The differential sensitivity of the hypothalamic-hypophysial-ovarian axis to 5-hydroxytryptophan alters the secretion of estradiol.

Serotonin [5-hydroxytryptamine (5-HT)] modulates ovarian function. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP), has been used to treat depression. However, the effects of 5-HTP on ovarian and reproductive physiology remain unknown. In this research, we analysed the impact of 5-HTP on the monoaminergic system and its interactions with the reproductive axis and ovarian estradiol secretion when administered by distinct routes. Female rats 30 days of age were injected with 5-HTP i.p. (100 mg/kg), into the ovarian bursa (1.5 µg/40 µL) or into the median raphe nucleus (20 µg/2.5 µL) and were killed 60 or 120 min after injection. As controls, we used rats of the same age injected with vehicle (0.9% NaCl). Monoamine, gonadotrophin and steroid ovarian hormone concentrations were measured. The injection of 5-HTP either i.p. or directly into the ovarian bursa increased the concentrations of 5-HT and the metabolite 5-hydroxyindole-3-acetic acid in the ovary. For both routes of administration, the serum concentration of estradiol increased. After i.p. injection of 5-HTP, the concentrations of luteinizing hormone were decreased and follicle-stimulating hormone increased after 120 min. Micro-injection of 5-HTP into the median raphe nucleus increased the concentrations of 5-HT in the anterior hypothalamus and dopamine in the medial hypothalamus after 120 min. Our results suggest that the administration of 5-HTP either i.p. or directly into the ovarian bursa enhances ovarian estradiol secretion.

Neural activation associated with maternal and aversive interactions with pups in the Mongolian gerbil (Meriones unguiculatus).

According to approach-avoidance model, virgin female laboratory rats display maternal behaviour when the tendency to approach and interact with the pup is stronger than avoiding it. A positive neural mechanism that includes the medial preoptic area (mPOA)/bed nucleus of the stria terminalis (BNST) and a negative mechanism that involves the anterior hypothalamic nucleus (AHN)/ventromedial nucleus (VMN)/ periaqueductal grey (PAG) underlie to these behaviours. Unlike virgin rats, which avoid the pups, virgin females Mongolian gerbils (Meriones unguiculatus) can be immediately either maternal or aggressive with the pups. Furthermore, the Mongolian gerbil is monogamous and biparental species. Despite these difference, we hypothesised that maternal and aggressive interaction with the pups could activate mPOA/BNST and AHN/VMH/PAG, respectively, and that maternal response could be associated with high concentrations of estradiol (E2). Twenty virgin maternal females and 20 aggressive toward the pups were selected. Ten maternal females interacted with the pups (MAT-pups) and 10 with candy (MAT-candy). Of the 20 aggressive females, 10 interacted with the pups (AGG-pups) and 10 with candy (AGG-candy). Immediately after the test, blood samples were taken to quantify E2. The brains were dissected for c-Fos immunohistochemistry. MAT-pups females had significantly higher activation in mPOA/BNST than MAT-candy females, while AGG-pups showed significant activation in AHN/VMH/PAG compared with AGG-candy females. The maternal response was associated with high concentrations of E2. These results suggested a positive and a negative mechanism in the regulation of maternal behaviour in the Mongolian gerbil, and that the immediate maternal response could be due to high E2 concentrations.

Commercial pesticides for urban applications induced population growth and sub-cellular alterations in Raphidocelis subcapitata (Chlorophyceae) at concerning environmental concentrations.

