Elisabetta Baldi
email: e.baldi@dfc.unifi.it

Michaela Luconi°

Monica Muratori

Sara Marchiani

Lara Tamburrino

Gianni Forti°

NONGENOMIC ACTIVATION OF SPERMATOZOA BY STEROID HORMONES: FACTS AND FICTIONS

The rapid effects of steroids on spermatozoa have been demonstrated for the first time two decades ago. Progesterone (P), which is present throughout the female genital tract with peaks of levels in the cumulus matrix surrounding the oocyte, stimulates several sperm functions, including hyperactivation and acrosome reaction. These effects are mediated by an extranuclear pathway, as P stimulates an influx of calcium, the tyrosine phosphorylation of sperm proteins and other signalling cascades in a rapid manner. Whether these effects are receptor mediated and which receptors mediate these effects are still a matter of discussion despite all the efforts of the scientific community aimed at identifying them during the last 20 years. Although responsiveness to P is related to sperm fertilizing ability, the physiological role of P during the process of fertilization is discussed, and recent evidence points for a role of the steroid as a chemotactic agent for sperm. A similar situation applies for estrogens (E), which have been shown to induce direct effects on sperm by an extranuclear pathway. In particular, E appear to decrease acrosome reaction in response to P, exerting a role in ensuring an appropriate timing for sperm exocytosis during the process of fertilization.

A c c e p t e d M a n u s c r i p t 3

INTRODUCTION

The first demonstration of an effect of steroid hormones on spermatozoa dates back 20 years, when [START_REF] Calzada | Effect of steroid hormones and capacitation on membrane potential of human spermatozoa[END_REF] demonstrated an increase of the membrane potential upon treatment with testosterone (T), estrogens (E) and progesterone (P). One year later, [START_REF] Osman | Steroid induced exocytosis: the human sperm acrosome reaction[END_REF] reported the rapid induction of acrosome reaction (AR) by P and 17α-P in human spermatozoa. Αt that time the occurrence of rapid, later on improperly named "nongenomic" or "membrane-initiated", effects of steroids, had been reported for neurosteroids, in particular for 5α-reduced, 3α-hydroxylated pregnane derivatives [START_REF] Mendelson | Sleep induction by an adrenal steroid in the rat[END_REF], and for vitamin D [START_REF] Nemere | Rapid action of 1,25-dihydroxyvitamin D3 on calcium transport in perfused chick duodenum: effect of inhibitors[END_REF]. The concept that steroid hormones may have effects besides the classic (genomic) ones has not been immediately accepted by the scientific community (the endocrinologists, in particular) and the demonstration that steroids may induce effects in genomically inert cells like spermatozoa has probably contributed to strengthen the idea that such effects can occur. In addition, there is an universal agreement that the effects of P on sperm are observed also when non permeable P conjugates are used and are not counteracted by classical P antagonists such as mifepristone (also known as RU487), strengthening the idea that these effects are "extranuclear" and "nongenomic" in nature [START_REF] Correia | Non-genomic steroid actions in human spermatozoa[END_REF]. The first report on the effects of P on sperm has been followed by publication of many papers (up to now almost 300 can be found in Medline using P, sperm and AR as keywords), attempting to define the molecular mechanisms underlying these actions as well as to identify the "putative" receptors involved and their role in the process of fertilization. Now we know which molecular pathways are activated by P in spermatozoa (Figure 1), but, despite many efforts, the identity of the sperm membrane receptor for the steroid remains elusive (see paragraph 4). Similarly, despite the demonstration that responsiveness to P is related to fertilization, whether the steroid is a physiological activator of sperm is still discussed by some scientists (see paragraph 3). Lately, the potential effect of estrogens (E) and endocrine disruptors (ED) on claimed decline of male fertility has been investigated. Even if the possible targets of actions of E and ED is spermatogenesis, the ability of E to interact with P in modulating acrosome reaction was revealed as A c c e p t e d M a n u s c r i p t 4 well (see paragraph 3). The present review summarizes and analyzes critically the main discoveries in the last years on the molecular pathways that are activated by P and E in sperm, the pathophysiological relevance of the effects of these hormones and the possible involvement of receptors. Owing important differences in the reproductive process and male gamete biology among mammalian species this review is focusing with results obtained in human sperm unless otherwise indicated.

MOLECULAR PATHWAYS AND FUNCTIONS ACTIVATED BY PROGESTERONE AND ESTROGENS IN SPERM

In the last two decades there have been reports demonstrating that P may in vitro stimulate several functions in human sperm (namely: hyperactivated motility, capacitation, chemotaxis and acrosome reaction). All these functions are involved in the process of fertilization of the oocyte (Figure 2) and occur physiologically in the female genital tract. Indeed, immediately after ejaculation spermatozoa are unable to fertilize, a quality that they acquire during transit in the female genital tract following interaction with local factors (reviewed in [START_REF] Muratori | Molecular markers of human sperm functions[END_REF]. In order to become able to fertilize spermatozoa must undergo a complex event known as capacitation at the end of which sperm acquire hyperactivated motility (a special type of progressive motility, characterized by disordered movement of the head and tail needed to penetrate oocyte vestments) as well as the ability to respond to stimuli leading to acrosome reaction, an exocytotic process which helps the sperm to penetrate the zona pellucida surrounding the oocyte. Chemotaxis is believed to occur in the proximity of the oocyte and is involved in directing the sperm in the last part of its journey. P has been shown to activate several signalling pathways [generation of cAMP, increase of intracellular calcium (Ca 2+ ), promotion of tyrosine phosphorylation of proteins, activation of phospholipases and many others], all of which have been demonstrated as being involved in the regulation of these functions. However, at present, the picture of the signalling pathways that are activated and, most importantly, which of them are required for induction of a particular sperm function, are not clear. The situation concerning direct effects of E (including environmental E) on spermatozoa is even more intricate. Indeed, although E have been shown to activate some signalling pathways in spermatozoa, whether they are associated with one or more sperm functions is discussed. P-induced sperm capacitation. As mentioned above, capacitation is a complex event occurring in sperm following their release in the female genital tract that ultimately prepares and allows sperm to reach the oocyte and to become reactive to stimuli that induce AR in vivo. In vitro, the process is achieved in the presence of both bicarbonate and albumin, inducing respectively soluble adenylate cyclase activation and generation of cAMP, and an increase of membrane fluidity by promoting the efflux of cholesterol from the plasma membrane. There are reports suggesting a positive effect of P on capacitation of human sperm [START_REF] Kay | Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction[END_REF][START_REF] De Lamirande | Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion[END_REF][START_REF] Thundathil | Different signal transduction pathways are involved during human sperm capacitation induced by biological and pharmacological agents[END_REF][START_REF] Yamano | Human spermatozoa capacitated with progesterone or a long incubation show accelerated internalization by an alkyl ether lysophospholipid[END_REF]. This effect is, however, obtained with high concentrations (1-30 µM) of the steroid and its physiological relevance is doubtful (discussed in the next paragraph). In addition, there is not a universally recognized marker or endpoint to define capacitation, making comparison between these studies very difficult, due to the fact that different markers have been used to markers to define it. Induction of capacitation by P appears to be related promotion of generation of oxygen species by sperm [START_REF] De Lamirande | Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion[END_REF], activation of the mitogen-activated protein kinase (MAPK) but not of the cAMP/PKA cascade [START_REF] Thundathil | Different signal transduction pathways are involved during human sperm capacitation induced by biological and pharmacological agents[END_REF]. Indeed, short-term application of µmolar concentrations of P activated MAPK in sperm (Luconi et al, 1998c), but, interestingly, this effect was not related to AR induction (Luconi et al, 1998c). Since a previous work from our laboratory demonstrated an involvement of MAPK in sperm capacitation (Luconi et al, 1998a), these studies seem to indicate that the in vitro effect of P on capacitation, if any, is mediated by this pathway.

P-induced sperm motility.

