analyzed using the trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. Addition of kisspeptin at lower doses (1 and 10 ng/mL) increased cell viability, the accumulation of PCNA and cyclin B1, decreased the accumulation of bax and caspase 3, and promoted release of progesterone, estradiol, and IGF-I, but not testosterone. A high dose (100 ng/mL) of kisspeptin had the opposite, inhibitory effect. The addition of FSH increased cell viability, proliferation, decreased apoptosis, and promoted progesterone, testosterone, estradiol, and IGF-I release. Kisspeptin at lower doses supported the stimulatory action of FSH on viability, PCNA and cyclin B1 accumulation, and release of progesterone and estradiol, promoted its inhibitory action on bax and caspase 3 accumulation, but did not modify its action on testosterone and IGF-I release. On the contrary, kisspeptin at a high dose inhibited and even reversed the FSH effect. FSH mimicked and promoted both the stimulatory and inhibitory action of kisspeptin on all examined ovarian functions besides IGF-I release. These observations show that kisspeptin can directly regulate basal ovarian cell functions. Furthermore, they demonstrate the functional interrelationships between kisspeptin and FSH in direct regulation of ovarian functions.

TNFα-Erk1/2 signaling pathway-regulated SerpinE1 and SerpinB2 are involved in lipopolysaccharide-induced porcine granulosa cell proliferation

Lipopolysaccharide (LPS) is an inhibitory factor that causes hormonal imbalance and subsequently affects ovarian function and fertility in mammals. Previous studies have shown that the exposure of granulosa cells (GC) to LPS leads to steroidogenesis dysfunction. However, the effects of LPS on the viability of GC remain largely unclear. In the present study, we aimed to address this question and unveil the underlying molecular mechanisms using cultured porcine GC. Results showed that GC proliferation and tumor necrosis factor α (TNFα) secretion were significantly increased after exposure to LPS, and these effects were completely reversed by blocking the TNFα sheddase, ADAM17. Moreover, GC proliferation induced by LPS was mimicked by treatment with recombinant TNFα. In addition, SerpinE1 and SerpinB2 expression levels increased in GC after treatment with LPS or recombinant TNFα, whereas blocking the Erk1/2 pathway completely abolished these effects and also inhibited GC proliferation. Further, consistent with the effects of blocking the Erk1/2 pathway, cell proliferation was completely inhibited by knocking down SerpinE1 or SerpinB2 in the presence of LPS or recombinant TNFα. Mitochondrial membrane potential (MMP) polarization in GC was increased by LPS or recombinant TNFα treatment, and these changes were completely negated by Erk1/2 inhibition, but not by SerpinE1 or SerpinB2 knockdown. Taken together, these results suggested that the TNFα-mediated upregulation of SerpinE1 and SerpinB2, through activation of the Erk1/2 pathway plays a crucial role in LPS-stimulated GC proliferation, and the increase in GC MMP may synergistically influence this process.

Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to “cell cycle”, “cell division”, “cell cycle process”, “cell cycle phase transition”, “cell cycle G1/S phase transition”, “cell cycle G2/M phase transition” and “cell cycle checkpoint” ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte—granulosa cells—are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h—before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: “Cell morphogenesis”, “cell differentiation” and “cell development” were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.

Quercetin directly inhibits basal ovarian cell functions and their response to the stimulatory action of FSH

