Pharmacologic blockade of nicotinic receptors in the suprachiasmatic nucleus increases ovarian atresia and inhibits follicular growth.

Reproduction in all mammalian species depends on the growth and maturation of ovarian follicles, that is, folliculogenesis. Follicular development can culminate with the rupture of mature follicles and the consequent expulsion of their oocytes (ovulation) or in atresia, characterized by the arrest of development and eventual degeneration. These processes are regulated by different neuroendocrine signals arising at different hypothalamic nuclei, including the suprachiasmatic nucleus (SCN). In the later, the activation of muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs) by acetylcholine is essential for the regulation of the pre-ovulatory signals that stimulate the rupture of mature follicles. To evaluate the participation of the nAChRs in the SCN throughout the oestrous cycle in the regulation of the hypothalamic-pituitary-ovarian axis. For this purpose, 90-day-old adult female rats in metoestrus, dioestrus, proestrus or oestrus were microinjected into the left- or right-SCN with 0.3 µL of saline solution as vehicle or with 0.225 µg of mecamylamine (Mec), a non-selective antagonist of the nicotinic receptors, diluted in 0.3 µL of vehicle. The animals were sacrificed when they presented vaginal cornification, indicative of oestrus stage, and the effects of the unilateral pharmacological blockade of the nAChRs in the SCN on follicular development, ovulation and secretion of oestradiol and follicle-stimulating hormone (FSH) were evaluated. The microinjection of Mec decreased the serum levels of FSH, which resulted in a lower number of growing and healthy follicles and an increase in atresia. The higher percentage of atresia in pre-ovulatory follicles was related to a decrease in the number of ova shed and abnormalities in oestradiol secretion. We also detected asymmetric responses between the left and right treatments that depended on the stage of the oestrous cycle. The present results allow us to suggest that during all the stages of the oestrous cycle, cholinergic signals that act on the nAChRs in the SCN are pivotal to modulate the secretion of gonadotropins and hence the physiology of the ovaries. Further research is needed to determine if such signals are generated by the cholinergic neurons in the SCN or by cholinergic afferents to the SCN.

Unilateral lesion of the suprachiasmatic nucleus impairs estradiol feedback, follicular development, estrous cycle and ovulation.

In brief: The SCN regulates ovulation by stimulating the preovulatory surge of gonadotropins. This study revealed an additional role in the sensitization of the hypothalamus to estradiol that changes along the estrous cycle and the side of the nucleus. Abstract: Ovulation is timed by neural signals originating at the suprachiasmatic nucleus (SCN) that trigger ovulation when converge with high estradiol levels, which indicates the maturation of ovarian follicles. We have shown that the hypothalamic regulation of ovulation is asymmetrical and we hypothesized that the paired SCN could contribute to such symmetries. We unilaterally lesioned the SCN of rats at each stage of the estrous cycle and evaluated the acute effects on the progression of their estrous cycle, follicular development and ovulation. Lesions prevented progression of the estrous cycle when performed in estrus/metestrus but not in diestrus/proestrus. Abnormalities in follicular development were observed in the nonovulating lesioned rats and this was independent of the side of the SCN destroyed and the stage of the cycle when surgery was performed. Groups of lesioned rats were then hormonally primed with GnRH or estradiol to assess the neuroendocrine pathway altered by the treatment. GnRH restored ovulation, suggesting that both SCN are needed for proper triggering of the preovulatory surge of GnRH and that unilateral lesion does not impair the sensitivity of the pituitary or the ovary to GnRH and gonadotropins, respectively. With regard to restoring ovulation, estradiol was asymmetrically effective in rats lesioned in estrous, partially effective in rats operated at diestrus and ineffective in rats at metestrus. Our results indicate that the SCN regulates the activity of the hypothalamic-pituitary-ovarian axis not only by modulating the preovulatory surge of GnRH/gonadotropins but also by promoting the hypothalamic integration of estrogenic signals from the ovaries in an asymmetric and stage-dependent fashion.

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

The content of gonadotropin-releasing hormone (GnRH), kisspeptin, and estrogen receptors (ERα/ERß) in the anteromedial hypothalamus displays daily variations throughout the rat estrous cycle.