Forward motility represents the necessary requisite for sperm to reach the oocyte. In the proximity of the latter, spermatozoa acquire a particular type of motility (namely hyperactivated motility) characterized by disordered movements of the head and flagellum [START_REF] Luconi | Pathophysiology of sperm motility[END_REF]. Many factors affect sperm motility, including calcium, cAMP, and constituents found A c c e p t e d M a n u s c r i p t 6 in the female genital tract [START_REF] Luconi | Pathophysiology of sperm motility[END_REF]. A positive effect of P (0.1-1 µm) on forward [START_REF] Contreras | Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties[END_REF] and hyperactivated (Uhler et al, 1992;[START_REF] Calogero | Effects of gammaaminobutyric acid on human sperm motility and hyperactivation[END_REF][START_REF] Jaiswal | Human sperm chemotaxis: is progesterone a chemoattractant?[END_REF] motility has been reported by some Authors but not confirmed by others [START_REF] Kay | Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction[END_REF][START_REF] Wang | Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro[END_REF][START_REF] Luconi | Human spermatozoa as a model for studying membrane receptors mediating rapid nongenomic effects of progesterone and estrogens[END_REF]. The effect of P on hyperactivated motility was found to be transient and was not counteracted by P antagonists (Uhler et al, 1992;[START_REF] Yang | Progesterone and RU486: opposing effects on human sperm[END_REF]. Recently, it has been found that P induces Ca 2+ oscillations, due to a release from intracellular stores (see also next) in the proximity of the flagellum, which are strictly related to the flagellar activity [START_REF] Harper | Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating[END_REF][START_REF] Bedu-Addo | Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone[END_REF], suggests that this mechanism, rather than the massive Ca 2+ entry stimulated by the steroid, is responsible for induction of hyperactivation. Such effects were observed at µmolar concentrations of P, in the presence of EGTA in the external medium, and, as mentioned above, were associated with changes in the flagellar beating, a typical feature of hyperactivated motility that occurs in the proximity of the oocyte [START_REF] Bedu-Addo | Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone[END_REF]. The fact that only a small fraction of spermatozoa shows Ca 2+ oscillations may provide an explanation for the conflicting results concerning the effects of the steroid on sperm motility reported in the previous papers.

It has been reported that P, at micromolar concentrations, stimulates an increase of cAMP intracellular levels in sperm (Parinaud and Mihlet, 1996), however, whether activation of soluble adenylate cyclase and thus cAMP generation are required for hyperactivated motility in human sperm is not clear [START_REF] Marquez | Soluble adenylyl cyclase is required for activation of sperm but does not have a direct effect on hyperactivation[END_REF] and the involvement of this system in the increase of motility stimulated by the steroid requires further studies.

P-induced sperm chemotaxis.

Sperm chemotaxis plays an essential role in species with external fertilization (Friedrich and Julicher, 2007) and has been demonstrated to occur also in mammals [START_REF] Sun | Human sperm chemotaxis: both the oocyte and its surrounding cumulus cells secrete sperm chemoattractants[END_REF]. The process takes place in the proximity of the fertilization site and it is initiated by the release of factors from the oocyte and the surrounding cumulus cells. Whether P or other steroids present in the follicular fluid or in the cumulus matrix may be responsible for this chemoattractant effect is still an open question. As for the effects on hyperactivated motility, A c c e p t e d M a n u s c r i p t 7 conflicting results have been reported also on the effects of P on sperm chemotaxis (Villanueva-Diaz et al, 1995;Jaiswal et al,1999;[START_REF] Wang | Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro[END_REF][START_REF] Teves | Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa[END_REF]. It must be remarked that evaluating sperm chemotaxis is very difficult and the use of videomicroscopy and computer image analysis appears to be the only most reliable and direct way to investigate directionality of sperm toward a chemoattractant. Using this approach, [START_REF] Teves | Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa[END_REF] recently showed that P is chemoattractant for human spermatozoa at nanomolar concentrations. Of interest, application of a P gradient (starting from 0.3 nM) induces slow Ca 2+ oscillations in the caudal part of the head of about 30% of sperm [START_REF] Harper | Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating[END_REF]. Ca 2+ oscillations in response to P do not appear to be dose-dependent and as mentioned above, occur in the absence of calcium in the external medium and seem to be involved in the modulation of flagellar activity [START_REF] Bedu-Addo | Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone[END_REF] producing a type of flagellar beating which is required for a chemiotactic response. However, a direct relationship between mdofication of motility parameters and chemotaxis is lacking and further studies are need to establish it.

P-induced acrosome reaction (AR).

AR occurs in vivo following sperm interaction with the oocyte zona pellucida [START_REF] Hoodbhoy | Insights into the molecular basis of sperm-egg recognition in mammals[END_REF]) and consists of fusion and fenestration of external and acrosomal membrane of sperm allowing the release of acrosomal content (mostly proteolytic enzymes). AR can be induced by many constituents present in the female genital tract. Among these, P has been intensively studied in the last decade, in view of its high concentrations in the genital tract fluids and in the oocyte cumulus mass, where the steroid reaches µmolar concentrations. In view of the role of Ca 2+ in induction of AR, there are no doubts that the massive influx of the ion stimulated by P can trigger the process [START_REF] Correia | Non-genomic steroid actions in human spermatozoa[END_REF]Muratori et al, 2008).

The wave of intracellular Ca 2+ stimulated by P in sperm is biphasic, composed of a transient increase followed by a sustained elevation [START_REF] Blackmore | Cell surface-binding sites for progesterone mediate calcium uptake in human sperm[END_REF][START_REF] Baldi | Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa[END_REF] and is obtained at concentrations ranging from 10 nM to 100 µM (Luconi et al, 1998b). The response is totally dependent on extracellular Ca 2+ and is clearly dose-dependent (Luconi et al, 1998b;[START_REF] Harper | Encoding of progesterone stimulus intensity by intracellular [Ca2+] ([Ca2+]i) in human spermatozoa[END_REF]. Investigating the involvement of membrane Ca 2+ channels has been the aim of many studies, A c c e p t e d M a n u s c r i p t 8 but at present, which channels (voltage-operated? receptor-operated?) are involved in responsiveness to P in human sperm remains elusive [START_REF] Bonaccorsi | Low-voltage-activated calcium channels are not involved in capacitation and biological response to progesterone in human sperm[END_REF]Guzman-Grenfell and Gonzalez-Martinez, 2004). Recently, evidence has been reported for mobilization of Ca 2+ from sperm intracellular stores in response to P [START_REF] Bedu-Addo | Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone[END_REF], indicating that multiple mechanisms are involved in the response. Indeed, in the absence of extracellular Ca 2+ (EGTAbuffered medium), a small percentage of cells (about 5%) shows a tiny Ca 2+ transient, suggesting mobilization from intracellular stores. The nature of such intracellular stores in spermatozoa is still discussed (Bedu-Addu et al, 2008).

Of interest, whereas the Ca 2+ response occurs in the vast majority of P-stimulated sperm, as demonstrated by image analysis of fura2-loaded spermatozoa (Kirkman-Brown et al, 2000), AR in response to the steroid occurs only in a minor percentage (10-15) of them [START_REF] Baldi | Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa[END_REF]. Such a difference is too high to reflect the diverse sensitivity of the methods used to detect sperm intracellular Ca 2+ and AR and appears to underlie the necessity for the simultaneous activation of other pathways for the attainment of sperm exocytosis. On the other hand, other pathways have been demonstrated as being activated by P, but their involvement in the induction of AR is still controversial. For instance, although some studies demonstrate an involvement of the cAMP/PKA pathway in the induction of AR in sperm [START_REF] Harrison | Involvement of protein kinase A and A kinase anchoring protein in the progesterone-initiated human sperm acrosome reaction[END_REF], others do not [START_REF] Moseley | Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation[END_REF]. Indeed, the increase in cAMP concentrations following P stimulation is very low [START_REF] Parinaud | Progesterone induces Ca++-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm[END_REF], raising doubts about the relevance of the pathway in the response to the steroid. A similar situation applies for the phospholipase C/inositol3phosphate/diacylglycerol/protein kinase (PLC/IP3/DAG/PKC) pathway: although the enhancing effect of P on inositol trisphophate [START_REF] Thomas | Phosphatidylinositol 4,5-bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent upon Ca2+ influx[END_REF] and diacylglycerol (O'Toole et al, 1996a) synthesis has been reported, blockers of phospholipase C do not appear to affect P-stimulated Ca 2+ fluxes [START_REF] Harper | Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating[END_REF], and whether the pathway is involved in the induction of AR remains controversial (O'Toole et al, 1996b;[START_REF] Luconi | Identification and characterization of functional nongenomic progesterone receptors on human sperm membrane[END_REF]. P at µM concentrations has been also shown to promote tyrosine phosphorylation of sperm proteins [START_REF] Tesarik | Stimulation of protein tyrosine phosphorylation by a progesterone receptor on the cell surface of human sperm[END_REF][START_REF] Luconi | Stimulation of protein tyrosine phosphorylation by platelet-activating factor and progesterone in human spermatozoa[END_REF] and this pathway is A c c e p t e d M a n u s c r i p t 9 involved in AR and Ca 2+ influx in response to the steroid [START_REF] Martinez | Stimulation of tyrosine phosphorylation by progesterone and its 11-OH derivatives: dissection of a Ca(2+)-dependent and a Ca(2+)-independent mechanism[END_REF][START_REF] Bonaccorsi | Tyrosine kinase inhibition reduces the plateau phase of the calcium increase in response to progesterone in human sperm[END_REF]. However, as for cAMP generation, tyrosine phosphorylation increase in response to P is small [START_REF] Luconi | Stimulation of protein tyrosine phosphorylation by platelet-activating factor and progesterone in human spermatozoa[END_REF][START_REF] Kirkman-Brown | Inhibitors of receptor tyrosine kinases do not suppress progesterone-induced [Ca2+]i signalling in human spermatozoa[END_REF] and its influence on Ca 2+ transients and AR is discussed [START_REF] Kirkman-Brown | Inhibitors of receptor tyrosine kinases do not suppress progesterone-induced [Ca2+]i signalling in human spermatozoa[END_REF]. Recently, we have demonstrated that the tyrosine kinase Src is involved in P-induced Ca 2+ influx and AR in human sperm [START_REF] Varano | Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa[END_REF]. In particular, we have shown that inhibition of Src activation decreases Ca 2+ influx in response to the steroid, affecting both the peack and the plateau phases of the response. As a consequence, AR in response to P is suppressed. These data suggest that Src may be stimulated by P upstream of Ca 2+ increase, in line with other results of our and other groups demonstrating that the plateau phase of P Ca 2+ response is inhibited by generic inhibitors of tyrosine kinases in spermatozoa [START_REF] Bonaccorsi | Tyrosine kinase inhibition reduces the plateau phase of the calcium increase in response to progesterone in human sperm[END_REF][START_REF] Tesarik | Single cell analysis of tyrosine kinase dependent and independent Ca2+ fluxes in progesterone induced acrosome reaction[END_REF]. Src, which is located in the sperm post-acrosomal region, may be involved in the release of Ca 2+ from sperm intracellular stores (such as the redundant nuclear envelop; [START_REF] Ho | Characterization of the intracellular calcium store at the base of the sperm flagellum that regulates hyperactivated motilità[END_REF], which are located in the same area. At present, the mechanism leading to Src activation by P in sperm is not known.