Information regarding the safety and environmental risks of pesticides intended for urban use remains limited. This study aimed to assess the effects of four common pesticides on the microalga Raphidocelis subcapitata: DIAZINON® 25% C. E., Roundup®, URBACIN® 20C. E., and VAPODEL® 20% C. E., which are commercial formulations of diazinon, glyphosate, dichlorvos, and cypermethrin, respectively. According to 96-h inhibition of population growth bioassays, the four pesticide toxicities exemplified the following order: DIAZINON® (diazinon) > Roundup® (glyphosate) > VAPODEL® (dichlorvos) > URBACIN® (cypermethrin). Increasing pesticide concentrations elicited alterations in the specific growth rates (µmax). The macromolecule contents and photosynthetic pigments increased in groups exposed to the highest concentrations of DIAZINON® 25%, Roundup®, and URBACIN® 20 compared to the control group, despite these treatments inducing lower population growth rates. VAPODEL® 20% induced higher growth rates and lower macromolecule content compared to the control. Since active ingredients were not quantified, certain comparisons may prove limiting, but it is important to assess the effects of the whole mixtures in the form that they enter the environment, especially for urban-intended applications or generic formulations with higher additive contents. Finally, this study demonstrated that commercial pesticide formulations designed for urban applications might pose a threat to freshwater microalgae due to their underestimated toxic potential, but further studies are required.

Acute effects of para-chloroamphetamine on testosterone and markers of apoptosis in seminiferous epithelium of prepubertal male rats.

Serotonin is a neurotransmitter that affects the secretion of gonadotropins and testosterone. In prepubertal male rats, serotonin has a stimulating role in testosterone secretion. Here, we used prepubertal male rats to study the effects of para-chloroamphetamine (pCA) on circulating testosterone and gonadotropins and markers of apoptosis in germ cells from day 1 to day 5 post-treatment. The intraperitoneal administration of pCA induced a significant reduction in concentrations of hypothalamic serotonin and circulating testosterone, but gonadotropins were not affected. In the seminiferous epithelium of pCA-treated rats, increased the number of germ cells positive to markers of apoptosis, concomitantly with alterations in morphometry and the presence of multinucleated germ cells. Levels of testosterone were reduced starting from 1 day after pCA was administered. The time window between the administration of the pCA and collection of samples was sufficient to detect changes in testosterone levels, in contrast with a previous work where no changes were found. There was a possible relationship between the reduction of testosterone and an increase in the number of germ cells positive to apoptosis markers. However, the mechanism that links pCA-testosterone-germ cell positive to markers of apoptosis is unknown. Our outcomes support the view that pCA exposure during the prepubertal stage has an acute impact on testosterone levels and affects the structure and physiology of seminiferous epithelium.

Feeding behavior of early life stages of the zebrafish Danio rerio is altered by exposure to glyphosate.

Glyphosate levels and the transfer of glyphosate across trophic levels have rarely been studied in zooplankton. The food preferences of zebrafish during the first-feeding stage (which is critical for the survival of organisms), were analyzed because of the requirement for live food. Larval survival begins to be affected when glyphosate intake exceeds 0.3666 µg/larvae/day, in the case that only the food is contaminated; if the medium is also contaminated, the effects on survival start from 0.2456 µg/larvae/day. It was shown that glyphosate was more likely to be incorporated through the medium than through the food (zooplankton), which supports the results of previous studies that have ruled out the potential for biomagnification. The bioconcentration factor (BCF) of glyphosate was determined using an ELISA tests specific to measure glyphosate in the fish D. rerio, the rotifers Brachionus calyciflorus and Lecane papuana, and the cladoceran Ceriodaphnia dubia. The experimental design consisted in exposing seven zebrafish adults per replica (four replicates) in three treatments 1, 5, and 10 mg/L of glyphosate for 96 h to obtain bioconcentration factors in the gills, liver, and muscle. These concentrations were selected as potential glyphosate concentrations right after application as double highest reported concentration. Glyphosate levels in zooplankton can represent up to 6.26% of the total weight of rotifers (BFC = 60.35) and in zebrafish adult organs were less than 8 µg/mg of tissue (BCF values < 6). Although glyphosate does not biomagnify, our results suggest that glyphosate affected the dynamics between zooplankton and zebrafish larvae, diminishing survival and feeding rates, given that zooplankton species bioconcentrate glyphosate in large quantities. The BCF values found in this contribution are higher than expected. Glyphosate exposure affected energy metabolism and feeding behavior of zebrafish larvae, which presented high mortality rates at environmentally relevant concentrations.

