Cryopreservation of pig granulosa cells: effect of FSH addition to freezing medium

We cryopreserved swine granulosa cells by a slow cooling rate system; FSH was added to the freezing medium to test its effectiveness in protecting the cells. After thawing, proliferative activity, viability, steroidogenesis and apoptosis were tested; moreover, we determined heat shock protein (HSP70) production, to investigate the recovery from stress and superoxide dismutase (SOD) and catalase activity to evaluate a possible impairment of the antioxidant pathway. E2 production was enhanced by cryopreservation in particular with FSH; on the contrary, P4 production was inhibited by the freezing process in particular without FSH. Only the higher FSH concentration (10 ng/ml) stimulated steroid secretion in freshly collected cells; P4 production by cells cryopreserved in the presence and in absence of FSH was increased by both 5 and 10 ng/ml while the lowest concentration was effective in stimulating E2 production only when FSH was added to freezing medium. Freezing did not modify proliferative activity, while apoptosis was higher in frozen than in fresh cells. HSP70 production was lower in cells cryopreserved in presence of FSH, whose antioxidant metabolism was also conserved: SOD and catalase activities were similar to control. In conclusion, cryopreservation does not seem to markedly affect granulosa cells, in particular if they are frozen in presence of FSH; the gonadotrophin somehow improves their performances after thawing, probably stimulating E2 production and the antioxidant metabolism.     

Isolation,proliferation and characterization of endometrial canine stem cells

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.     

The impact of the phyto‐oestrogen genistein on swine granulosa cell function

Soya and soybean products used in swine feeding contain genistein, a non‐steroidal phyto‐oestrogen which has been demonstrated to influence endocrine functions. This observation leads us to design this study to evaluate the effect of genistein on swine granulosa cell steroidogenesis and proliferation. In the attempt to unravel the genistein signal transduction mechanisms, we verified the effect of lavendustin, a Tyrosine Kinase (TK) inhibitor, and the potential involvement of NO/cGMP pathway. Finally, as angiogenesis is essential for follicle development, we tested the effect of the phyto‐oestrogen on vascular endothelial growth factor production and on granulosa cell redox status, because free‐radical species modulate neovascularization. Our data provide evidence that genistein interferes with granulosa cell steroidogenesis while it does not modulate cell growth: this effect could be at least partially produced by inhibiting TK‐dependent signalling systems. On the contrary, NO/cGMP pathway or vascular endothelial growth factor production can be excluded as signalling mechanism involved in phyto‐oestrogen effects. Remarkably, genistein stimulates hydrogen peroxide production thus potentially inhibiting follicular angiogenesis. Collectively, these results suggest that genistein consumption could potentially negatively impact swine reproductive function.     

Bisphenol A disrupts granulosa cell function

Because of its widespread use and potential adverse biological effects, bisphenol A (BPA) represents one of the most studied endocrine-disrupting compounds. Within the reproductive system, ovarian granulosa cells have been documented as a target of BPA action, but no consensus has been reached about functional modifications induced by BPA. On these bases, we studied the potential disrupting effects of BPA on the main granulosa cell functional activities, also taking into account a potential interference with the ovarian angiogenic process. Ovarian granulosa cells were isolated from porcine follicles and cultured in the presence or absence of BPA at different concentrations for 48 h. Cell proliferation was studied by measuring adenosine triphosphate content. Progesterone (P4) and estradiol 17β (E2) production was determined by radioimmunoassay. Vascular endothelial growth factor (VEGF) output was quantified by an enzyme-linked immunosorbent assay. Redox status was monitored by measuring superoxide anion and hydrogen peroxide, and by determining the activities of the scavenging enzymes superoxide dismutase, catalase, and peroxidase by colorimetric methods. Granulosa cell proliferation as well as redox status resulted unaffected by BPA. Concentrations of E2 were stimulated by the lower BPA concentration, whereas they were inhibited by the larger doses tested. P4 output was decreased by all BPA concentrations. To the contrary, VEGF production was stimulated. Data indicate that BPA can interfere with reproductive activity by affecting granulosa cell steroidogenesis in vitro; furthermore, BPA can exert a promoting effect on the ovarian angiogenic process by increasing VEGF output in pigs. A disruption of this finely tuned process seems particularly relevant because of the risk of uncontrolled neovascularization.     