Effects of estrogens and xenoestrogens on sperm.

That E are important for male reproductive functions became clear following the development of E receptor knock out (ERΚΟ) mice, which show important alterations of spermatogenesis and infertility [START_REF] Eddy | Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility[END_REF] due to modifications in the expression of a battery of genes involved in spermatogenesis and fluid reasorbption in the reproductive tract [START_REF] Zhou | Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function[END_REF]. In addition, it has been recently shown that a polymorphism in the promoter of the ERα is related to a lower sperm count in the ejaculate [START_REF] Guarducci | Estrogen receptor alpha promoter polymorphism: stronger estrogen action is coupled with lower sperm count[END_REF]. Besides the genomic effects, evidence has emerged concerning direct, extranuclear, effects of E (Figure 1) on mouse spermatogenetic cells, where the steroid rapidly stimulates a transient increase in ERK activation [START_REF] Vicini | 17-beta-estradiol elicits genomic and non-genomic responses in mouse male germ cells[END_REF]. Studies concerning extranuclear effects of E in mature spermatozoa are conflicting. Our group [START_REF] Luconi | Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects[END_REF] has shown that a short time in vitro treatment of human sperm with E induces a small rise of intracellular Ca 2+ which interferes with the subsequent Ca 2+ wave stimulated by P. Ultimately, this A c c e p t e d M a n u s c r i p t 10 effect determines a reduction in P-induced AR, although E themselves do not appear to affect the process. These results have been recently confirmed by another group [START_REF] Vigil | Physiological action of oestradiol on the acrosome reaction in human spermatozoa[END_REF]. Similar results were obtained with the ER antagonists tamoxifen and ICI 164384 [START_REF] Luconi | Effects of estrogenic compounds on human spermatozoa: evidence for interaction with a nongenomic receptor for estrogen on human sperm membrane[END_REF], suggesting that the putative receptor involved in the estrogenic response in spermatozoa shows different characteristics respect to classical ERs (see section 4). Conversely, no effects on spermatozoa were observed with two xenoestrogens, namely bisphenol A and octylphenol polyethoxilate [START_REF] Luconi | Effects of estrogenic compounds on human spermatozoa: evidence for interaction with a nongenomic receptor for estrogen on human sperm membrane[END_REF]. In a subsequent study, [START_REF] Francavilla | Evaluation of the effect of 17alphaOH-progesterone and 17beta-oestradiol on human sperm ability to fuse with oocytes: comparison and possible interference with the effect of progesterone[END_REF] reported that pre-incubation with E does not alter the ability of human sperm to fuse with the oocyte nor interferes with the enhancing effect of P on the process. Conversely, Adeoya-Osiguwa et al (2003) reported a stimulatory effect of E and different xenoestrogens on both capacitation, acrosome reaction and fertilizing ability of mouse spermatozoa. Using mycotoxins with E-like action, [START_REF] Tsakmakidis | In vitro effect of zearalenone and alpha-zearalenol on boar sperm characteristics and acrosome reaction[END_REF] recently reported an inhibitory effect on spontaneous boar sperm AR.

Finally, [START_REF] Aquila | Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway[END_REF] hypothesize a role for E in human sperm survival by stimulation of the PI3K/Akt and ERK pathways. Interestingly, at difference with the other studies, this study reported inhibition of the effects by classical ER antagonists.

PATHOPHYSIOLOGICAL RELEVANCE OF THE STEROID EFFECTS ON SPERM

As many of the maturation processes and functional events that characterize sperm life after release from the testis occur in the female genital tract, there is no doubt that factors present in this environment, and steroid hormones in particular, have a potential role in their modulation.

Capacitation, development of hyperactivated motility, chemotaxis and AR are all events occurring in the female reproductive tract and potentially modulated in this contest.

As capacitation is needed to prepare sperm to undergo AR, it is believed to occur in the uterus or in the fallopian tube just before spermatozoa reach the oocyte. In this situation, capacitation should be induced by P at the concentration found in the tubal fluid, which is lower than the one found in the cumulus matrix [START_REF] Munuce | Comparative concentrations of steroid hormones and proteins in human peri-ovulatory peritoneal and follicular fluids[END_REF]. Conversely, the effects of the steroid on capacitation are At a first glance, induction of AR by P (which is present throughout the female genital tract, with its maximal levels in the cumulus matrix) may be surprising. An acrosome-reacted sperm does not retain the ability to fertilize even if AR occurs in the proximity of the oocyte, and thus premature AR should be avoided. However, at the concentrations found in the cumulus mass, P induces AR and a fertilizing sperm needs to pass through the cumulus to fertilize an egg. In this light, the inhibiting effect of E on P-induced AR [START_REF] Luconi | Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects[END_REF][START_REF] Vigil | Physiological action of oestradiol on the acrosome reaction in human spermatozoa[END_REF] may have a physiological relevance: the interplay between the two steroids during sperm transit in the cumulus mass may be important to ensure a correct timing of the process. In addition, the effects of P present in the cumulus matrix may be involved in priming the sperm to the subsequent effects of the zona pellucida proteins [START_REF] Roldan | Exocytosis in spermatozoa in response to progesterone and zona pellucida[END_REF]. Finally, a physiological effect of P in removing poor quality spermatozoa by inducing premature AR [START_REF] Harper | Reassessing the role of progesterone in fertilization-compartmentalized calcium signalling in human spermatozoa?[END_REF]) cannot be excluded. It is important to mention that P-induced AR is related to the sperm fertilizing ability [START_REF] Krausz | Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization[END_REF][START_REF] Jacob | Human sperm non-nuclear progesterone receptor expression is a novel marker for fertilization outcome[END_REF], and that responsiveness to P is decreased in subfertile patients [START_REF] Falsetti | Decreased responsiveness to progesterone of spermatozoa in oligozoospermic patients[END_REF], strongly suggesting that the ability of a spermatozoon to respond to the steroid has to be considered a pre-requisite for fertilization.

If the physiological relevance of induction of capacitation and AR by P is a matter of discussion, the effects on motility and chemotaxis could be considered the primary physiological functions of the steroid. As mentioned above, there are reports showing that the effects of P on the three processes occur in vitro at concentrations of the steroid similar to those found in the areas of the female genital tract where they occur. In addition, P-stimulated hyperactivated motility and chemotaxis appear to occur in a small fraction of spermatozoa [START_REF] Harper | Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating[END_REF][START_REF] Teves | Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa[END_REF], suggesting that a sperm selection is needed and takes place during the journey to the oocyte. In this light, it is possible that P may induce hyperactivation only in those sperm that possess all the requisites to fertilize the oocyte.