Plants, fruits, and vegetables containing the bioflavonoid quercetin are widely used in food, beverages, and medicines; however, the effects of quercetin on reproductive processes and the possible mechanisms of quercetin action require extensive investigation. The aim of our study was to examine the direct effects of quercetin on basic ovarian cell functions and their response to follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I), known hormonal stimulators of reproduction. We analyzed the effects of quercetin alone (0, 1, 10, and 100 ng/ml) on cultured porcine ovarian granulosa cells or isolated ovarian follicles; or of quercetin (10 ng/ml) in combination with FSH (0, 0.01, 0.1, or 1 IU/ml) or IGF-I (0, 1, 10, or 100 ng/ml) on cultured porcine granulosa cells. The expression of proliferative (PCNA, cyclin B1) and apoptotic (BAX) markers, as well as markers for release of progesterone (P4), testosterone (T), and leptin (L), were measured by quantitative immunocytochemistry, Western immunoblotting, RT-qPCR, and EIA/RIA. Addition of quercetin reduced the accumulation of PCNA and cyclin B1, as well as their transcript levels, promoted the accumulation of BAX, decreased the release of P4 and L, and increased the release of T in cultured granulosa cells. In ovarian follicles, quercetin reduced the levels of both P4 and T. Exposure to FSH stimulated PCNA and decreased BAX accumulation, and increased the release of P4, T, and L. Quercetin inhibited and even reversed the effects of FSH. Like FSH, IGF-I also promoted granulosa cell proliferation and suppressed apoptosis. Quercetin did not modify IGF-I effects. These data suggest that the plant molecule quercetin can directly down-regulate basal ovarian cell functions (proliferation, apoptosis, and release of ovarian steroid and peptide hormones) and their response to the stimulatory activity of the upstream hormonal stimulator FSH.

Conserved microRNA mediates heating tolerance in germ cells versus surrounding somatic cells

ABSTRACT Mammalian fertility is reduced during heat exposure in the summer, but is regained as temperatures decrease in the autumn again. However, the mechanism underlying the phenomenon remains unknown. We investigated heat stress tolerance of germ cells and their surrounding somatic cells, and discovered that microRNA ssc-ca-1 was upregulated after heat stress in cultured porcine granulosa cells (GCs), but not in serum-starved GCs. Ssc-ca-1 inhibited heat shock protein 70 (Hsp70) expression through its 3′- and 5′-UTRs. Although Hsp70 mRNA transcription was induced in GCs by in vivo exposure to heat in the summer, ssc-ca-1 inhibited Hsp70 expression. In ovarian cultures, heat stress-induced Hsp70 expression in primordial but not in growing follicles; ssc-ca-1 expression did not change in primordial follicles, but increased in growing follicles. Consistently, ssc-ca-1 was present in testicular cells and exhibited the same function as in ovarian cells. It modulated the different Hsp70 expression between spermatogonial stem cells and Sertoli cells after scrotal heat stress. This mechanism is of relevance to mammalian fertility in parts of the world dominated by heat stress associated with global climate change.

The phytoestrogen, diosgenin, directly stimulates ovarian cell functions in two farm animal species

The present in vitro study was conducted to examine the direct action of the plant steroidal sapogenin, diosgenin, on basic farm animal ovarian cell functions. As models, we used cultured porcine ovarian granulosa cells, porcine whole follicles, and rabbit ovarian fragments. The effects of diosgenin (0, 1, 10, or 100 μg/mL medium) on the markers of proliferation, cytoplasmic apoptosis, steroid (progesterone: P4, testosterone: T, and estradiol: E2) release, and peptide hormone (insulin-like growth factor I: IGF-I) release were analyzed by quantitative immunocytochemistry and radioimmunoassay. Diosgenin promoted proliferation, apoptosis, and T and E2 release and inhibited P4 output in cultured porcine granulosa cells. Similarly, cultured porcine ovarian follicles showed diosgenin-induced inhibition of P4 and stimulation of T release. In cultured rabbit ovarian fragments, diosgenin stimulated P4 and IGF-I release. This is the first study showing that diosgenin can promote basic ovarian cell functions such as proliferation, apoptosis, and steroid and peptide hormone release. The similar effects of diosgenin on porcine granulosa cells and ovarian follicles suggest that granulosa cells are the primary ovarian target of diosgenin. The contrasting effects of diosgenin on porcine and rabbit ovarian P4 output suggest that diosgenin functions in a species-specific manner. These observations indicate that diosgenin has potential applications for improving female reproduction.

