<br/>The influence of stress on reproductive functions has been subject of much research. Various kinds of stress are known to affect reproductive functions. In females, the complex regulation of the ovarian cycle relies on a series of neuroendocrine events whose temporal relationship is so critical that any disruption in the orderly sequence may jeopardize reproductive success. During the oestrous cycle, especially the mechanisms which induce oestrous behaviour, the LH and FSH surge and ovulation seem to be vulnerable to stress. The evidence that stress influences reproductive functions is mainly based on studies performed with intact and castrated male rats, and with ovariectomized and ovariectomized, oestrogenprimed female rats, and mainly concern the effect of stress on basal LH secretion. The hormonal status of these animal models differs essentially from those of intact pro-oestrous female rats, and this can of course be of influence on the response to the stressor. However, only a few studies have been performed on the effects of stress on the surge of gonadotropins in the intact cyclic female rat. This thesis focusses on the effect of restraint stress on the surge of gonadotropins and ovulation in the intact cyclic female rat and the underlying mechanisms.<p>In <strong>Chapter 1</strong> a review is given of stress and reproductive functions. In addition, possible pathways via which stress may influence reproduction are briefly discussed.<p><strong>Chapter 2</strong> describes experiments on the effects of different periods of restraint stress on preovulatory LH and FSH surge profiles and ovulation in the intact cyclic female rat. In literature, the few studies performed with intact female rats have shown that: 1) the LH surge and subsequent ovulation are suppressed by footshock stress , and that ovulation is inhibited by immobilization stress ; 2) the gonadotropin surge can be delayed by one day if on pro-oestrus the surge is blocked, and 3) a partially suppressed LH surge can induce alterations in the ovulation process, since treatment of rats of which the endogenous LH surge is blocked with a small amount of LH on the morning of pro-oestrus can cause meiotic resumption and induce precocious follicle luteinization. In the experiments described in chapter 2, groups of pro-oestrous rats were subjected to restraint which was started 0, 1 or 2 h before the presumed onset of the LH surge and continued during the surge until the beginning of the dark period, thereby covering most of the period during which the surge appears. The presumed onset of the LH surge is approximately 2 h after the middle of the light period. Exposure to restraint resulted in a partial or total inhibition of the LH and FSH surge (no significant difference between the three restraint groups). In rats with a partially suppressed LH surge, ovulation occurred or the ovaries contained unaffected graafian follicles on the day following the stress. In restraint rats with a completely blocked LH surge (≤detectable levels), LH and FSH levels were not elevated the next day, indicating that the surge of gonadotropins had not been delayed by one day. The ovaries of these rats indeed contained unaffected graafian follicles. The results of this study indicate that, during pro-oestrus, restraint stress suppresses (and does not delay) the release of preovulatory gonadotropins in the intact female rat. Partial suppression of the LH surge by restraint induces ovulation or ovulation had not occurred and the graafian follicles were unaffected.<p>The following chapters are focused on the central mechanisms involved in the inhibition of the pro-oestrous surge of gonadotropins by restraint stress. An interaction of hormones and neuropeptides of the HPA axis (e.g. CRH, AVP and βEND) with those of the HPG axis (e.g. GnRH, LH and FSH) has been suggested. It has been reported that central administration of CRH (the major neuromodulator of the HPA axis) to male or ovariectomized female rats results in a suppression of LH-, but not of FSH-release. In addition, prolonged administration of CRH can cause downregulation of CRH receptors which may alter its effect on the LH and FSH surge. Therefore, <strong>Chapter 3</strong> deals with the effects of central administration of CRH on the pre-ovulatory surge of LH and FSH in the intact female rat. An icv injection (bolus) or infusion (6-h) with CRH resulted in an inhibition of the LH surge and to a lesser degree, the FSH surge. The inhibition of pro-oestrous LH surge levels lasted 3-4 h; subsequently LH increased to control surge levels. A 9-h infusion of CRH which was started 4 h before the presumed onset of the surge, however, did not affect the surge of LH and FSH. This indicates that CRH can inhibit the LH surge (and to a lesser extent the FSH rise) for only 3-4 h and that thereafter adaptive mechanisms are activated that restore LH secretion to surge levels even in the presence of CRH. Inhibition of LH by CRH can occur via activation of the endogenous opioid βEND, and AVP may mediate the CRH-induced βEND release. Therefore, we investigated if the inhibitory effect of CRH on the LH and FSH surge was modified by icv pretreatment with an AVP-antiserum. Pretreatment (icv) with AVP-antiserum did not prevent but, on the contrary, prolonged the inhibitory effect of CRH on the pro-oestrous LH surge. Pretreatment with AVP-antiserum before the start of the 9-h CRH infusion resulted in a suppression of the LH surge. AVP-antiserum alone did not influence pro-oestrous LH surge levels. These results indicate that AVP-antiserum pretreatment potentiates the inhibitory effect of CRH on the pro-oestrous LH surge. Therefore, we suggest that during high central levels of CRH, - as are obtained after icv injection and may occur during stress - AVP pathways become activated that mitigate the suppression of LH release.