A c c e p t e d M a n u s c r i p t not appear to have a direct role on sperm-oocyte interaction [START_REF] Francavilla | Evaluation of the effect of 17alphaOH-progesterone and 17beta-oestradiol on human sperm ability to fuse with oocytes: comparison and possible interference with the effect of progesterone[END_REF]. However, it is worth mentioning that aromatase mRNA have been found in sperm and their levels are lower in the immotile sperm fraction [START_REF] Carreau | Aromatase and estrogens in man reproduction: a review and latest advances[END_REF], suggesting a possible role of E produced by sperm from androgens present in the male genital tract in the development of motility. This effect, if any, does not appear to be mediated by a genomic pathway, as the observed actions of E are rapid and the presence of classical ERs in mature sperm is uncertain [START_REF] Luconi | Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects[END_REF][START_REF] Aquila | Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway[END_REF][START_REF] Carreau | Aromatase and estrogen receptors in male reproduction[END_REF]). E derived from testosterone conversion by aromatase present in spermatozoa, may be also involved in the regulation of ER-activated pathways in the epididymis or in the testis (Figure 3), which have the potential to regulate sperm maturation at these levels [START_REF] Hess | Oestrogen, ist receptor and function in the male reproductive tract -a review[END_REF].

Moreover, a direct autocrine as well as paracrine effect on modulation of sperm activation may be exerted by the self production of E in the sperm from T conversion (Figure 3).

The effects of endocrine disruptors (EDs) on male reproductive functions appear to be even more complex. There are reports that consider EDs responsible for the claimed global decrease in semen quality. It must be considered that there is no agreement on this point (see also [START_REF] Fisch | Declining worldwide sperm counts: disproving a myth[END_REF], and that it is probably a localized more than worldwide phenomenon. Decline in semen quality has been suggested to be associated to male pre-natal exposure to EDs, and in vitro and in vivo laboratory data seem to confirm such an association [START_REF] Giwercman | Influence of endocrine disruptors on human male fertility[END_REF], although further studies are awaited. Human beings are increasingly exposed to EDs (for instance to compounds like cadmium and bisphenol A, which have been demonstrated to bind estrogenic receptors) and there is evidence that some EDs may disrupt steroidogenesis in the Leydig cells [START_REF] Takiguchi | New aspects of cadmium as endocrine disruptor[END_REF][START_REF] Kumar | Disruption of LH-induced testosterone biosynthesis in testicular Leydig cells by triclosan: probable mechanism of action[END_REF], which may be reflected in impaired spermatogenesis. In particular, triclosan [START_REF] Kumar | Disruption of LH-induced testosterone biosynthesis in testicular Leydig cells by triclosan: probable mechanism of action[END_REF] appears to reduce testosterone production by Leydig cells by decreasing the activity of a membrane adenylate cyclase, whereas cadmium [START_REF] Takiguchi | New aspects of cadmium as endocrine disruptor[END_REF] affects androgens production by directly binding to androgen and estrogen receptors. EDs have been also demonstrated to rapidly disrupt the integrity of the blood-testis barrier in the seminiferus A c c e p t e d M a n u s c r i p t 13 epithelium [START_REF] Fiorini | Sertoli cell junctional proteins as early targets for different classes of reproductive toxicants[END_REF][START_REF] Fisch | Declining worldwide sperm counts: disproving a myth[END_REF]. However, it is not clear whether these effects are mediated by binding to steroid receptors and/or a non genomic mechanism. The recent demonstration that E exerts rapid effects on germ cells [START_REF] Vicini | 17-beta-estradiol elicits genomic and non-genomic responses in mouse male germ cells[END_REF], suggests that an interplay between the rapid and classical actions may occur during spermatogenesis, and EDs may thus interfere with the process both by classical and non genomic mechanisms.

ARE THE STEROID EFFECTS ON SPERM RECEPTOR-MEDIATED?

The involvement of a receptor-mediated mechanism in the action of P has been proposed based on different findings, including the increase of sperm responsiveness to P following capacitation [START_REF] Baldi | Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa[END_REF] and the lack of a response to a second challenge with the steroid (Luconi et al, 1998).

The ability of membrane-impermeant conjugates of P to exert similar effects as the native compound seems to indicate that this receptor is located at the membrane. However, the use of protein-conjugated steroids has been criticized because they can release the free compound in unknown amounts. Moreover, big proteins, such as BSA, can have effects on sperm per se [START_REF] Meizel | Molecules that initiate or help stimulate the acrosome reaction by their interaction with the mammalian sperm surface[END_REF].

Several hypothesis can be made concerning the type of E and P receptor mediating the rapid effects of the steroid on spermatozoa (Figure 1), namely: the classical P or ER located at the membrane (B1), a new receptor located at the membrane (B2) or occurrence of membrane-perturbing effects of the steroids (C).

Although the classical P receptors have not been detected in spermatozoa [START_REF] Sabeur | Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction[END_REF]Luconi et al, 1998b), an antibody directed against the C-terminal region of the P receptor, able to reduce AR and Ca 2+ influxes induced by the steroid, recognizes protein bands of 54-60 kDa in human spermatozoa [START_REF] Sabeur | Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction[END_REF]Luconi et al, 1998;[START_REF] Shah | Coexistence of intracellular and membrane-bound progesterone receptors in human testis[END_REF] but so far, whether these proteins represent the sperm receptors for the steroid remains unclear. A G-protein coupled, membrane P receptor (mPR) has been cloned in different species including the human [START_REF] Zhu | Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor[END_REF], but attempts to link this receptor to the one responsible for the membrane effects of the A c c e p t e d M a n u s c r i p t 14 steroid in sperm have been unsuccessful. In addition, the human homolog of the mPR has been recently demonstrated as localizing to the endoplasmic reticulum and being unresponsive to P [START_REF] Krietsch | Human homologs of the putative G protein-coupled membrane progestin receptors (mPRalpha, beta, and gamma) localize to the endoplasmic reticulum and are not activated by progesterone[END_REF]. More recently, an extranuclear P receptor has been identified in human sperm by proteomic analysis [START_REF] Baker | Identification of gene products present in Triton X-100 soluble and insoluble fractions of human spermatozoa lysates using LC-MS/MS analysis[END_REF], although its involvement in induction of AR or other P effects is awaited. Given the numerous candidates for the receptor, the possible involvement of a molecular complex, as recently suggested by Correira et al ( 2007), is attractive. For a more complete updated review on membrane progesterone receptor see [START_REF] Fernandez | Honey, we need to talk about the membrane progestin receptors[END_REF].

The situation concerning E is similar: as mentioned above, results about the presence of classical ERs are conflicting, and although some E binding proteins have been found in mature spermatozoa [START_REF] Luconi | Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects[END_REF][START_REF] Carreau | Aromatase and estrogen receptors in male reproduction[END_REF], their involvement in the rapid effects of the steroid has not been unequivocally demonstrated. Finally, the possibility that a non-receptoral mechanism may be involved for both steroids cannot be excluded. The high concentrations needed to obtain the effects and the observation that membrane perturbating effects occur in sperm following challenge with P or E (Shivaji and Jaganndham, 1992) represent a convincing alternative to a receptor mechanism.

CONCLUDING REMARKS

After 20 years from the discovery of rapid effects of P on spermatozoa, most of the signalling pathways involved have been disclosed, but still many questions remain to be solved. As discussed above, the main physiological role of P in sperm may be related to stimulation of hyperactivated motility and chemotaxis: however, further studies are needed to confirm these effects as well as to better clarify the signalling pathways involved in these specific effects. Concerning the effects of P on induction of AR, which occur at µmolar concentrations of the steroid, it must be considered that spermatozoa must cross the cumulus mass before reaching the zona pellucida, being here exposed to elevated concentrations of the steroid. It remains to be established which is the final effect of this situation on sperm. The time needed to cross the zone could be long enough to provoke an untimely induction of AR (possibly excluding that sperm from the process of fertilization). On the contrary, A c c e p t e d M a n u s c r i p t 15 the exposition to P could simply sensitizes the sperm for the subsequent action of zona proteins (Roldan et al, 2004). In both cases, a physiological role of the effect of P cannot be excluded: indeed, those sperm that eventually undergo AR during crossing of the cumulus may be considered as kamikaze sperm facilitating the passage of the one committed to fertilize.

Although most of the current evidence suggests the involvement of a receptor mechanism in the action of P, identification of this receptor, which may give hints to studies on contraceptive vaccines and on therapeutical approaches to male infertility, is still lacking. Alternative mechanisms (for instance, effects on membrane fluidity, which cannot be excluded when stimulating a cell with such high concentrations of a steroid molecule) should be also taken into consideration.

