Regulatory Peptides 120 (2004) 69 – 75 www.elsevier.com/locate/regpep The effects of reduced oxygen tension on swine granulosa cell Giuseppina Basini a,*, Federico Bianco a, Francesca Grasselli a, Martina Tirelli a, Simona Bussolati a, Carlo Tamanini b a Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualità e Sicurezza degli Alimenti-Sezione di Fisiologia Veterinaria, Università di Parma, Via del Taglio 8, Parma 43100, Italy b DIMORFIPA, Università di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy Received 11 June 2003; received in revised form 10 February 2004; accepted 23 February 2004 Available online 25 March 2004 Abstract Follicular growth is characterized by an augmented vascularization, possibly driven by a fall in the oxygen supply. The present study was undertaken to investigate the effects of hypoxia on swine granulosa cells. At first, we quantified oxygen partial pressure ( pO2) in follicular fluid from different size follicles; the granulosa cells collected from large follicles (>5 mm) were subjected for 18 h to normoxia (19% O2), partial (5% O2) or total hypoxia (1% O2). The effects of these conditions were tested on the main parameters of granulosa cell function, steroidogenesis and cell proliferation, and on vascular endothelial growth factor (VEGF), nitric oxide (NO) and superoxide anion (O2 ) production. Oxygen tension in follicular fluid was negatively related to follicular size, pointing out a gradual reduction during follicular growth. Severe hypoxic conditions determined a reduction of both 17h estradiol and progesterone production, while partial hypoxia did not seem to affect them. Hypoxia increased VEGF as well as O2 production in swine granulosa cells without impairing cell growth; in addition, it decreased NO output. We may conclude that physiological hypoxia could play a pivotal role in the follicular angiogenic process stimulating VEGF synthesis by granulosa cells. ROS are possibly involved in hypoxic signalling. D 2004 Elsevier B.V. All rights reserved. Keywords: VEGF; Ovary; Nitric oxide; Superoxide anion; Steroid hormones 1. Introduction The knowledge on angiogenesis, the process by which new capillaries develop from preexisting vessels, has greatly improved in the last years thanks to the research on cancer [1]. Most of efforts have been addressed to the inhibition of neovascularization in tumors, which always represents a pathological process [2]. The only chance to study the physiological pattern of the angiogenic process in the adult is offered by the female reproductive system [3]. In particular, the ovary is distinctive since it represents a site of active angiogenesis that takes place both in the growing follicle and in the corpus luteum formation. The vascular sheath that develops during follicular maturation in the thecal compartment expands with ongoing folliculogenesis, * Corresponding author. Tel.: +39-521-902775; fax: +39-521-902770. E-mail address: basini@unipr.it (G. Basini). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.02.013 but the thecal capillaries do not penetrate the membrana propria; therefore, granulosa layer remains avascular until breakdown of the basement membrane at ovulation [4,5]. The vasculature of the follicle is necessary for supplying hormones and their precursors, oxygen and nutrients [6]; the preferential delivery of gonadotropins via a more highly developed vascular system in individual follicles has been suggested to play an instrumental role in the selection and growth of the dominant follicle [7]. It is worth noticing that the increased thickness of the avascular granulosa layer during follicular growth should result in a decrease of pO2 within the follicle, since these cells depend exclusively on diffusion processes for nutrients and oxygen supply [8]. Extensive angiogenic responses in other tissues have been shown to be elicited under circumstances in which oxygen homeostasis is impaired [9]. Among the substances regulating the angiogenic process, a key role is played by the vascular endothelial growth 70 G. Basini et al. / Regulatory Peptides 120 (2004) 69–75 factor (VEGF), also known as vascular permeability factor (VPF), which is mitogen for endothelial cells and stimulates vascular permeability [10 – 12]. The involvement of VEGF in follicular development is