Enseñanza asistida por pares en una facultad de medicina de Lima, Perú. El caso de la Sociedad Estudiantil de Ciencias Clínicas

La enseñanza asistida por pares es una metodología educativa donde interactúan grupos de estudiantes. Una parte toma el rol de educador, y la otra toma el rol de alumno. Esto trae varios beneficios a ambas partes. Se presenta el caso de un programa donde se utiliza esta metodología, en el cual estudiantes de una facultad de Medicina en Lima, Perú toman el rol de tutores de estudiantes de años menores de la misma facultad. Se describen también sus actividades y los recursos académicos utilizados.

SUMMARY Peer teaching is an educational methodology in which groups of students interact among each other. One group takes on the role of the educator and the other one takes on the role of the student, which benefits both of them. We report here the case of a program where this methodology was used by students in a medical school in Lima, Peru, who were tutors of junior students from the same faculty. The activities and resources used in this methodology are also described.

Intraocular toxicity caused by MEROCTANE perfluorocarbon liquid.

Serious intraocular toxicity cases have been reported worldwide after the use of different perfluorocarbon liquids. The current study reports for the first-time the clinical pictures of cases of acute intraocular toxicity caused by MEROCTANE, a perfluoro-octane commercialized by a Turkish company and distributed in many countries. A series of 18 cases from Chile and Spain was retrospectively analysed. To evaluate the impurity profile, a suspicious MEROCTANE sample (lot OCT.01.2013) was analysed by gas chromatography mass spectrometry and compared with a non-suspicious sample of the same commercial perfluoro-octane (lot OCT 722011). Cytotoxicity was tested following a direct-contact method, taking into consideration the high volatility and hydrophobicity of perfluoro-octane and following the ISO 10993 guideline. Cytotoxicity test showed clear cytotoxic effects of the analysed batch (less than 9% of cell viability). Moreover, chemical analysis demonstrated the presence of many contaminants, some highly toxic (acids and alcohols). Perfluorocarbon liquids are useful tools for intraocular surgery but companies and Agencies of Medical Devices must implement measures that guarantee the safety of these products based on both chemical and cytotoxicity analysis for every batch. Medical staff should be encouraged to report any suspected case to their respective National Agencies.

Evaluation of the environmental impact of magnetic nanostructured materials at different trophic levels.

Safety on the use of magnetic nanomaterials (MNMs) has become an active topic of research given all the recent applications of these materials in various fields. It is known that the toxicity of MNMs depends on size, shape, and surface functionalization. In this study, we evaluate the biocompatibility with different aquatic organisms of engineered MNMs-CIT with excellent aqueous dispersion and long-term colloidal stability. Primary producers (the alga Pseudokirchneriella subcapitata), primary consumers (the rotifer Lecane papuana), and predators (the fish, Danio rerio) interacted with these materials in acute and sub-chronic toxicity tests. Our results indicate that P. subcaptita was the most sensitive taxon to MNMs-CIT. Inhibition of their population growth (IC50 = 22.84 mg L-1) elicited cell malformations and increased the content of photosynthetic pigments, likely due to inhibition of cell division (as demonstrated in AFM analysis). For L. papuana, the acute exposure to MNMs shows no significant mortality. However, adverse effects such as decreased rate of population and altered swimming patterns arise after chronic interaction with MNMs. For D. rerio organisms on early life stages, their exposure to MNMs results in delayed hatching of eggs, diminished survival of larvae, altered energy resources allocation (measured as the content of total carbohydrates, lipids, and protein), and increased glucose demand. As to our knowledge, this is the first study that includes three different trophic levels to assess the effect of MNMs in aquatic organisms; furthermore, we demonstrated that these MNMs pose hazards on aquatic food webs at low concentrations (few mgL-1).

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.

Assessment of Renal Osteodystrophy via Computational Analysis of Label-free Raman Detection of Multiple Biomarkers.