Concerning E, there is evidence that estrogenic compounds with endocrine disrupting properties may affect spermatogenesis in animals exposed during their uterine life, however, at the moment, it is not clear the relative contribute of rapid vs classical pathways. For some of the EDs studied, evidence for direct binding to estrogen receptors is lacking. A direct effect of E on sperm appears to be important for ensuring an appropriate timing for sperm activation during the process of fertilization, however, the mechanisms and the receptor involved are still quite obscure and require further studies. The recent demonstration that G protein receptor 30 (GPR30) mediates the proliferative effects of E in spermatogonial cells in the mouse [START_REF] Sirianni | The novel estrogen receptor, G protein-coupled receptor 30, mediates the proliferative effects induced by 17β-estradiol on mouse spermatogonial GC-1 cell line[END_REF], opens new perspectives in the field prompting scientists to investigate the involvement of such receptor in the physiology of human sperm.

  pathways and functions activated by steroids in spermatozoa 3. Pathophysiological relevance of the steroid effects in sperm 4. Are the steroid effects on sperm receptor mediated? 5. Concluding remarks A c c e p t e d M a n u s c r i p t 2 ABSTRACT

  concentrations of the steroid, similar to those found in the cumulus matrix, raising doubts about their physiological significance in the process.

Fig. 2 :

 2 Fig.2: Sperm activation phases required for fertilization". During their transit through the

Fig. 3 :

 3 Fig.3: The sperm as a "mobile endocrine unit". Spermatozoa can convert testosterone (T) into


Peng Kang

Justyna Rogalska

Catherine A Walker

Matthew Burke

Jonathan R Seckl

Malcolm R Macleod

Maggie Lai
email: m.lai@ed.ac.uk

Injury-induced mineralocorticoid receptor expression involves differential promoter usage: a novel role for the rat MRβ variant

Keywords: Mineralocorticoid receptor, transcript, variant, oxygen-glucose deprivation, hypothermia

Neuronal injury results in increased mineralocorticoid receptor (MR) expression and is associated with increased neuronal survival, suggesting that enhancing MR signalling may have therapeutic implications. MR has a complex gene structure with at least three untranslated exons (α, β, γ) each with unique promoters and a common coding region. We examined whether distinct cellular stressors differentially regulate exon-specific MR transcripts. MRβ transcript was specifically upregulated in rat primary cortical cultures undergoing hypothermic oxygen-glucose deprivation (OGD/H) through activation of its own promoter. This effect was mediated in part by ERK signalling as blockade with PD98059 inhibited OGD/H-induced MRβ promoter activity. A specific increase in MRβ transcript expression was also found in vivo in hypothermic anoxic neonatal rat hippocampus. These results demonstrate a novel key role for the MRβ transcript in response to injury and suggest that some of the known neuroprotective effects of hypothermia may be mediated through increased MR expression.

Glucocorticoid hormones exert multiple effects within the central nervous system amongst which they are potent regulators of neuronal survival. Their actions are mediated by two closely related intracellular receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) which once activated function as transcriptional regulators of target genes [START_REF] Evans | A molecular framework for the actions of glucocorticoid hormones in the nervous system[END_REF]. Under normal conditions high affinity MRs are substantially occupied by physiological levels of glucocorticoids; in contrast the lower affinity GRs only become activated when glucocorticoid levels rise either during the circadian peak or in response to stress [START_REF] De Kloet | Brain corticosteroid receptor balance in health and disease[END_REF]. MR signalling is therefore predominantly regulated by receptor abundance whereas GR signalling is more dependent on hormone concentrations. This has important implications and may explain why MR and GR can have opposite actions in neuronal viability where predominant MR activation appears to have a survival effect for instance in dentate gyrus neurons [START_REF] Woolley | Effects of aldosterone or RU28362 treatment on adrenalectomy-induced cell death in the dentate gyrus of the adult rat[END_REF] while GR overactivation increases vulnerability to for instance excitotoxicity [START_REF] Reagan | Controversies surrounding glucocorticoid mediated cell death in the hippocampus[END_REF][START_REF] Sapolsky | Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders[END_REF].

We have previously shown a rapid induction of MR (mRNA and protein) in vitro in rat primary hippocampal and cortical neuronal cultures following sub-lethal cell stress, and in vivo in rat hippocampus following hypothermic transient global cerebral ischemia [START_REF] Macleod | Mineralocorticoid receptor expression and increased survival following neuronal injury[END_REF]. In each case pharmacological antagonism with spironolactone increased neuronal death suggesting that MR induction is an endogenous neuroprotective mechanism and may mediate some of the effects of hypothermia. Indeed, in vitro overexpression of human MR (hMR) protects PC12 cells from oxygen-glucose deprivation (OGD) [START_REF] Lai | Overexpression of the mineralocorticoid receptor protects against injury[END_REF]. Crucially hippocampal neurons in transgenic mice overexpressing MR in forebrain (MR-Tg) have reduced neuronal loss following transient global cerebral ischemia [START_REF] Lai | Forebrain mineralocorticoid receptor overexpression enhances memory, reduces anxiety and attenuates neuronal loss in cerebral ischaemia[END_REF]. Furthermore hippocampal MR is increased in post-mortem samples from patients who had a brief cardiac arrest, and presumed cerebral hypoxia, some weeks prior to death (Lai et al., in press), implying that MR is also increased in human brain following neuronal stress. Taken together these findings suggest that increasing neuronal MR expression might have important therapeutic uses for example in patients at high risk of cerebral ischemic insult.

The regulation of MR gene expression is complex and any clinical exploitation of the neuroprotective effects of increased MR will require an understanding of its regulation following cell stress. The rat MR gene gives rise through alternate splicing to three distinct MR mRNA transcripts (MRα, β, γ) which differ in their 5'-untranslated region [START_REF] Kwak | Heterogeneity of the mineralocorticoid receptor messenger ribonucleic acid: differential expression and regulation of splice variants within the rat hippocampus[END_REF] but encode the same mature protein. Each corresponding 5' flanking region acts as a functional promoter allowing independent regulation of each mRNA isoform. Similarly, the human MR gene gives rise to two MR mRNA species controlled by two promoters P1 and P2 [START_REF] Zennaro | Characterization of the human mineralocorticoid receptor gene 5'-regulation region: evidence for the differential hormonal regulation of two alternative promoters via non-classical mechanisms[END_REF] which correspond to rat MRα and MRβ respectively. These alternate promoters appear to determine tissue-and development-specific expression of the MR gene [START_REF] Vazquez | Alpha, beta, gamma mineralocorticoid receptor messenger ribonucleic acid splice variants: differential expression and rapid regulation in the developing hippocampus[END_REF] and suggest separate mechanisms for upregulation of MR in response to different stimuli. Indeed, endogenous glucocorticoids appear to regulate the different MR transcripts but other regulators have yet to be identified.

Here, we have investigated the mechanism(s) of cell stress-induced MR upregulation at the molecular and cellular level. We examined the effects of oxygen/glucose deprivation (OGD) and/or hypothermia on MR mRNA variant expression in rat primary neuronal cultures. To study the regulation of these different promoters in response to different cell stressors, we used the luciferase reporter system in rat PC12 cells, and to establish if in vitro findings were of biological relevance in vivo we have also analysed the MR mRNA variant involved in mediating the MR response to anoxia and/or hypothermia in hippocampal neurons of the neonatal rat brain.

Materials and Methods

All animals were maintained in accordance with the guidelines of the UK Home Office Animals (Scientific Procedures) Act and the Polish Local Committee on the Use and Care of Laboratory Animals.

Primary cortical culture

A c c e p t e d M a n u s c r i p t 4 Primary neuronal cultures were prepared from cerebral cortices of E18 Wistar rat fetuses as described previously [START_REF] Macleod | Mineralocorticoid receptor expression and increased survival following neuronal injury[END_REF]. Briefly, dissected cortices were dissociated in Hanks balanced salt solution (HBSS) containing trypsin (1800U/ml) and DNAse I (200U/ml) for 10 minutes at 37°C. Cells were plated onto poly-D-lysi ne coated 6-well multiwell plates (BD Biosciences, Oxford, UK) at a density of 2x10 5 cells/cm 2 in Neurobasal™ medium (Invitrogen, Paisley, UK) supplemented with B27 (Invitrogen) and 0.5mM L-glutamine (Invitrogen). Cells were grown under humidified conditions of 95% air/5% CO 2 at 37°C with medium changed after 7 days in vitro (d.i.v.). Under these conditions the neuronal population was >90% as determined by microtubule-associated protein 2 immunohistochemistry (data not shown). Experiments were performed at 14 d.i.v.