Epigallocatechin-3-gallate from green tea negatively affects swine granulosa cell function

The use of herbs as additives in livestock nutrition as an alternative to antibiotics is becoming a new goal in animal production. It is known that green tea exerts antimicrobial activity owing to specific flavonoid compounds named catechins, primarily represented by epigallocatechin-3-gallate (EGCG). Remarkably, despite many potential benefits of green tea and EGCG consumption, it is also important to get an insight on the possible reproductive-related consequences of feeding supplementation. To this purpose, granulosa cells were harvested from follicles > 5mm and treated with 5 and 50 microg/ml of EGCG in order to evaluate the effects on the main parameters of granulosa cell function: steroidogenesis, by measuring progesterone and estradiol-17beta production, and proliferation, one of the major feature of ovarian follicular growth. Moreover, as the genesis of new vessels has been demonstrated to be fundamental for follicle development, we evaluated the effect of EGCG on the production of the main angiogenetic factor, VEGF, by swine granulosa cells. Finally, since reactive oxygen species (ROS) might be involved in the control of female reproductive activity, we studied the effect of EGCG on superoxide anion (O2-) and hydrogen peroxide (H2O2) production by swine granulosa cells and on the activity of the scavenging enzyme superoxide dismutase (SOD). EGCG significantly (p < 0.05) inhibited proliferation, steroidogenesis, VEGF and O2- production by swine granulosa cells; on the contrary, H2O2 levels and SOD activity were stimulated (p < 0.05) by the catechin. Therefore, since our data demonstrate that EGCG has a negative effect on reproductive performances in swine, feeding supplementation should be carefully considered.     

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) 17β, 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.     

Angiogenic Activity of Swine Granulosa Cells: Effects of Hypoxia and the Role of VEGF

Veterinary Research Communications -     

Gossypol,a polyphenolic aldehyde from cotton plant,interferes with swine granulosa cell function

Gossypol is a polyphenol isolated from the seed, roots and stem of cotton plant (Gossypium sp.) It has been associated with adverse effects on female reproduction, but recently also shown having promising effects against several malignancies. Its mechanisms of action are however still not fully understood. This study was therefore conducted to investigate the effect of 5 or 25 μg/mL gossypol on swine granulosa cell steroidogenic activity, redox status and Vascular Endothelial Growth Factor (VEGF) production. Study demonstrated that gossypol significantly (P < 0.001) inhibited granulosa cell estradiol 17β and progesterone production, an effect that could be at least partially mediated by an increase (P < 0.05) of nitric oxide and superoxide anion production as a consequence of superoxide dismutase inhibition. Moreover, gossypol stimulates (P < 0.001) VEGF production. In conclusion, study has demonstrated effecs of gossypol on swine granulosa cell function in vitro. Effects on female swine fertility can not be excluded.     

The plant alkaloid sanguinarine is a potential inhibitor of follicular angiogenesis

Sanguinarine (SA), a phytobiotic from Sanguinaria Canadensis, has been demonstrated to inhibit vessel growth. Current restrictions on the use of antibiotic growth promoters have motivated addition of this alkaloid as a naturally appetizing feed additive for farm animals. However, concern may araise since angiogenesis is a fundamental event in ovarian follicle growth. Therefore, the aim of this study was to evaluate the potential negative role of SA in follicular angiogenesis. For this purpose, we studied the effect of 300 nM SA on the production of vascular endothelial growth factor (VEGF) by swine granulosa cells from follicles >5 mm and on the activation of Akt, the main effector of the VEGF signalling pathway. In addition, the potential interference of SA in vessel development was tested in an in vitro angiogenesis bioassay. SA inhibited both VEGF production and VEGF-induced Akt activation in swine granulosa cells. Moreover, it was able to block vessel growth induced by VEGF. Taken together, our results suggest that SA could be detrimental to follicular angiogenesis, and therefore supplementation of feed with this alkaloid should be carefully considered.