supported by experimental data showing its increase in follicular fluid during follicle growth [13] as well as a positive relationship between VEGF production by granulosa cells and original follicular size [11,14]. Hypoxia has been shown to modulate VEGF production [15] through the production of hypoxia-inducible factor (HIF-1) [16], a transcription factor that functions as a master regulator of oxygen homeostasis. HIF-1 is not only regulated by oxygen tension, but also by various other stimuli, among which it is worth remembering nitric oxide (NO) [17]. Even though evidence exists on the antiangiogenic properties of NO in the follicle [18], its involvement in hypoxia-mediated angiogenesis is not clear [19,20]. On this basis, we have undertaken the present study to verify whether a relationship exists between hypoxia and VEGF production within the ovarian follicle. To do this, we quantified oxygen tension in swine follicles at different stages of development; moreover, we tested the effects of two hypoxic conditions on the production of VEGF, NO, O2 and steroids as well as on proliferation of granulosa cells collected from large follicles. streptomycin (100 Ag/ml), amphotericin B (2.5 Ag/ml), selenium (5 ng/ml) and transferrin (5 Ag/ml). Once seeded, cells were incubated at 37 jC under humidified atmosphere (5% CO2) for 24 h and then subjected for 18 h to normoxic (19% O2), hypoxic (5% O2) or anoxic (1% O2) conditions. Total hypoxia was achieved employing an AnaerocultR A mini while partial hypoxia was obtained by means of an AnaerocultR C mini (Merck, Darmstadt, Germany). In both cases, the system consisted of plastic pouches and a paper gas-generating sachet. 2.2. pO2 in follicular fluid Follicular fluid was collected separately from individual large and medium follicles containing enough fluid to fill a cartridge (Samed, Merlino, Italy), while follicular fluid from three small follicles was pooled in order to obtain a sufficient amount. A total of 20 determinations for each follicular class were carried out. Fluid was assayed for pO2 by means of an i-STAT R Portable Clinical Analyzer (iSTAT, Princeton, NJ, USA) [23] after ensuring quality control with the electronic simulator as recommended by the manufacturer. The integrity of the cartridges and the calibration were verified before commencement of each determination. The intra-assay coefficient of variation for pO2 measurement was < 5%. 2. Materials and methods 2.3. Cell proliferation All the reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise specified. Viable cells (104) were cultured in 200 Al CM at different O2 tensions (as described above) and cell proliferation was evaluated by 5-bromo-2V-deoxyuridine (BrdU) incorporation assay test (Roche, Mannheim, Germany). Briefly, 20 Al BrdU were added to each well during the last 4 h of incubation, then culture media were removed and a DNA denaturating solution was added in order to improve the accessibility of the incorporated BrdU for antibody detection. Thereafter, 100 Al anti-BrdU antibody were added to each well. After a 1.5 h incubation at 21 jC the immune complexes were detected by the subsequent substrate reaction. The reaction product was quantified by measuring the absorbance at 450 nm against 690 nm using a Spectra Shell Microplate reader (SLT Spectra, Milan, Italy). To establish viable cell number, absorbance was related to a standard curve prepared by culturing in quintuplicate granulosa cells at different plating densities (from 103 to 105/200 Al) for 48 h. The curve was repeated in four different experiments. The relationship between cell number and absorbance resulted linear (r = 0.92). Cell number/well was estimated from the resulting linear regression equation. The assay detection limit was 103 cells/well and the variation coefficient was less than 5%. 