Toxic effects and possible mechanisms following malathion exposure in porcine granulosa cells

Malathion is a wide spectrum organophosphorothionate insecticide that is frequently found in drinking water, food and foodstuffs. Ovarian granulosa cells modulate oogenesis by providing metabolic nutrients to oocytes. They can decide the fate of folliculogenesis and oocyte maturation by supplying regulatory cues that help in reproduction. However, little is known about the underlying mechanisms of malathion as a reproductive toxicant in porcine granulosa cells. In the present study, we found that malathion has obvious toxic effects on cultured porcine granulosa cells in a dose-dependent manner. Malathion exposure resulted in significantly increased oxidative stress levels and DNA damage response, which was measured by the mRNA expression levels of homologous recombination (HR) pathway and non-homologous end-joining (NHEJ) pathway-related genes. Subsequently, it was found that malathion exposure could induce apoptosis and autophagy by qRT-PCR and fluorescence intensity analysis. In conclusion, malathion is a reproductive toxicant by inhibiting granulosa cell proliferation by multiple pathways connected to oxidative stress, DNA damage, apoptosis and autophagy.

The mTOR System Can Affect Basic Ovarian Cell Functions and Mediate the Effect of Ovarian Hormonal Regulators

Both reproductive hormones and the mammalian target of rapamycin (mTOR) intracellular signalling system, including mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2) and its key enzyme sirtuin 1 (SIRT 1) are involved in the control of ovarian processes but the interrelationship between hormones and mTOR has been studied insufficiently. The aim of our in vitro experiments was to elucidate the involvement of mTOR in the control of basic ovarian cell functions and in mediating the action of upstream hormonal stimulators. In the first series of experiments, we examined the effect of the known hormonal regulators of ovarian functions, Follicle-Stimulating Hormone (FSH), oxytocin (OT) and insulin-like growth factor I (IGF-I) (all at 0, 1, 10 and 100 ng mL-1 doses), on the accumulation of SIRT1 in porcine ovarian granulosa cells. In the second series of experiments, we examined the effects of mTOR blockers, PF 046 (an inhibitor of mTORC1) and WYE 687 (inhibitor of both mTORC1 and mTORC2) (both at a dose of 1 μg mL-1) on both basal and FSH-induced (0, 1, 10 and 100 ng FSH mL-1 doses) basic ovarian functions (proliferation, apoptosis and steroidogenesis) of cultured porcine granulosa cells. The accumulation of SIRT1, PCNA (a proliferation-related peptide) and Bax (an apoptosis-related peptide) was detected by immunocytochemistry. The release of progesterone (P4) and testosterone (T) was analysed by EIA. It was observed that either FSH or OT additions increased the SIRT1 accumulation in ovarian cells, whilst IGF-I addition decreased it. The PF 046, when given alone, inhibited ovarian cell proliferation but did not affect apoptosis or the release of P4 and T. The WYE 687, when added alone did not affect proliferation and apoptosis but inhibited the P4 and T release by ovarian cells. The FSH, when given alone, stimulated proliferation did not affect apoptosis and increased the release of both P4 and T. In the presence of PF 046, FSH did not significantly alter proliferation, induced apoptosis and suppressed the P4 and T release, i.e., this inhibitor of mTORC1 prevented the proliferation-stimulating effect induced by the pro-apoptotic action of FSH and inverted the stimulatory action of FSH on steroidogenesis. The WYE 687 prevented the effect of FSH on both proliferation and apoptosis, promoted the FSH-induced P4 release but did not affect the FSH action on the T output, i.e., the inhibition of both mTORC1 and mTORC2 inverted the FSH action on ovarian cell proliferation and apoptosis but not the action on steroidogenesis. Our observations suggest the following: (1) mTORC1 can be involved in the up-regulation of ovarian cell proliferation but not that of apoptosis and steroidogenesis (2) mTORC2 can be involved in the up-regulation of the release of both P4 and T but not the up-regulation of ovarian cell proliferation and apoptosis, (3) FSH is involved in the stimulation of ovarian cell proliferation and P4 and T release and (4) hormones (FSH, OT and IGF-I) can regulate ovarian mTOR and the ability of mTOR to modify FSH effects suggests that mTOR can mediate the actions of hormone on ovarian cells. It is hypothesized that both mTORC1 and mTORC2 may be mediators of the FSH action on ovarian cell proliferation, apoptosis and steroidogenesis.