Accurate clinical evaluation of renal osteodystrophy (ROD) is currently accomplished using invasive in vivo transiliac bone biopsy, followed by in vitro histomorphometry. In this study, we demonstrate that an alternative method for ROD assessment is through a fast, label-free Raman recording of multiple biomarkers combined with computational analysis for predicting the minimally required number of spectra for sample classification at defined accuracies. Four clinically relevant biomarkers: the mineral-to-matrix ratio, the carbonate-to-matrix ratio, phenylalanine, and calcium contents were experimentally determined and simultaneously considered as input to a linear discriminant analysis (LDA). Additionally, sample evaluation was performed with a linear support vector machine (LSVM) algorithm, with a 300 variable input. The computed probabilities based on a single spectrum were only marginally different (~80% from LDA and ~87% from LSVM), both providing an unacceptable classification power for a correct sample assignment. However, the Type I and Type II assignment errors confirm that a relatively small number of independent spectra (7 spectra for Type I and 5 spectra for Type II) is necessary for a p < 0.05 error probability. This low number of spectra supports the practicality of future in vivo Raman translation for a fast and accurate ROD detection in clinical settings.

Estradiol and estrogen receptor α in the mPOA and MeA in dwarf hamster (Phodopus campbelli) fathers.

E2 and its alpha receptor (ERα) have an essential role in the regulation of maternal behavior. In dwarf hamster (Phodopus campbelli), E2 facilitates the display of paternal care, and it is possible that ERα is part of the neuroendocrine mechanisms that regulate this behavior. The aim of this study was to analyze the influence of copulation, cohabitation with the pregnant mate and the presence of the pups on paternal behavior, circulating E2 levels and the presence of ERα in the medial preoptic area (mPOA) and medial amygdala (MeA) in dwarf hamsters. Eight males were mated with intact females (IFs), 8 with tubally ligated females (TLFs) and 8 with ovariectomized females (OFs). In males mated with IFs, paternal behavior tests were performed after copulation, halfway through pregnancy and 24 h after the birth of their pups. Males mated with TLFs were subjected to paternal behavior tests at equivalent periods as the males mated with IFs. In males mated with OFs, paternal behavior tests were performed on days 1, 5 and 10 of cohabitation. After the last paternal behavior tests, blood samples were taken for quantification of E2 by radioimmunoassay (RIA), and the brains were dissected to determine ERα immunoreactivity (ir) in the mPOA and MeA. Fathers mated with IFs had higher serum E2 concentrations and more ERα-ir cells in the mPOA than those of males mated with TLFs and OFs. These results suggest that E2 and its ERα may be associated with paternity in the dwarf hamster.

Estrogen Receptors Alpha and Beta in POA-AHA Region Regulate Asymmetrically Ovulation.

We examined the role of the estrogen receptors alpha (ERα) and beta (ERß) in of the preoptic-anterior hypothalamic area (POA-AHA) in the regulation of ovulation in rats. The number of ERα- and ERß-immunoreactive (-ir) cells was determined at 09:00, 13:00, and 17:00 h of each stage of the estrous cycle in intact rats. Additionally, the effects of blocking ERα and ERß on ovulation rate at 09:00 h on diestrus-2 or proestrus day through the microinjection of methyl-piperidino-pyrazole (MPP) or cyclofenil in either side of POA-AHA were evaluated. The number of ERα-ir and ERß-ir cells in POA-AHA varied in each phase of estrous cycle. Either MPP or cyclofenil in the right side of POA-AHA on diestrus-2 day reduced the ovulation rate, while at proestrus day it was decreased in rats treated in either side with MPP, and in those treated with cyclofenil in the left side. MPP or cyclofenil produced a decrease in the surge of luteinizing hormone levels (LH) and an increase in progesterone and follicle stimulating hormone (FSH). Replacement with synthetic luteinizing hormone-releasing hormone in non-ovulating rats treated with MPP or cyclofenil restored ovulation. These results suggest that activation of estrogen receptors on the morning of diestrus-2 and proestrus day asymmetrically regulates ovulation and appropriately regulates the secretion of FSH and progesterone in the morning and afternoon of proestrus day. This ensures that both, the preovulatory secretion of LH and ovulation, occur at the right time.

