VEGF,bFGF and Swine Granulosa Cells: Proliferation,Steroidogenesis and NO Production

Veterinary Research Communications -     

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.     

Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure

This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir.     

Molecular mechanisms of the biological clock in cultured fibroblasts

In mammals, the central circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN), but circadian oscillators also exist in peripheral tissues. Here, using wild-type and cryptochrome (mCry)-deficient cell lines derived from mCry mutant mice, we show that the peripheral oscillator in cultured fibroblasts is identical to the oscillator in the SCN in (i) temporal expression profiles of all known clock genes, (ii) the phase of the various mRNA rhythms (i.e., antiphase oscillation of Bmal1 and mPer genes), (iii) the delay between maximum mRNA levels and appearance of nuclear mPER1 and mPER2 protein, (iv) the inability to produce oscillations in the absence of functional mCry genes, and (v) the control of period length by mCRY proteins.     

Angiogenesis in Developing Follicle and Corpus Luteum

Angiogenesis is a process of vascular growth that is mainly limited to the reproductive system in healthy adult animals. The development of new blood vessels in the ovary is essential to guarantee the necessary supply of nutrients and hormones to promote follicular growth and corpus luteum formation. In developing follicles, the pre-existing endothelial cells that form the vascular network in the theca layer markedly develop in response to the stimulus of several growth factors, mainly produced by granulosa cells, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The angiogenic factors also promote vessel permeability, thus favouring the antrum formation and the events inducing follicle rupture. After ovulation, newly formed blood vessels cross the basement membrane between theca and granulosa layers and continue a rapid growth to sustain corpus luteum development and function. The length of luteal vascular growth varies in cycling and pregnant animals and among species; both angiogenesis and subsequent angioregression are finely regulated by systemic and local factors. The control of angiogenic development in the ovary could be a useful tool to improve animal reproductive performances.     

Factors Modulating Apoptosis: an in-vitro Study in Swine Granulosa Cells

The aim of this study was to investigate the effects of some endocrine and intra‐ovarian factors on the activation/inhibition of apoptosis in swine granulosa cells. Upon incubation in a 10% FCS‐supplemented M199, granulosa cells from small (< 3 mm) follicles programmed their death after 24–48 h of culture; in the absence of FCS, apoptosis was reduced after 24 h of culture. Cells cultured in the presence of FCS were treated with db‐cAMP, LH, FSH, Insulin‐like Growth Factor‐I IGF‐I or PMSG to verify the role of these substances in apoptotic death: all these molecules inhibited apoptosis after 48 h of incubation. A further aim of the study was to investigate the possible involvement of nitric oxide (NO), an intra‐ovarian modulator, in the regulation of granulosa cell apoptosis and its possible role in the modulation of steroidogenesis. After a 48 h incubation with a substrate of NO synthesis ( l ‐arginine, 0.1 and 1 m m ), a NO donor [S‐nitroso‐N‐acetyl‐penicillamine (SNAP) , 0.2 and 1 m m ] or a NO synthase inhibitor [N^ω‐nitro‐ l ‐arginine‐methyl‐ester (NAME, 1 and 5 m m )], the onset of apoptotic death was evaluated: l . arginine and NAME did not induce any significant variation of apoptosis, whereas 1 m m SNAP exerted a protective action. A significant stimulatory effect of l ‐arginine on NO production, associated with a suppressive action on estradiol 17β concentrations was observed; NAME exerted an inhibitory effect on NO production, associated with an increase in estradiol secretion; estradiol 17β production was markedly inhibited by SNAP. In summary, the depletion of FCS could induce a cell cycle arrest in G₀ whereas apoptosis could be the consequence of cell cycle progression mediated by FCS; gonadotropins and IGF‐I could also act as survival factors. NO appeared to represent a ‘trophic’ signal for the follicle, whose involvement in the regulation of ovarian function is substantiated by its modulatory action on steroidogenesis.     

Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin‐3‐gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2‐h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm , 500 nm and 5 μm ) and EGCG (10, 20 and 60 μm ), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm ) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm , the presence of EGCG at all the concentrations tested (10, 20 and 60 μm ) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.     

Immunodetection of hexose transporters in mammalian spermatozoa

Veterinary Research Communications -     

Low Molecular Mass Factors from Follicular Fluid Inhibit Steroidogenesis in Bovine Granulosa Cells

Gonadotropins are required for follicular growth and differentiation, but increasing amounts of evidence indicate that intrafollicular factors modulate their effects at granulosa cell level. In order to study the effect of factors present in bovine follicular fluid, a partial purification of low molecular mass factors from fluids collected from small (< 5 mm), medium (5–8 mm) and large (> 8 mm) follicles was performed and the biological activity of these peptides on steroidogenesis of granulosa cells from small and large follicles was examined. The purification was carried out by filtration through membranes in 25 and 10 kDa molecular weight cutoffs. After filtration, samples were analysed by polyacrylamide gel electrophoresis and protein concentration was measured by spectrophotometric analysis. Granulosa cells from small and large follicles were cultured in serum‐free DMEM/Ham's F12 (1 : 1) plus transferrin (5 mg/l) and selenium (5 µg/l) for 2 days. At the end of the culture period, media were renewed and follicular extracts from two different preparations (< 25 and < 10 kDa) were added at the concentrations of 1–10–100–1000 ng/ml. After 24 h the media were collected and stored until estradiol 17β (E2) and progesterone (P4) determination by validated radio‐immuno‐assays. Basal P4 production was 18.3 ± 1.4 (mean ± SEM)and 9.8 ± 1.8 ng/24 h per 3 × 10⁴ cells from small and large follicles, respectively. Both < 10 and < 25 kDa extracts reduced P4 production by cells from both the types of follicles (p < 0.05). Basal E2 release was 671.8 ± 21.4 and 5500 ± 800 pg/24 h per 3 × 10⁴ cells from small and large follicles, respectively. Both extracts reduced E2 production by either cells from small and large follicles (p < 0.05). No differences were observed in the inhibition of steroidogenesis by purifications obtained from large, medium or small follicles. Results of this study indicate that factors present in bovine follicular fluid can reduce steroidogenesis in granulosa cells in vitro.     

Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea

This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected. VEGF and steroid levels in luteal tissue were determined by ELISA and RIA, respectively; VEGF, VEGFR-2, ET-1, ET-A and ECE-1 mRNAs expression was measured by real-time PCR. VEGF protein levels were similar in the two groups, while all steroid (progesterone, testosterone, estradiol 17beta) concentrations were significantly (P < 0.001) higher in CLs collected from fasted animals compared with those from normally fed gilts. VEGF, VEGFR-2, ET-1 and ECE-1 (but not ET-A) mRNA expression was significantly lower (P < 0.05) in fasted versus normally fed animals. The overall conclusion is that all the parameters studied are affected by feed restriction, but the mechanisms activated at luteal level are possibly not fully adequate to compensate for nutrient shortage.