The Effects of Phytoestrogen Genistein on Steroidogenesis and Estrogen Receptor Expression in Porcine Granulosa Cells of Large Follicles.

Genistein is a biologically active isoflavone with estrogenic or antiestrogenic activity which can be found in a variety of soy products. Since in pigs' diet soy is the main source of protein, genistein may affect the reproductive/endocrine systems in these animals. Genistein has been shown to alter porcine ovarian and adrenal steroidogenesis but the mechanism of this action is still not clear. It is known that genistein binds to both estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), although it has a higher affinity to ERβ. Moreover, this phytoestrogen was demonstrated to posses the activity of protein tyrosine kinase (PTK) inhibitor. The aim of the study was to examine the in vitro effects of genistein on: (1) progesterone (P4) and estradiol (E2) secretion by porcine luteinized granulosa cells harvested from large follicles, and (2) the mRNA and protein expression of ERa and ERβ in these cells. In addition, to verify the role of PTK-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. Genistein significantly inhibited P4 and did not affect E2 secretion by porcine luteinized granulosa cells isolated from large follicles. Lavendustin C did not affect basal steroids secretion by examined cells. Genistein did not alter ERa but increased ERβ mRNA levels in the cultured porcine granulosa cells. In contrast to medium follicles, the expression of ERβ protein was unaffected by genistein in granulosa cells of large follicles. To conclude, the soy phytoestrogen genistein acts directly on the porcine ovary to decrease progesterone production and to increase the expression of ERβ mRNA. Moreover, genistein-induced changes in follicular steroidogenesis and granulosal sensitivity to estrogens in pigs may depend on maturity of the follicles.

Biochanin A affects steroidogenesis and estrogen receptor-β expressionin porcine granulosa cells

Biochanin A, similar to other isoflavones, is present in soy and soy-based food, but predominantly in red clover. Red clover extract and biochanin A were reported to affect reproductive processes as well as to demonstrate menopause relief and anticancerogenic properties. Because porcine granulosa cells provide a suitable in vitro model for studying the intracellular mechanism of phytoestrogen action in the ovary, the objective of the study was to evaluate the in vitro effects of biochanin A on the following: (1) progesterone (P4) and estradiol (E2) secretion by granulosa cells, (2) viability of the granulosa cells, and (3) mRNA and protein expression of estrogen receptors α (ERα) and β (ERβ) in the granulosa cells harvested from both medium (3–6 mm) and large (≥8 mm) porcine ovarian follicles. RIA, alamarBlue assay, reverse transcriptase–polymerase chain reaction, and immunocytochemistry were used in the study to address the objectives. Biochanin A significantly inhibited P4 and did not affect E2 secretion by porcine granulosa cells regardless of the size of follicles that served as the source of the cells. Cell viability was not affected by the treatment. Biochanin A did not alter ERα and ERβ mRNA levels in the cultured porcine granulosa cells. In contrast, this isoflavone increased (P < 0.05) the immunoexpression of ERβ in the cells from both follicle types. In summary, biochanin A, similar to genistein and daidzein, affects follicular steroidogenesis and ER expression. Its effect on ERβ protein was more intense compared with other previously examined phytoestrogens.

Production of cloned pigs with targeted attenuation of gene expression.