2.1. Follicular fluid and granulosa cell collection Swine ovaries were collected at a local slaughterhouse, placed into cold PBS (4 jC) supplemented with penicillin (500 IU/ml), streptomycin (500 Ag/ml) and amphotericin B (3.75 Ag/ml), maintained in a freezer bag and transported to the laboratory within 1 h. After a series of washing with PBS and ethanol (70%) follicles were divided into three classes corresponding to the diameter: small ( < 3mm), medium (3– 5 mm) and large follicles (>5 mm). Follicular fluid was collected with a 26-gauge needle from follicles of each class to determine the O2 tension. Granulosa cells from large follicles were aseptically harvested by aspiration with a 26-gauge needle and released in medium containg heparin (50 IU/ml), centrifuged for pelleting and then treated with 0.9% prewarmed ammonium chloride at 37 jC for 1 min to remove red blood cells. Cell number and viability were estimated using a haemocytometer under a phase contrast microscope after vital staining with trypan blue (0.4%) of an aliquot of the cell suspension. Cells were seeded at different plating densities (see below) in serum-free culture medium (CM) [21,22] composed by M199 supplemented with sodium bicarbonate (2.2 mg/ml), bovine serum albumin (BSA 0.1%), penicillin (100 IU/ml), 2.4. WST-1 assay for superoxide (O2 ) production O2 production was evaluated by the cell proliferation WST-1 test (Roche) applied to the same culture protocol 71 G. Basini et al. / Regulatory Peptides 120 (2004) 69–75 Fig. 1. pO2 levels in swine follicular fluid. Data are expressed as mean F S.E.M. Different letters indicate a significant difference ( p < 0.05). used above, since evidence also exists that tetrazolium salts can be used as a reliable measure of intracellular O2 production [24,25]. Briefly, 20 Al WST-1 were added to the cells during the last 4 h of incubation and absorbance was then determined using a Spectra Shell Microplate reader at 450 nm against 620 nm. 2.5. VEGF production Cells (106) in 1 ml CM + 1% FCS were seeded in 24-well plates and incubated for 48 h. VEGF content in culture media was quantified by an ELISA (Quantikine, R&D System, Minneapolis, MN, USA); this assay, developed for human VEGF detection, has been validated for pig VEGF [26]. The assay sensitivity was 0.23 pmol/l, the inter- and intra-assay CVs were always less than 7%. A Spectra Shell microplate reader, set to read 450 nm emission, was used to quantify the reaction product. 2.6. NO production A total of 105 cells/200 Al CM were seeded in 96well plates and incubated in the conditions described Fig. 3. Effect of partial and total hypoxia on O2 generation by 104 swine granulosa cells. Data are expressed as mean F S.E.M. Different letters indicate a significant difference ( p < 0.05). above. NO was assessed by measuring nitrite levels in culture media by the microplate method based on the formation of chromophore after reaction with Greiss reagent, which was prepared fresh daily by mixing equal volumes of stock A (1% sulfanilamide, 5% phosphoric acid) and stock B (0.1% N-[naphthyl] ethylenediamine dihydrochloride) [27]. 2.7. Steroid production A total of 104 cells/well were seeded in 96-well plates in 200 Al CM supplemented with androstenedione (28 ng/ml). Culture media were then collected, frozen and stored at 20 jC until progesterone (P4) and 17h estradiol (E2) determination by validated RIAs [28]. P4 assay sensitivity and ED50 were 0.24 and 1 nmol/l, respectively; E2 assay sensitivity and ED50 were 0.05 and 0.2 nmol/l. The intra- and inter-assay coefficients of variation were less than 12% for both assays. 2.8. Statistical analysis Each experiment was repeated at least four times (six replicates per treatment). Experimental data are presented as mean F S.E.M.; statistical differences between treatments were calculated with ANOVA using Statgraphics package (STSC, Rockville, MD, USA). When significant differences were found, means were compared by Scheffè’s F-test. Table 1 Effect of partial and total hypoxia on VEGF and NO production by 106 and 105 swine granulosa cells, respectively VEGF (pg/ml) NO (AM) Fig. 2. Effect of partial and total hypoxia on swine granulosa cells number. Data are expressed as mean F S.E.M. Control Partial hypoxia Total hypoxia 1061 F 90 a 3.16 F 0.3 a 2333 F 179.6 b 1.93 F 0.2 b 2729 F 107.1 b 1.47 F 0.1 b Data are expressed as mean F S.E.M. Different letters indicate a significant difference ( p < 0.05). 72 G. Basini et al. / Regulatory Peptides 120 (2004) 69–75 3. Results 3.4. VEGF production 3.1. pO2 in follicular fluid VEGF concentrations in control group (1061 F 90 pg/ ml) were significantly ( p < 0.05) increased by both partial and total hypoxia (Table 1); no differences were observed between these two conditions. pO2 levels were 126.9 F 2 mm Hg (means F S.E.M.) in small follicles and decreased to 93.4 F 3 and 67.7 F 2 mm Hg in medium and large follicles respectively (Fig. 1); the differences between the three follicular classes were significant ( p < 0.05). 