PC12 cell culture

Rat pheochromocytoma PC12 cells were kindly provided by Professor Karen Chapman (University of Edinburgh) and used at passage 8-16. Cells were plated onto collagen-coated tissue culture flasks and cultured in Dulbecco's modified Eagle's medium (DMEM; Lonza, Slough, UK) supplemented with 10% foetal bovine serum, 5% horse serum, 2mM L-glutamine, 50U/ml penicillin, 50g/ml streptomycin (all Invitrogen) at 37°C in a hum idified 95% air/5% CO 2 standard cell culture incubator.

Plasmid constructs

All reporter plasmids are based on the pGL3 basic vector system (Promega) containing the luciferase reporter gene. A fragment of the rat MRα promoter ranging from -1440 to +90 (designated +1 transcription start of MR exon 1α, Genbank™ accession number NM 013131) originally in pBL luc 5'-1500 (-1400/+117; gift from Dr M. Castren, University of Kuopio, Finland) was subcloned into the pGL3 basic vector (MR-Pα). Promoter fragments of MRβ (-280/+28; MR-Pβ) and MRγ (-1719/+17; MR-Pγ) were amplified from rat genomic DNA by PCR using the primers as shown in Table 1. PCR products were obtained using Pfx polymerase (Invitrogen) under the following cycling conditions: 95°C for 2 min; 30 cy cles of 95°C for 15 sec, 59°C for 30 sec, and 68°C for 30 sec; and extension at 68°C for 20 min, then purified for blunt-end cloning into the SmaI site of the pGL3 basic vector.

Exon-1 specific DNA fragments were amplified following reverse transcription of rat hippocampal total RNA using primers specific for each alternate exon 1 (Table 1). For probe fragments for ribonuclease protection assay (RPA) a common reverse primer complementary to exon 2 was used to amplify each MR variant. PCR products were obtained using Taq polymerase (Promega) under the following conditions: 95°C for 8 min; 30 cycles of 95°C for 1 min; 55°C for 1 min; 72°C for 1 min; and final extension at 72°C fo r 10 min. Purified fragments were then cloned into pGEM-T Easy vector (Promega) and used as templates for the generation of exon-1 specific cRNA probes.

Transient transfection assay

PC12 cells were plated onto collagen type IV-coated 6 well plates and at 50-60% confluency were transiently transfected with 0.5µg promoter insert-containing pGL3 plasmid and 0.5µg of the β-galactosidase expression plasmid pCH110 for normalization of transfection efficiency, using lipofectamine 2000™ (Invitrogen). Cells transfected with the promoterless pGL3 vector served as the baseline control. Transfected cells were differentiated 24 hr later by culturing in serum-free DMEM medium containing 50ng/ml nerve growth factor (NGF, Millipore, Watford, UK). Differentiated cells displayed neurite extensions and tyrosine hydroxylase immunoreactivity and were exposed to cell stress 3 days after NGF exposure.

Cell stress

A c c e p t e d M a n u s c r i p t 5 Transfected PC12 cells and primary cortical cultures were subjected to hypothermia (HYPO, 33°C), oxygen-glucose deprivation (OGD) or hypother mic OGD (OGD/H) for 6 hrs then returned to normal incubation conditions for a further 18 hrs before analysis. To induce HYPO, cells were incubated in a standard tissue culture incubator at 33°C and then returned to 37°C for 18 hrs. For the OGD experiments, cells were washed with glucose-free DMEM medium and incubated with preequilibrated oxygen-and glucose-free medium in a custom made temperature (37°C) and CO 2 (5%)-controlled anaerobic glove box (Coy Laboratories Ltd, Grass Lake, MI, USA) for 6 hrs then allowed to recover under normal conditions of oxygen and glucose for 18 hrs to mimic ischemic reperfusion. For OGD/H experiments, cells were treated as for the OGD experiments except that the temperature in the glove box was set at 33°C. F or experimental controls, cells were subjected to the same procedures but incubated in glucose-containing media under normoxic and normothermic conditions in a standard cell culture incubator. The duration of OGD incubation was initially determined from time course experiments using lactate dehydrogenase (LDH) release as a measure of cell damage: a 6 hr incubation period which caused approximately 20% LDH release (data not shown) was taken to be representative of a sub-lethal challenge. The same time-point was used for OGD/H and HYPO alone although in pilot experiments LDH release was lower after 6 hrs presumably due to a protective effect of hypothermia.

To disrupt signal transduction pathways potentially mediating MR promoter activity, the selective MEK1 inhibitor PD98509 (25µM), JNK inhibitor SP600125 (2µM), p38 inhibitor SB203580 (1-10µM) and cAMP-dependent protein kinase A inhibitor KT 5720 (1µM; all Merck Biosciences, Nottingham, UK) were added to fresh culture medium 2 hr prior to cell stress.

Luciferase and β-galactosidase assays

After exposure to relevant stimuli, culture medium was removed and cells were washed with PBS then lysed in 300µl cell lysis buffer. Cell lysates were centrifuged and 40µl supernatant was assayed in triplicate for luciferase activity using a microplate luminometer (Berthold, Alrewas, UK) with beetle luciferin (Promega) as the substrate. β-galactosidase activity was determined to correct for variation in transfection efficiency using the Tropix/Galacto Light kit (Cambridge Bioscience, Cambridge, UK). Luciferase activity was expressed relative to β-galactosidase activity.

Ribonuclease Protection Assay

Total RNA was isolated from primary cortical cultures by Trizol (Invitrogen) extraction according to the manufacturer's instructions. The concentration and integrity of each RNA preparation was estimated spectrophotometrically and by ethidium bromide staining after agarose gel electrophoresis. Exon 1 specific antisense and sense RNA probes were synthesized from corresponding linearized plasmids (described above) by in vitro transcription (Promega) with T 7 or SP 6 polymerase as appropriate in the presence of [α-32P]-GTP (3000Ci/mmol; GE Healthcare, Amersham, Bucks, UK). The antisense probe of the β-actin gene served as the internal control and was synthesized in the same manner. RPAs were performed using the HybSpeed™ RPA kit (Ambion, Warrington, UK) according to the manufacturer's instructions. Total RNA (20µg) was coprecipitated with 5 x 10 5 cpm riboprobe, resuspended in hybridization buffer and incubated at 68°C for 1 hr. Following RNase digestion (RNAse A/T1 1:25 dilution), protected probes were separated on polyacrylamide/urea gels and quantified by Phosphorimage (FujiFilm FLA-2000) analysis in conjunction with AIDA v2.0 software (Raytek, Sheffield, UK). The quantity of protected probe was normalized with reference to simultaneously hybridized  β-actin mRNA expression.

Neonatal anoxia

Anoxia was induced in newborn Wistar rats of both sexes at postnatal day (PND) 2, as previously described [START_REF] Caputa | Effect of temperature on postanoxia, potentially neurotoxic changes of plasma pH and free iron level in newborn rats[END_REF][START_REF] Rogalska | Stress-induced behaviour in juvenile rats:effects of neonatal asphyxia, body temperature and chelation of iron[END_REF]. Briefly, pups were placed in temperature controlled plethysmographic chambers at either 33°C (normal body temperature for PND 2 rats) or 31°C (hypothermia). Rectal temperatu re was measured and as soon as the desired body temperature was reached, anoxia was induced by flushing 100% nitrogen into the chambers.

A c c e p t e d M a n u s c r i p t 6 Respiratory effort was constantly monitored using the barometric method and animals were maintained under anoxic conditions for 25 min. After anoxia, animals were exposed to atmospheric air at unchanged temperature for 120 min then sacrificed. Brains were dissected and frozen on dry ice. Control rats were placed into the chambers and exposed to atmospheric air over the same period of time (145 min) under the respective temperature conditions.

In situ hybridization

Coronal sections (12µm) of fresh frozen brains were processed for in situ hybridization using 35 S-UTP (GE Healthcare) labelled exon 1 specific RNA probes as described above. Probes were annealed at a temperature of 50°C for 16 hrs a nd sections were washed, RNAse digested, dehydrated and exposed to photographic liquid emulsion (LM-1; GE Healthcare). Slides were developed three weeks later with Kodak 19 developer and hybridization signal was assessed by computer assisted quantitative grain counting (MCID Research Imaging, Canada).

Statistical analysis

All analyses were performed by an investigator blinded to experimental condition. Measurements from individual PC12 and primary cortical preparations were performed in triplicate for each experiment. All data are represented as the mean ± SEM. for the number of experiments indicated. Statistical analyses of experiments were performed by ANOVA followed by post-hoc comparisons using Tukey's multiple test (to compare values against each other) or Bonferoni's comparison test (to compare values against control). Values of p<0.05 were considered statistically significant.