The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45–82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

Effects of testosterone and 2-hydroxyflutamide on progesterone receptor expression in porcine ovarian follicles in vitro

The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T+2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6–8mm in diameter) were cultured for 48h. Experimental cultures were carried out with the addition of T (10−7M), 2-Hf (1.7×10−4M) or both T and 2-Hf for the last 24h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6–8mm in diameter) were cultured for 24h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P4) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P4 secretion, but simultaneous addition of 2-Hf and T increased P4 secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.

The Influence of the Antiandrogen 2‐Hydroxyflutamide on the Androgen Receptor Expression in the Porcine Ovarian Follicles – An In Vitro Study

Androgens are one of the most important agents influencing ovarian follicles growth and development. The biological action of androgens is primarily exerted through transcriptional regulation by the androgen receptor (AR), a member of the steroid hormone receptor superfamily. The purpose of this study was to test the role of androgen receptor agonist testosterone (T) or antagonist 2-hydroxyflutamide (2-Hf) and in combination on AR expression in cultured porcine granulosa cells (GC) or whole follicles. Granulosa cells isolated from mature pig follicles were cultured for 48h. During the last 12 and 24h of culture, they were incubated in the presence of T (10(-7) m/ml), 2-Hf (1.7×10(-4) m) or both T and 2-Hf (T+2-Hf, at the same concentrations as when added separately). To better imitate in vivo conditions, whole follicles (6-8mm in diameter) isolated from porcine ovaries have been incubated (for 12 and 24h) in an organ culture system with the addition of the same factors. Thereafter, cells or sections obtained from cultured follicles were processed for AR detection by immunocytochemistry or immunohistochemistry. Moreover, expression of AR mRNA and protein was determined by real-time PCR and Western blot analysis. It was shown that the addition of 2-Hf in the presence of T had a positive effect on AR mRNA and protein expression in porcine GC and ovarian follicles. Moreover, the addition of 2-Hf influenced AR distribution in GC cultures which is seen as change of its localization from nuclear to perinuclear. Our results suggest that androgens acting through AR could be involved in the control of AR expression in porcine GC in vitro and in vivo.

Does 2-hydroxyflutamide Inhibit Apoptosis in Porcine Granulosa Cells? — An &lt;i&gt;In Vitro&lt;/i&gt; Study

In mammalian ovaries, the majority of follicles are lost before ovulation by atresia. This degenerative process is initiated or caused by granulosa cell apoptosis. To reveal the androgen-dependent mechanism of selective follicular atresia, the culture model system for agonism and antagonism of the androgen receptor has been established. We examined the influence of an androgen receptor antagonist, 2-hydroxyflutamide (2-Hf), on the incidence of apoptosis in cultured porcine granulosa cells. They were incubated (6 and 12-h) in the presence of testosterone (T, 10⁻⁷M), 2-Hf (1.7×10⁻⁴ M) or both T and 2-Hf (T+2-Hf), and then analyzed by flow cytometry with fluorescein labelled annexin V. To better imitate in vivo conditions, the intact porcine follicles (6-8 mm in diameter) have been incubated in an organ culture system with the addition of the same factors. Sections obtained from follicles fixed after culture were stained with hematoxylin and eosin, and the presence of apoptosis-related DNA strand breaks was evaluated by the TUNEL method. Estradiol and progesterone concentrations in the culture media were measured by radioimmunoassays. The addition of T or 2-Hf to the culture media caused an increase in the number of apoptotic granulosa cells, while treatment with T+2-Hf decreased it in both in vitro and organotypic models. Follicles cultured with the addition of T or 2-Hf exhibited morphological changes indicating follicular atresia. Granulosal estradiol secretion was considerably stimulated by T+2-Hf. The highest increase in follicular estradiol secretion was observed after the anti-androgen addition. In both granulosal and follicular cultures, the production of progesterone declined in the presence of T or 2-Hf but increased after their simultaneous addition. In conclusion, androgen receptor antagonist 2-Hf attenuates induction of granulosa cell apoptosis in the presence of a high T level. The nature of this protective mechanism as yet is unknown and requires further research.