Energy budget in Alona guttata (Chydoridae: Aloninae) and toxicant-induced alterations.

Although chydorids are the most diverse cladocerans in the world, there is still little information available related to their biology and even less with respect to their susceptibility to toxicants. Therefore, this work aimed to implement protocols with Alona guttata for acute, chronic, and sublethal toxicity tests, using the environmental concern toxicants deltamethrin (DM) and lead (Pb2+), which are commonly used due to agriculture and vector control or for the automotive industry, respectively. Once the results of LC50 (0.1160 ± 0.0107 µg/L for DM and 1.5797 ± 0.1605 mg/L for Pb2+) were obtained, sublethal concentrations (0.01 to 0.2 LC50) were used for the evaluation of biomarkers and chronic toxicity. Concentrations as low as 0.01 LC50 reduced Alona's survival and fecundity, negatively affecting demographic parameters, and decreased the energy reserves. A significant correlation was found between the natural rate of population increase and the caloric content, which demonstrates the suitability of these biomarkers as endpoints of early warning that allow inferring alterations at higher biological levels. Subsequently, this work could constitute the first report on the evaluation of the energy budget in a non-daphnid species, its alterations due to exposure to toxic substances and the correlation with demographic responses.

Paternal behavior in the Mongolian gerbil, and its regulation by social factors, T, ERα, and AR.

The present study evaluates the role of social factors in the transition from infanticidal to paternal male behavior and its association with T concentration, presence of estrogen receptor alpha (ERα) and androgen receptor (AR) in the olfactory bulb (OB), medial preoptic area (mPOA) and medial amygdala (MeA) of Mongolian gerbils (Meriones unguiculatus). This study included thirty-six sexually inexperienced males displaying aggressive behavior toward foreign pups. The selected animals were mated and organized into four groups. The paternal behavior tests were performed on the day of copulation (DCOPUL), during cohabitation with a pregnant female (CPREG), on the day of birth (DBIRTH), and on day 6 postpartum (DPP6). Eight sexually inexperienced males (CTL (male-male cohabitation) were used as control. After paternal behavior tests, blood samples were obtained to quantify T by radioimmunoassay; the brains were removed and analyzed for immunoreactivity (ir) of ERα and AR. All males of the DCOPUL, DBIRTH, and DPP6 groups exhibited paternal behavior, whereas the males of CPREG and CTL groups were aggressive with the pups. Paternal behavior was associated with high T concentrations, and the presence of ERα-ir and AR-ir in the OB, MeA, and mPOA. These results suggest that the transition from aggressive to paternal response to pups is facilitated by copulation, and that in this transition is involved an increase in T concentration. Moreover, the presence of ERα-ir and AR-ir in the OB, mPOA, and MeA could indicate that estrogenic and androgenic pathways participate in the regulation of paternal behavior of the Mongolian gerbils.

Neuronal activation associated with paternal and aversive interactions toward pups in the Mongolian gerbils (Meriones unguiculatus).

Approach/avoid model is used to analyze the neural regulation of maternal behavior in the laboratory rat. This model proposes that the medial preoptic area (mPOA) and bed nucleus of stria terminalis (BNST) are brain regions involved in facilitating mechanisms. By contrast, anterior hypothalamic nucleus (AHN), ventromedial hypothalamic nucleus (VMH), and periaqueductal gray participate in the inhibiting mechanisms of neural regulation of maternal behavior. We hypothesized that there are also facilitating and inhibiting mechanisms in the neural regulation of paternal behavior. Here, we determined which neural areas are activated during paternal and aversive interactions with pups in the Mongolian gerbils (Meriones unguiculatus). By testing paternal behavior, we selected 40 males aggressive toward pups and 20 paternal males. These males were organized into six groups of 10 animals in each group: aggressive males that interacted with pups (AGG-pups) or candy (AGG-candy), paternal males that interacted with pups (PAT-pups) or candy (PAT-candy), and males with testosterone (T)-induced paternal behavior that interacted with pups (IPAT-pups) or candy (IPAT-candy). After interacting with pups or candy, the brains were extracted and analyzed for immunoreactivity (ir) with c-fos. Males that interacted with pups had significantly higher c-fos-ir in the mPOA/BNST than males that interacted with candy. Males that displayed aggression had significantly higher c-fos-ir in the AHN, VMH, and periaqueductal gray than aggressive males that interacted with candy. These results suggest that in the neural regulation of paternal behavior in the Mongolian gerbil underlie positive and negative mechanisms as occurs in maternal behavior.