Results

Cell stress stimulates differential expression of variant MR mRNA transcripts

To investigate how injury-induced MR might be regulated we initially used dissociated embryonic cortical neuronal cultures. These cultures express MR at levels approximately half that of hippocampal neuronal cultures but respond to cell stress in a similar manner [START_REF] Macleod | Mineralocorticoid receptor expression and increased survival following neuronal injury[END_REF] and therefore provide a suitable model for the purpose of this study.

The effects of HYPO, OGD or OGD/H on the abundance of the three specific MR mRNA variants MRα, MRβ and MRγ, were determined by ribonuclease protection assay with specific riboprobes targeted to each variant exon 1 and complementary to the first 180 nucleotides of exon 2 (common to all MR mRNA variants). All three MR mRNA variants were detected in primary cortical culture with MRα and MRβ present in roughly equal proportion and MRγ approximately fourfold less abundant (data not shown). Neither HYPO nor OGD altered the expression of the individual MR mRNA variants (Fig. 1). In contrast, OGD/H led to a significant induction of MRβ mRNA levels (p<0.05), but not those of MRα or MRγ.

MR promoter activity in response to cell stress

To examine further the upregulation of MR mRNA following cell stress we prepared luciferase reporter constructs containing the proximal promoter for each MR exon 1 variant and measured transcriptional activity at the promoter level in differentiated pC12 cells in response to HYPO, OGD and OGD/H. Although differentiated PC12 cells do not express endogenous MR, both MRα and MRγ promoters showed significantly greater basal activity compared to pGL3 (p<0.001) whereas MRβ promoter activity was not significantly different to pGL3 (Fig. 2) suggesting at least some of the signal pathways for MR promoter activity are active. Exposure to cell stress led to differential activation of the MR promoters: both MRα (p<0.01) and MRβ (p<0.05) promoter activity was increased by OGD/H but not by OGD or HYPO alone. However we found no effect on MRγ promoter activity. Cell stress per se did not affect β-gal activity (data not shown).

A c c e p t e d M a n u s c r i p t 7

ERK signalling pathway mediates cell stress activation of MR promoter

We previously reported an association between increased MR mRNA and activation of the extracellular signal regulated kinase (ERK) pathway in primary neuronal cultures subjected to cell stress [START_REF] Macleod | Mineralocorticoid receptor expression and increased survival following neuronal injury[END_REF]. To assess the involvement of this signalling pathway in OGD/H induced MRα and MRβ promoter activity, we treated transfected differentiated PC12 cells with the selective ERK pathway inhibitor PD98059 prior to exposure to OGD/H. To test if other components of the mitogen activated kinase (MAPK) pathway were involved in promoter activation, we also separately treated cells with the JNK pathway inhibitor SP600125, the p38 MAPK inhibitor SB203580 or the cAMP dependent protein kinase A (PKA) inhibitor KT5720.

OGD/H-induced MR promoter activation appears to be mediated by specific signalling pathways (Fig. 3): we found no effect of inhibitor on basal MRα activity other than the p38 kinase inhibitor SB203580 which caused an apparent increase in basal MRα activity. However SB203580 did not inhibit OGD/H induced MRα promoter activity implying that p38 kinase is not involved in this activation. Similarly there was no inhibitory effect of KT5720. In contrast, both PD98059 and SP600125 attenuated OGD/H-induced activation of MRα promoter activity to levels comparable to that seen under control conditions (p<0.05) suggesting a requirement for ERK1/2 and JNK signalling. Interestingly PD98059 inhibition reduced OGD/H-induced MRα promoter activity to twofold lower than control. We found no effect of inhibitor on β-gal promoter activity (data not shown).

For the MRβ promoter there was no effect of inhibitor on basal MRβ promoter activity; PD98059 significantly attenuated OGD/H-induced MRβ promoter activity (p<0.001), but SP600125, KT5720 and SB203580 had no effect. This suggests that OGD/H-induced MRβ induction is mediated by ERK1/2 signalling alone (Fig. 3).

Differential regulation of variant MR mRNA transcripts by hypothermia and anoxia in neonatal rat brain

To investigate whether the changes in the expression profiles of the variant MR mRNA transcripts in vitro were reflected in vivo and therefore of biological importance, we used a well characterized neonatal rat model of anoxia where body temperature can be manipulated [START_REF] Caputa | Effect of temperature on postanoxia, potentially neurotoxic changes of plasma pH and free iron level in newborn rats[END_REF][START_REF] Rogalska | Stress-induced behaviour in juvenile rats:effects of neonatal asphyxia, body temperature and chelation of iron[END_REF]. We measured changes in the expression of MR variant transcripts and total MR mRNA in the hippocampus because this region has high basal expression of MR and is particularly vulnerable to ischemia.

Firstly, in situ hybridization using a probe complementary to the coding region which does not distinguish between the MR variants showed that total MR mRNA in neonatal hippocampus under control conditions was most highly expressed in the CA3 and DG with lower expression in CA1, CA2 and CA4 (Fig. 4). Results revealed that total MR mRNA was significantly induced by hypothermia (p<0.05) but not by anoxia in all hippocampal regions except CA4 where total mRNA levels were induced in response to anoxia (p<0.05). However there was no synergistic effect of hypothermia and anoxia (n.s.).

To determine which variant MR transcript might be responsible for the upregulation of total MR mRNA, exon-1 specific probes were used to analyse adjacent brain sections. Under control conditions, all three MR mRNA transcripts were detectable in rat hippocampus at PND 2 with the same regional pattern of expression as seen for total MR mRNA (Fig. 4). Of the three variant transcripts, only MRβ was significantly altered by the experimental procedure: across all regions, the MRβ transcript was significantly increased under hypothermic compared to normothermic conditions (p<0.05) irrespective of oxygen status.

Discussion

Here we show, both in vitro and in vivo, that MR transcripts are differentially regulated in response to injury. Under clinically relevant conditions, the MRβ transcript is specifically upregulated in neurons via the MRβ promoter, an effect which appears to be mediated through the ERK1/2 pathway and which appears not to involve other MAP kinase pathways. We found no changes in MRγ and while MRα promoter activity was enhanced by OGD/H exposure in A c c e p t e d M a n u s c r i p t 8 differentiated PC12 cells, this was not associated with elevated transcript levels either in vitro or in vivo. To our knowledge this is the first demonstration of the differential regulation of alternate MR promoters in response to cell stress and implicates the MRβ promoter as a major regulatory point for MR mRNA expression in response to cell injury.

Variation in expression and differential regulation of the MR transcripts has previously been reported to occur during brain development where both the MRβ and MRγ are expressed during development but decline in adulthood [START_REF] Vazquez | Alpha, beta, gamma mineralocorticoid receptor messenger ribonucleic acid splice variants: differential expression and rapid regulation in the developing hippocampus[END_REF]. Cellular responses to brain injury often appear to recapitulate events that occur during development [START_REF] Chen | Differential expression of cell fate determinants in neurons and glial cells of adult mouse spinal cord after compression injury[END_REF] and so activation of the MRβ promoter with increased MRβ expression is a plausible response to injury. Our data suggests that this occurs only in response to specific cell stressors. Mild to moderate hypothermia is generally held to have modest effects under normal conditions but the combination of hypothermia and ischemia generally leads to a reduction in the extent of ischemic damage [START_REF] Buslo | Post-ischemic moderate hypothermia inhibits CA1 hippocampal ischemic neuronal injury[END_REF][START_REF] Green | Protective effect of brain hypothermia on behaviour and histopathology following global cerebral ischemia in rats[END_REF][START_REF] Kollmar | Neuroprotective effect of delayed moderate hypothermia after focal cerebral ischemia: an MRI study[END_REF][START_REF] Van Der Worp | Hypothermia in animal models of acute ischemic stroke: a systematic review and meta-analysis[END_REF]. While the exact mechanism by which hypothermia exerts this protective effect is not clear, changes in gene expression have been reported [START_REF] Ohta | Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia[END_REF][START_REF] Kobayashi | Global profiling of influence of intra-ischemic brain temperature on gene expression in rat brain[END_REF]. Our present findings support an important role for hypothermia-induced changes in gene expression and also corroborate our previous observations of increased hippocampal total MR mRNA expression in adult rats subjected to hypothermic but not normothermic transient global cerebral ischemia [START_REF] Macleod | Mineralocorticoid receptor expression and increased survival following neuronal injury[END_REF] where blockade of MR exacerbated ischemic damage.