3.2. Cell proliferation BrdU incorporation assay did not evidence any significant effect of hypoxia on cell proliferation: in fact, a fivefold increase in cell number was observed both in normoxia and hypoxia (Fig. 2). 3.3. O2 production Hypoxia significantly stimulated ( p < 0.05) O2 generation: both total and partial hypoxic conditions exerted similar effects (Fig. 3). 3.5. NO production In the control group, NO levels were 3.16 F 0.33 AM; a marked decrease was observed both in total and partial hypoxia ( p < 0.05) with no significant differences between the two hypoxic conditions (Table 1). 3.6. Steroid production E2 and P4 levels in the control group were 5.5 F 0.5 and 84.7 F 8.9 ng/ml, respectively. Both E2 and P4 levels were significantly ( p < 0.05) reduced in total hypoxia (Fig. 4), while no significant effect was observed under partial hypoxic condition. 4. Discussion Fig. 4. Effect of partial and total hypoxia on E2 (a) and P4 (b) production by 104 swine granulosa cells. Data are expressed as mean F S.E.M. Different letters indicate a significant difference ( p < 0.05). Several authors [29 – 31] evidenced that a sustained increase in oxygen consumption or a functional impairment of existing vasculature, both resulting in insufficient perfusion, triggers neovascularization. On this basis, our first goal was to measure oxygen partial pressure ( pO2) in follicular fluid which resulted negatively related to follicular size, in accordance with the observations by Fischer et al. [4]. It has long been recognized that tissues respond to ischemia by producing angiogenic factors that recruit new blood vessels to the ischemic area. However, despite considerable progress has been made in the understanding of the pathways which are activated during cellular hypoxia, no consensus has been reached on the mechanism by which O2 sensing is achieved [32,33]. Since the mitochondrion is the major oxygen-consuming organelle, it might be expected to play a central role in oxygen-sensitive processes by varying the production of reactive oxygen species (ROS) during hypoxia [34,35]. There is a growing consensus that the cellular O2 sensors are redox-based. Among the variety of possible molecular sensors, NADH oxidase and NADPH oxidase have been indicated as the most important [36,37], presumably as a result of the increased availability of NADH during hypoxia. Several studies [38,39] indicate that the response involves an increase of superoxide (O2 ) production by NADH and NADPH oxidase. Tetrazolium salts are widely used to measure cell proliferation and viability. Their function has been shown to depend on cellular production of reduced pyridine nucleotides: these cofactors act as substrates for cellular oxidoreductases that reduce tetrazolium salts (such as G. Basini et al. / Regulatory Peptides 120 (2004) 69–75 MTT, XTT, MTS and, more recently, WST-1) to formazan end-products [40]. Our data on WST-1 reduction suggest that low oxygen may cause an increase of O2 production in granulosa cells without affecting cell proliferation, as indicated by thymidine uptake. Cells subjected to hypoxic environment have been demonstrated to exhibit a variety of biological responses, including the activation of signalling pathways that regulate proliferation [41]. To our knowledge, the effect of hypoxia on granulosa cell proliferation has never been examined; hypoxia has been found to exert a stimulatory effect on the growth of both endothelial cells from human umbilical vein [42] and neurons [43] and to inhibit osteoblasts [44] and bone marrow cell proliferation [45]; the reasons for these discrepancies are unclear, but the response likely depends on the cell type. Yoshino et al. [46] demonstrated that hypoxia is a major inducer of angiogenic factors such as interleukin-8 and VEGF, a peptide described both as a specific mitogen for vascular endothelial cells and as a factor that increases vascular permeability [47,48]. Wang et al. [49] reported that hypoxia-induced transcription of VEGF mRNA involves a specific transcription factor known as hypoxiainducible