An increase in estradiol facilitates the onset of paternal behavior in the dwarf hamster (Phodopus campbelli).

In the dwarf hamster (Phodopus campbelli), activational effects of testosterone (T) and estradiol (E2) in the regulation of paternal behavior have been repeatedly rejected because peripheral concentrations of E2 do not change across the reproductive cycle of males. Further, castration no affected paternal behavior despite that both T and E2 concentrations decreased significantly. However, the role of these hormones has not been evaluated in models of castration and hormonal replacement in virgin males. Here, we analysed the effects of E2 and T in paternal behavior in virgin male dwarf hamster (Phodopus campbelli). Thirty paternal (PAT) males were bilaterally castrated; of them, 10 were implanted with T, 10 with E2 and 10 males received no treatment. Other 10 PAT males underwent sham-castration. Seventeen aggressive (AGG) males were also bilaterally castrated; of these, 10 AGG received E2 replacement, 7 were not treated. Other 7 AGG males were submitted to sham-castration. Following treatments, paternal behavior tests were conducted again. T and E2 levels in plasma were quantified by radioimmunoassay (RIA). The results showed that the treatments did not affect the paternal behavior of males that were initially paternal. Neither castration nor sham-castration surgery affected the behavior of AGG males. However, when these males were treated with E2 and the concentrations of this hormone increase significantly they became paternal. Our data suggest that an increase in E2 levels shifted infanticidal behavior to paternal behavior in dwarf hamster.

Toxicity evaluation of cypermethrin, glyphosate, and malathion, on two indigenous zooplanktonic species.

In Aguascalientes, Mexico, there is a special concern about pesticides because of their intensive use on guava production areas, which are located in the vicinity of water reservoirs; thus, non-target organisms could be exposed. Thereafter, the aim of this work was to assess the effect of cypermethrin, Faena® (glyphosate), and malathion, which are the most used pesticides in Aguascalientes' guava production, on the indigenous freshwater species Alona guttata (cladoceran) and Lecane papuana (rotifer). Acute 48-h toxicity tests were carried out, and LC50 values were calculated. Then, five sublethal concentrations (1/80, 1/40, 1/20, 1/10, and 1/5 of the respective LC50) were selected for the chronic assays: (a) intrinsic growth rate analysis in the rotifer and (b) partial life table analysis in the cladoceran. The results of the acute toxicity tests showed that A. guttata was more sensitive to malathion (LC50 = 5.26 × 10-3 mg/L) at concentrations found in natural environments with continuous application on guava fields, whereas L. papuana was more sensitive to Faena® (LC50 = 19.89 mg/L). The somatic growth of A. guttata was inhibited for the chronic exposure to cypermethrin. In addition, cypermethrin and Faena® seemed to exert endocrine disruptive effects on A. guttata. Moreover, malathion chronic exposure significantly decreased the survival of A. guttata. Moreover, L. papuana was affected chronically for the three pesticides.

The participation of the muscarinic receptors in the preoptic-anterior hypothalamic areas in the regulation of ovulation depends on the ovary.