Therapeutic cooling is used in the management of hypoxic-ischemic encephalopathy in neonates (Hoehn et al., 2008) but to date the effects in adult ischemic stroke or traumatic brain injury have yet to be established. Importantly there may be age-dependent differences in the response to hypothermia. Indeed, the regulation of MR appears to be different in neonatal rat brain where MR expression is increased, again via the MRβ transcript, in response to hypothermia but not to anoxia. In contrast to primary cortical culture, there was no significant increase in MRβ expression with hypothermic anoxia compared with hypothermia alone. The lack of an injury (anoxia) induced increase in MR may be due to the fact that normal body temperature in neonates is 33°C and this in itself may be an adaptive respo nse to take advantage of protective responses occurring at this temperature. Indeed we saw little evidence of neuronal damage in normothermic anoxia in contrast to our observations in the adult rat [START_REF] Macleod | Mineralocorticoid receptor expression and increased survival following neuronal injury[END_REF] and human brain (Lai et al., 2008) where such changes are clearly seen, and may explain why MR is not additionally induced by anoxic hypothermia. Intriguingly we have also observed MRβ transcript unregulation in hyperthermic (39°C) neonatal rat brain [START_REF] Rogalska | Effect of hyperthermia and anoxia on glucocorticoid and mineralocorticoid receptor expression in neonatal rat hippocampus[END_REF] in the absence of any effect on neuronal viability and the most plausible explanation is that MR induction occurs as an endogenous response to thermal stress to protect the brain from subsequent injury. Confirmation of specific MRβ upregulation in the adult brain needs to be sought but nonetheless, these data lend further support our hypothesis that increased MR expression forms a component of the response to hypo-(and hyper-) thermia and that MRβ is the major driver of this response.

We investigated further the upstream regulation of injury-induced MRβ promoter activity and found involvement of the ERK member of the MAP kinase signalling pathway. ERK signalling is primarily activated by mitogens such as growth factors, phorbol esters and serum [START_REF] Rossler | Neuroprotection of immortalized hippocampal neurons by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extra-cellular signal regulated protein kinase and phosphatidylinositol 3-kinase[END_REF][START_REF] Hetman | Neuroprotection by brain derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase[END_REF][START_REF] Gause | Effect of phorbol ester on mitogen activated protein kinase kinase activity in wild type and phorbol ester resistant EL4 thymoma cells[END_REF][START_REF] Whitmarsh | Intergration of MAP kinase signal transduction pathways at the serum response element[END_REF] but can also be stimulated by various cell stressors [START_REF] Lewis | Signal transduction through MAP kinase cascade[END_REF][START_REF] Ji | Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity[END_REF]. Phosphorylated ERK is found to a greater degree in the penumbra than in the ischemic core [START_REF] Irving | Differential activation of MAPK/ERK and p38/SAPK in neurons and glia following focal cerebral ischemia in the rat[END_REF][START_REF] Ferrer | Signaling of cell death and cell survival following focal cerebral ischemia, life and death struggle in the penumbra[END_REF] and crucially is present at much higher levels in ischemia resistant regions of the hippocampus [START_REF] Gu | Extracellular signal-regulated kinase 1/2 activation in hippocampus after cerebral ischemia may not interfere with post-ischemic cell death[END_REF][START_REF] Zablocka | Opposite reaction of ERK and JNK in ischemia vulnerable and resistant regions of hippocampus: involvement of mitochondria[END_REF]. Hypothermic neuroprotection also coincides with ERK activation [START_REF] Hicks | Hypothermia differentially increases extracellular signal-regulated and stress activated protein kinase/c-Jun terminal kinase activation in the hippocampus during reperfusion after asphyxial cardiac arrest[END_REF] and taken together this body of evidence generally supports a protective role for ERK signalling. Furthermore in silico analysis shows that there is a putative binding site for the ERK downstream effector, Elk-1, within the MRβ promoter. Interestingly, it has been shown that injury enhances Elk-1 phosphorylation [START_REF] Ferrer | Active, phosphorylation-dependent MAP kinases MAPK/ERK, SAPK/JNK and p38 and specific transcription factor substrates are differentially expressed following systematic administration of kainic acid to the adult rat[END_REF]. Our present findings suggest that ERK activation favours neuronal survival through enhancing MR expression. Although we previously showed that blockade of ERK inhibited injury-induced total MR mRNA expression in primary neuronal culture, we have yet to confirm that this is due to a reduction in MRβ transcript expression. Whether Elk-1 plays a role in regulating MRβ remains to be investigated.

9

  In summary, we provide evidence for specific transcriptional regulation of the MR gene by different conditions of neuronal stress. Therapeutic hypothermia during ischemia increases MR, primarily via an increase in the transcription of MRβ, which enhances survival in injured neurons. Since hypothermia is difficult to deliver clinically, targeted upregulation of MRβ in neurons may provide a suitable target for future therapeutic strategies in conditions characterized by cerebral ischemia including stroke and cardiac arrest.

  Figure LegendsFig. 1. Effect of cell stress on variant MR mRNA expression in rat primary cortical culture. (A) Schematic representation of the rat MR gene and its mRNA transcripts. The MR gene consists of 11 exons including three untranslated first exons, 1α, 1β and 1γ (hatched boxes) and 8 translated exons (2-9; white boxes. Each variant MR transcript comprises an untranslated exon spliced to exon 2. The black lines represent the location of each exon 1specific probe. Relative abundance of each variant MR mRNA following (B) hypothermia (HYPO), (C) oxygen-glucose deprivation (OGD) or (D) a combination of OGD and hypothermia (OGD/H). Results (black bars) are expressed as percent change of untreated controls (white bars). Data represent mean ± SEM of six independent experiments, with three replicates per experiment. *P<0.05 OGD/H vs non-treated control.

Fig. 2 .

 2 Fig. 2. Effect of cell stress on variant MR promoter activity in differentiated rat PC12 cells. (A) Schematic representation of the MR promoter constructs used in the luciferase reporter assays (B) Relative luciferase activity under basal conditions for each MR variant promoter relative to pGL3. Relative luciferase activity was calculated for cells exposed to (C) HYPO, (D) OGD or (E) OGD/H (black bars) and expressed as a percent change of untreated controls (white bars). Data represent mean ± SEM of six independent experiments, with three replicates per experiment. *P<0.05, **P<0.01 vs pGL3 or untreated control.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Comparison of OGD/H-induced MRα promoter activity in the presence of MAP kinase pathway inhibitors in differentiated rat PC12 cells. The effect of each inhibitor on (A) MRα and (B) MRβ promoter activity expressed as relative luciferase activity either in the presence (black bars) or absence (white bars) of OGD/H. Data represent mean ± SEM of seven independent experiments, with three replicates per experiment. For MRα promoter: *P<0.05 vs OGD/H control; #P<0.05 vs stressed vehicle control. For MRβ promoter: *P<0.001 vs OGD/H control; #P<0.001 vs stressed vehicle control Fig. 4. Effect of hypothermic anoxia on variant MR mRNA expression in neonatal rat hippocampus. Quantitative analysis of (A) MRα, (B) MRβ, (C) MRγ and (D) total MR transcript abundance by grain counting under: normothermic/normoxic (white bars); normothermic/anoxic (hatched bars); hypothermic normoxic (black bars) and hypothermic anoxic (shaded bars) conditions. Data represent the mean ± SEM with six animals per group. *P<0.05, **P<0.01, ***P<0.001. Representative autoradiographic images showing distribution of MR (E, F); MRβ (G, H); MRγ (I, J) and total MR mRNA (K, L) under normothermic/normoxic (E, G, I, K) or hypothermic/normoxic conditions (F, H, J, L).

  Figure 1

  Figure 4

Table 1 .

 1 Primer sequences used for PCR amplification of specific DNA sequences

		Forward Primer	Reverse Primer
	MR-P	5'TGCACTGGAGTTCCGTTTCC	5'CGGCGATAGAGAGTGGTTAG
	MR-P	5'ACCTTGAGCCCTGTGTTTGG	5'GAAGACCAAGAGCGGACTAG
	MR (RPA)	5'AGGAAGAGAGCCAACTTCAGGCTG	5'GTACTGTTGTTCGGAATAGCACCG
	MR	5'AGCCTCCCTAACATGTCCTAG	5'GTACTGTTGTTCGGAATAGCACCG
	MR	5'TCCTAGTCCGCTCTTGGTCTTC	5'GTACTGTTGTTCGGAATAGCACCG
	MR (in situ)	5'CGGGAGAAGAGAGCCAACTT	5'GTCGTCCTCTCGCCGTCTAC
	MR	5'GGGTCTTACCGCTCGACTG	5'AGTCGCTGCACTCACCTTTT
	MR	5'TTCCCTCCTAGTCCGCTCTT	5'ACGCACCCTGTATGCTTCTC

Table 1

 1

Acknowledgements

We thank Ms Jessie Hong for excellent technical assistance. This work was supported by the Cunningham Trust and Research into Ageing. ML is a holder of a Research into Ageing Personal Fellowship. MB was a recipient of a British Neuroendocrinology Society Undergraduate Summer Vacation studentship.