Upregulation of P-Glycoprotein Activity in Porcine Oocytes and Granulosa Cells During In Vitro Maturation

Multidrug resistance P-glycoprotein (Pgp), coded by the multidrug resistance type I (MDR1/ABCB1) gene, is an energy-dependent efflux pump and functions in systemic detoxification processes. In the present study, the expression and development of Pgp were evaluated in the porcine oocyte during in vitro maturation to compare with the expression of Pgp in cultured granulosa cells. As revealed by Western blotting using anti-human Pgp antibody, a single band of Pgp with an apparent molecular size of 170 kDa was detected in the germinal vesicle stage oocytes. The surface of GV oocyte was positively labeled by immunostaining. In the second metaphase oocyte after culture in the maturation medium containing porcine follicular fluid and human chorionic gonadotropin, the level of Pgp was increased. The elevation of the oocyte Pgp level was associated with increased activity of rhodamine 6G efflux from the oocyte, and its efflux was suppressed by verapamil, an inhibitor of Pgp. Removal of porcine follicular fluid from the maturation medium resulted in little alteration of the oocyte Pgp level. Expression of Pgp was also elevated in cultured porcine granulosa cells during cell maturation when stimulated with follicle-stimulating hormone or luteinizing hormone for 24-48 h. Collectively, the present results indicate that the transporting activity of P-glycoprotein upregulates in porcine oocytes and granulosa cells during exposure to gonadotropins or prior to ovulation.

334. PROGESTERONE PRODUCTION FROM GRANULOSA CELLS OF SOWS IS ENHANCED EQUALLY BY OMEGA-3 DERIVED PROSTAGLANDIN E3 AND OMEGA-6 DERIVED PROSTAGLANDIN E2

Caughey et al (2005) reported that prostaglandins derived from omega 3 sources eicsapentaenoic acid (EPA C20:5) and docosahexaenoic acid (DHA C22:6) have different properties to those derived from the preferred substrate, arachidonic acid (ARA C20:4; n-6). Armstrong et al (2006) demonstrated that PGE2 increased progesterone when porcine granulosa cells were cultured in vitro with hCG. We hypothesized that PGE3 which is derived from EPA will produce a lower steroidogenic response as progesterone from isolated granulosa cells collected from pre-ovulatory sow ovaries. Ovaries were collected from slaughtered sows and follicles between 3–8 mm were aspirated and through a series of wash steps in HTCM (Hepes TCM 199). Mid-sized granulosa cells were recovered in a solution of BTCM (bicarbonate TCM) containing IGF-1. 0.5 × 106 cells/mL were cultured in 250 µL of Control (BTCM only), PGE2 or PGE3 (320 ng/mL in BTCM, Cayman Chemical Co.) treatments with IGF1 at 25 ng/mL. Cultures were incubated for 22 h at 38oC. Cultures were centrifuged and the supernatant was analysed in duplicate for progesterone. Data was analysed by Univariate GLM ANOVA. There was no significant difference between PGE2 and PGE3 treatments, however the main effect of PGE significantly increased progesterone production relative to the control (P = 0.017). Granulosa cells cultured with omega 3 derived PGE3 did not produce significantly lower progesterone levels than those with PGE2. We conclude that both PGE2 and PGE3 promote a steroidogenic response in cultured porcine granulosa cells. (1) Armstrong DT, Formosa, ER, Amato F, Schultz SJ. 2006. Prostaglandin E2 up-regulates luteinizing hormone receptor (LHR) expression and enhances steroidogenic responses of follicle cells.(2) Caughey GE, James MJ, Cleland LG. 2005. Prostaglandins and leukotrienes. pp. 42–49. In ‘Encyclopaedia of Human Nutrition. Vol. 4’. (Eds B Caballero, L Allen, A Prentice).