<p>In <strong>Chapter 4</strong> experiments are described in which we investigated the role of endogenous CRH and AVP in the inhibitory effect of restraint stress on the pro-oestrous LH surge. Many behavioural and physiological effects observed during stress can be mimicked by central administration of CRH, and CRH release is observed during stress. Additionally, in chapter 3 we showed that central administration of CRH can inhibit the LH surge in the intact cyclic female rat. A possible involvement of endogenous CRH in the restraint-induced inhibition of the LH surge was investigated by icv administration of the CRH antagonist α-helical CRH before application of restraint stress. In pro-oestrous rats, pretreatment with α-helical CRH did not affect the induced suppression, whereas the restraint-induced corticosterone response was partially prevented by both doses of the antagonist. These data suggest that CRH pathways activated during restraint stress do not mediate the inhibitory effect on LH surge levels. As suggested before, high central CRH levels may activate AVP pathways and several studies suggest some role of AVP in the control of LH secretion during stress. Therefore, we pretreated pro-oestrous rats with AVP-antiserum and then subjected them to restraint stress. In control rats, restraint stress partially inhibited the LH surge. Pretreatment (icv) with AVP-antiserum potentiated this inhibition. This result indicates that the residual surge seen in control rats is the result of a stimulatory AVP neuronal pathway activated by restraint stress. In an additional experiment, iv treatment with the GnRH agonist Ovalyse <sup><font size="-2">®</font></SUP>during restraint resulted in a steep and transient rise of LH levels, indicating that the pituitary was not rendered refractory to GnRH.<p>During the oestrous cycle opioids tonically inhibit secretion of gonadotropins through inhibition of GnRH release, except for the period during the preovulatory surge of gonadotropins. Morphine or βEND administration before the onset of the LH surge can inhibit the spontaneous or steroid-induced LH surge. Blockade of opioids receptors with the antagonist naloxone before the presumed onset of the LH surge, induces a premature surge. Several studies have suggested a role for opioids in the effects of stress on reproductive functions (inducing the inhibitory effects on LH secretion) but only a few have used intact female rats . In <strong>Chapter 5</strong> experiments are described which investigate the role of opioids in the restraint-induced inhibition of the LH surge in the intact cyclic female rat. The aim of our experiments was to distinguish between the putative role of opioids in mediating the inhibitory effect of restraint on the LH surge and their role in the cyclic regulation of LH secretion. Repeated iv injections with the opioid antagonist naloxone during restraint did not affect the restraint-induced suppression of the pro-oestrous LH surge, neither did pretreatment with naloxone or naltrexone, a longer acting opioid antagonist. This indicates that opioids are not critically involved as mediators in the restraint-induced suppression of the LH surge in the intact female rat. Pretreatment with naltrexone did induce a premature rise of LH levels, indicating, in line with results from others, that there is an inhibitory tone of endogenous opioids at that time, and that withdrawal of this inhibition can lead to initiation of an LH surge.<p>GABA is a major inhibitory neurotransmitter in the brain, and under non-stress conditions it is involved in the regulation of GnRH release. For instance, it appears to play a role in the negative feedback action of oestrogen on the GnRH release . Additionally, there is evidence that stress can alter GABAergic activity in the brain. Yet, a relationship between the stress effects on reproductive functions and GABAergic activity has not been established. In <strong>Chapter 6</strong> we investigated the role of GABA in the restraint-induced inhibition of the preovulatory LH surge. First, we determined the effect central GABA mechanisms on the prooestrous LH secretion. Indeed, icv administration of the GABA <sub>A</sub> receptor agonist muscimol as well as the GABA <sub>B</sub> receptor agonist baclofen inhibited the LH surge. These data accord with studies obtained by others in ovariectomized and ovariectomized/steroid-primed female rats. To investigate the conceivable involvement of GABA in the inhibitory effect of restraint on the LH surge, prooestrous rats were given an icv injection with the GABA <sub>A</sub> receptor antagonist bicuculline, or with the GABA <sub>B</sub> receptor antagonist phaclofen, and were subsequently subjected to restraint stress. Neither of these pretreatments, however, prevented the restraint-induced suppression of the LH surge. These data suggest that activation of GABA receptors does likely not mediate the inhibitory effect of restraint on the LH surge of the intact female rat.<p>In summary, the experiments performed with intact cyclic female rats described this thesis yielded the following conclusions;<br/>- restraint stress during pro-oestrus can lead to (partial or complete) suppression of the release of preovulatory gonadotropins. Even when the surge is largely suppressed, the residual LH surge is sufficient to induce ovulation; in case of complete suppression, the graafian follicles remain unaffected and ovulation does not occur.<p>- exogenous CRH can inhibit the pro-oestrous LH surge (and to a lesser extent the FSH surge), but this effect is only temporary even when prolonged icv infusions with high doses are given ("escape" after 3-4 h). Pretreatment with AVP-antiserurn prolongs the inhibitory effect of CRH on LH.<p>- exogenous CRH can activate a vasopressinergic mechanism that counteracts the inhibitory effects of CRH on the surge, and underlies the "escape".<p>- during restraint stress CRH pathways are activated that result in a corticosterone response, but appear not to be critically involved in the restraint-induced inhibition of the pro-oestrous LH surge.<p>- during restraint stress, a vasopressinergic mechanism is activated in the brain, that apparently stimulates LH secretion and can sustain an LH surge.<p>- endogenous opioids do likely not mediate the effect of restraint. Yet, an inhibitory opioid tone exists (also under non-stress conditions) in the period preceding the LH surge. Withdrawal of this tone can yield a premature surge.<p>- pharmacological stimulation of GABA receptors in the brain can inhibit the LH surge in the intact cyclic female rat via GAB <sub>A</sub> and GABA <sub>B</sub> receptors. GABA receptors, however, appear not to be involved in the inhibitory effect of restraint stress on the pro-oestrous LH surge of the intact female rat.