factor (HIF). In a previous work [14] we showed that porcine granulosa cells represent a site of VEGF production which appears to be maximal in cells from large follicles. The present observations suggest that hypoxia can be responsible for these high levels. In fact, the increase in follicular diameter during maturation is accompanied by a decreased oxygen tension in follicular fluid which appears to negatively affect VEGF production by granulosa cells. Collectively, these results point out the important role of hypoxia in the development of an extensive vascular network driven by VEGF within the follicle wall; nevertheless, others [50] reported conflicting data, which do not support a model for hypoxic stress promoting VEGF production by granulosa cells. By the way, hypoxia is not the only factor that regulates VEGF production. Nitric oxide (NO), a highly diffusible gas synthesized by NO synthases (NOS) from L-arginine and molecular O2, has been recognized as a positive or negative modulator of VEGF synthesis [51]. In a previous work [18], we showed that the treatment with S-nitroso-L-acetyl penicillamine (SNAP), a potent NO donor, reduces VEGF production by granulosa cells, thus suggesting that NO may play an anti-angiogenic role within the ovary; a negative loop between VEGF and NO is also confirmed by the finding that VEGF inhibits NO production by granulosa cells [14]. Several studies have examined the regulation of NOS activity by O2 tension in cultured cells: McCormick et al. [52] found that although hypoxia increases inducible NOS gene expression in macrophages, NO synthesis is markedly reduced. Whorton et al. [53] demonstrated that exposure to hypoxia reduces NO produc- 73 tion by endothelial NOS. Our results are in agreement with those of Steiner et al. [54] who showed that hypoxia results in oxidative stress due to a change in the ROS –NO balance. However, the mechanisms underlying these changes are unclear. Two alternative explanations are possible: (1) excess ROS generated during hypoxia consume NO, since it acts as an antioxidant; (2) the reduced availability of O2 substrate negatively affect NOS, thus decreasing NO production and increasing in the meanwhile ROS levels. Further studies are needed in order to clarify this important point. The adaptation to hypoxia involves an integrated response among different cell functions [55]. The effect of reduced oxygen tension on ovarian steroidogenesis has been examined by Koos and Feiertag [56] who demonstrated that hypoxia inhibits P4 accumulation in rat granulosa cell culture. Low oxygen tension has been demonstrated to prevent E2 secretion in ED27 cells [57], P4 production by placental villous cells [58] and P4 and E2 synthesis in isolated trophoblast in culture [59]. Our results show that both P4 and E2 production are reduced in total but not in partial hypoxia as recently reported by Martinez-Chequer et al. [50]. It is worth noticing that a severe hypoxic milieu is not representative of the in vivo situation, as confirmed by our present data about pO2 tension in follicular fluid; partial hypoxic conditions are possibly more comparable to those characterizing the well developed ovarian follicle, which relies on E2 and P4 production for its survival [60]. Severe hypoxia has been suggested to inhibit P450 side chain cleavage [61], for which O2 represents an essential co-factor in different cell types [62,63]. We may argue that ROS could play some role in inhibiting steroidogenesis: low oxygen tension appears to increase ROS generation [64], which have been demonstrated to exert antisteroidogenic effect in granulosa cells [65,66]. Taken together, our current data suggest that large swine follicles are physiologically exposed to oxygen shortage. This condition would activate a mechanism which, via VEGF, ensures a proper blood supply to the growing follicle. ROS likely play a crucial role in mediating hypoxic signal. The reduction of O2 milieu, if not maximal, does not seem to impair steroid production by granulosa cells. Acknowledgements This research was supported by a MIUR COFIN grant. References [1] Teo NB, Shoker BS, Martin L, Sloane JP, Holcombe C. Angiogenesis in pre-invasive cancers. Anticancer Res 2002;22:2061 – 72. [2] Folkman J. 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