The content of gonadotropin-releasing hormone (GnRH), its mRNA, and estrogen receptor alpha (ERα) and beta (ERß) in the hypothalamus varies throughout the estrous cycle. Furthermore, the abundance of these molecules displays asymmetry between the right and left side. In the present study, we investigated the changes in the content of ERα, ERß, kisspeptin, and GnRH by western blot in the left and right anteromedial hypothalamus, at four different times during each stage of the rat estrous cycle. The serum levels of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were also measured. ERα and ERß levels changed depending on the stage of the estrous cycle, meanwhile that of kisspeptin was modified according to both the hour of the day and the stage of the cycle. Except in estrus day, ERß was higher in the right hypothalamus, while ERα was similar in both sides. During both proestrus and estrus, the content of kisspeptin and GnRH was higher in the right hypothalamus. The highest levels of FSH and LH occurred at 17:00 h of proestrus. But at estrus, the highest FSH levels were observed at 08:00 h and the lowest at 17:00 h. Thus, the current results show that the content of ERα, ERß, kisspeptin, and GnRH in the anteromedial hypothalamus are regulated as a function of the stage of the estrous cycle and the hour of the day. Furthermore, the content of these proteins is regularly higher in the right anteromedial hypothalamus, regardless of the stage of the cycle or time of the day.

Clock control of mammalian reproductive cycles: Looking beyond the pre-ovulatory surge of gonadotropins.

Several aspects of the physiology and behavior of organisms are expressed rhythmically with a 24-h periodicity and hence called circadian rhythms. Such rhythms are thought to be an adaptive response that allows to anticipate cyclic events in the environment. In mammals, the circadian system is a hierarchically organized net of endogenous oscillators driven by the hypothalamic suprachiasmatic nucleus (SCN). This system is synchronized by the environment throughout afferent pathways and in turn it organizes the activity of tissues by means of humoral secretions and neuronal projections. It has been shown that reproductive cycles are regulated by the circadian system. In rodents, the lesion of the SCN results on alterations of the estrous cycle, sexual behavior, tonic and phasic secretion of gonadotropin releasing hormone (GnRH)/gonadotropins and in the failure of ovulation. Most of the studies regarding the circadian control of reproduction, in particular of ovulation, have only focused on the participation of the SCN in the triggering of the proestrus surge of gonadotropins. Here we review aspects of the evolution and organization of the circadian system with particular focus on its relationship with the reproductive cycle of laboratory rodents. Experimental evidence of circadian control of neuroendocrine events indispensable for ovulation that occur prior to proestrus are discussed. In order to offer a working model of the circadian regulation of reproduction, its participation on aspects ranging from gamete production, neuroendocrine regulation, sexual behavior, mating coordination, pregnancy and deliver of the product should be assessed experimentally.

A neural circadian signal essential for ovulation is generated in the suprachiasmatic nucleus during each stage of the oestrous cycle.

NEW FINDINGS: What is the central question of this study? Is the suprachiasmatic nucleus the structure that generates the neural circadian signals that occur during every stage of the oestrous cycle, not only pro-oestrus, and are these signals essential for proper regulation of ovulation? What is the main finding and its importance? Transient inhibition of Na+ -dependent action potentials in the suprachiasmatic nucleus by tetrodotoxin microinjection at 14.00 h inhibits ovulation irrespective of the stage of the oestrous cycle at which the procedure is performed. Microinjection of saline solution into the suprachiasmatic nucleus has a disruptive effect on ovulation that depends on the stage of the cycle at which it is administered. ABSTRACT: Reproduction is a highly timed process that depends on both the reproductive and circadian systems. The core oscillator of the latter resides at the suprachiasmatic nuclei (SCN) and it is pivotal for the regulation of the pro-oestrus pre-ovulatory surge of gonadotropins in females. There is evidence to suggest that this system may be involved in the regulation of neuroendocrine events that are essential for ovulation and that occur prior to pro-oestrus. We explored this possibility by transiently inactivating the SCN. Female rats were implanted with guide cannulas aimed at the SCN. After recovery of the oestrous cycle, animals were injected with tetrodotoxin (TTX), artificial cerebrospinal fluid (ACSF) or saline solution while freely moving. Injections were performed at 14.00 h of each stage of the oestrous cycle. Animals were killed on the next predicted oestrus day, the number of ova shed was counted and intact rats at oestrus stage were used as absolute control. ACSF did not modify ovulation. Saline solution blocked ovulation in oestrus- and dioestrus-injected rats. Irrespectively of the stage of the oestrous cycle, TTX blocked ovulation. These results lead us to suggest that a neural circadian signal, pivotal for triggering the gonadotropin pre-ovulatory surge, arises from the SCN during the critical window of pro-oestrus. We also suggest that a similar signal, needed for the regulation of other events that are indispensable for proper regulation of ovulation, is also generated in this nucleus during the other stages of the cycle at a similar time.