BACKGROUND: Muscarinic receptors (mAChRs) of the preoptic and anterior hypothalamus areas (POA-AHA) regulate ovulation in an asymmetric manner during the estrous cycle. The aims of the present study were to analyze the effects of a temporal blockade of mAChRs on either side of the POA-AHA performed in diestrus-2 rats on ovulation, the levels of estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) and the mechanisms involved in changes in ovulation. METHODS: Cyclic rats on diestrus-2 day were anesthetized and randomly assigned to the following groups: 1) microinjection of 1 µl of saline or atropine solution (62.5 ng) in the left or right POA-AHA; 2) removal (unilateral ovariectomty, ULO) of the left (L-ULO) or right (R-ULO) ovary, and 3) rats microinjected with atropine into the left or right POA-AHA plus L-ULO or R-ULO. The ovulation rate and the number of ova shed were measured during the predicted estrus, as well as the levels of estradiol, FSH and LH during the predicted proestrus and the effects of injecting synthetic LH-releasing hormone (LHRH) or estradiol benzoate (EB). RESULTS: Atropine in the left POA-AHA decreased both the ovulation rate and estradiol and LH levels on the afternoon of proestrus, also LHRH or EB injection restored ovulation. L- or R-ULO resulted in a lower ovulation rate and smaller number of ova shed, and only injection of LHRH restored ovulation. EB injection at diestrus-2 restored ovulation in animals with L-ULO only. The levels of estradiol, FSH and LH in rats with L-ULO were higher than in animals with unilateral laparotomy. In the group microinjected with atropine in the left POA-AHA, ovulation was similar to that in ULO rats. In contrast, atropine in the right POA-AHA of ULO rats blocked ovulation, an action that was restored by either LHRH or EB injection. CONCLUSIONS: These results indicated that the removal of a single ovary at noon on diestrus-2 day perturbed the neuronal pathways regulating LH secretion, which was mediated by the muscarinic system connecting the right POA-AHA and the ovaries.

Unilaterally blocking the muscarinic receptors in the suprachiasmatic nucleus in proestrus rats prevents pre-ovulatory LH secretion and ovulation.

BACKGROUND: The suprachiasmatic nucleus (SCN) and the cholinergic system of various regions of the hypothalamus participate in the regulation of gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, which are necessary for the occurrence of ovulation. In the present study, our goal was to analyse the effects of unilaterally blocking the muscarinic receptors in the SCN on ovulation and steroid secretion. METHODS: Cyclic rats were randomly allotted to one of the experimental groups. Groups of 8-14 rats were anaesthetized and microinjected with 0.3 µl of saline or a solution of atropine (62.5 ng in 0.3 µl of saline) into the left or right SCN at 09.00 or 19.00 h during diestrus-1 or on the proestrus day. The rats were euthanized on the predicted day of oestrus, and evaluated ovulation and levels of progesterone and oestradiol. Other groups of 10 rats were microinjected with atropine into the left or right SCNs at 09.00 h on the proestrus day, were euthanized eight h later, and luteinizing hormone (LH) was measured. RESULTS: At 09.00 or 19.00 h during diestrus-1, atropine microinjections into the SCNs on either side did not modify ovulation. The atropine microinjections performed at 09.00 h of proestrus into either side of the SCN blocked ovulation (right SCN: 1/9 ovulated vs. 9/10 in the saline group; left SCN: 8/14 ovulated vs. 10/10 in the saline group). The LH levels at 17.00 h in the rats that were microinjected with atropine at 09.00 h of proestrus were lower than those of the controls. In the non-ovulating atropine-treated rats, the injection of synthetic LH-releasing hormone (LHRH) restored ovulation. Atropine treatment at 19.00 h of proestrus on either side of the SCN did not modify ovulation, while the progesterone and oestradiol levels were lower. CONCLUSION: Based on the present results, we suggest that the cholinergic neural information arriving on either side of the SCN is necessary for the pre-ovulatory secretion of LH to induce ovulation. Additionally, the regulation of progesterone and oestradiol secretion by the cholinergic innervation of the SCN varies with the time of day, the day of the cycle, and the affected SCN.