Sex differences in neural projections of fear memory processing in mice and humans.

It remains unexplored in the field of fear memory whether functional neuronal connectivity between two brain areas is necessary for one sex but not the other. Here, we show that chemogenetic silencing of centromedial (CeM)-Tac2 fibers in the lateral posterior BNST (BNSTpl) decreased fear memory consolidation in male mice but not females. Optogenetic excitation of CeM-Tac2 fibers in the BNSTpl exhibited enhanced inhibitory postsynaptic currents in males compared to females. In vivo calcium imaging analysis revealed a sex-dimorphic fear memory engram in the BNSTpl. Furthermore, in humans, the single-nucleotide polymorphism (SNP) in the Tac2 receptor (rs2765) (TAC3R) decreased CeM-BNST connectivity in a fear task, impaired fear memory consolidation, and increased the expression of the TAC3R mRNA in AA-carrier men but not in women. These sex differences in critical neuronal circuits underlying fear memory formation may be relevant to human neuropsychiatric disorders with fear memory alterations such as posttraumatic stress disorder.

CRISPR/Cas9 as a Simple Technique for the Generation of Murine Knockout Models for Neuropsychiatric Diseases.

The development of schizophrenia-like rodent models is still a major challenge for the study of this mental disorder. Schizophrenia and other neuropsychiatric disorders are thought to be triggered by multiple factors, and furthermore, the genetic component of schizophrenia is highly complex. The edition of one single gene for mimicking some of the symptoms of the disorder could cause unintended mutations that could influence animal's behavior making it difficult to study. Since 2013, CRISPR-Cas gene-editing technology has been a great improvement in the specificity of transgenic model generation because of its speed, efficiency, cost, and apparent ease. This protocol describes a simple method to generate a knockout mouse model using CRISPR technology, which can be applied to any gene presumably involved in the development of schizophrenia and other neuropsychiatric disorders.

Delta and kappa opioid receptors in human endometrium during the menstrual cycle: Expression and localization.

OBJECTIVE: Endogenous opioid peptides were reported to be involved in the regulation of reproductive physiology and their precursors and receptors were described in many of the male and female reproductive tissues. Mu opioid receptor (MOR) was described in human endometrial cells and its expression and localization changed during the menstrual cycle. However, there is no data from the distribution of the other opioid receptors: Delta (DOR) and Kappa (KOR). The objective of the present work was to analyze the dynamics of expression and localization of DOR and KOR in human endometrium throughout the menstrual cycle. STUDY DESIGN: Human endometrial samples from different menstrual cycle phases were analyzed by immunohistochemistry. RESULTS: DOR and KOR were present in all samples analyzed and the protein expression and localization changed throughout the menstrual cycle. Both receptor expression increased during the late proliferative phase and decreased during the late secretory-one, especially in the luminal epithelium. DOR expression was generally higher than KOR expression in all cell compartments. CONCLUSIONS: The presence of DOR and KOR in human endometrium and their dynamic changes during the menstrual cycle join the results previously obtained in MOR suggesting a possible role of opioids in reproduction events related to the human endometrium.

The endocannabinoid system modulates the ovarian physiology and its activation can improve in vitro oocyte maturation.

The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by analyzing the meiotic competence of the oocytes and their evolution toward embryos. Thus, when CB1 and CB2 receptors were missed, the ovary area and volume was significantly less and the action of the equine chorionic gonadotropin (eCG) hormone was diminished. In addition, the mutant genotypes had fewer ovarian follicles and they were less competent after eCG administration compared with wild-type mice, and this lack of CB receptors showed a mismatch of oocyte maturation. However, the in vitro use of THC showed improvements in oocytes IVM after a Pre-IVM step for 48 hr, as those oocytes reached a significantly higher polar body rate, a larger diameter and the best result on blastocysts rate was achieved when THC was used during the IVM step.

Delta and kappa opioid receptors on mouse sperm cells: Expression, localization and involvement on in vitro fertilization.

The endogenous opioid peptides have been reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with sentences around the harmful effect of opioids in male fertility but, actually, there is only one study regarding the real fertility potential of spermatozoa that have been exposed to mu specific opioids. The aim of the present study was to see if the modulation of delta (OPRD1) and kappa (OPRK1) opioid receptors in mouse sperm during capacitation was able to vary the embryo production after in vitro fertilization (IVF). The presence of OPRD1 and OPRK1 in mouse mature spermatozoa was analyzed by RT-PCR and immunofluorescence. Incubating the sperm with, on one hand, the delta specific agonist DPDPE and/or antagonist naltrindole, and, on the other hand, the kappa specific agonist U-50488 and antagonist nor-binaltorphimine, we analyzed the involvement of OPRD1 and OPRK1 on IVF and preimplantational embryo development. We verified the presence of OPRD1 and OPRK1 in mouse mature spermatozoa, not only at the mRNA level but also at protein level. Moreover, the sperm incubation with DPDPE, before the IVF, had an effect on the fertilization rate of sperm and reduced the number of reached blastocysts, which was reverted by naltrindole. Instead, the use of the kappa agonist U-50488 and the antagonist nor-binaltophimine did not have any effect on the amount and the quality of the achieved blastocysts. Although nowadays the pure delta or kappa opioid ligands are not used for the clinic, clinical trials are being conducted to be used in the near future, so it would be interesting to know if the modulation of these receptors in sperm would generate any consequence in relation to fertilization capacity.

Mu opioid receptor expression and localisation in murine spermatozoa and its role in IVF.

The endogenous opioid peptides are reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with statements on the harmful effect of opioids on male fertility but, in fact, there are no studies regarding the real fertilisation potential of spermatozoa that have been exposed to opioids. The aim of the present study was to examine if modulation of mu opioid receptor (OPRM1) in murine spermatozoa during capacitation influenced embryo production after IVF. The presence of OPRM1 in murine mature spermatozoa was analysed by reverse transcription-polymerase chain reaction and immunofluorescence. We analysed the involvement of OPRM1 on IVF and pre-implantational embryo development by incubating the spermatozoa with the opioid agonist morphine and/or antagonist naloxone. We verified the presence of OPRM1 in murine mature spermatozoa, not only at the mRNA level but also the protein level. Moreover, incubation of the spermatozoa with morphine, before IVF, had an effect on the fertilisation rate of the spermatozoa and reduced the numbers of blastocysts, which was reversed by naloxone. Considering that opioids are widely used clinically, it is important to take into account their effect, via OPRM1, on the fertility of patients.

Tetrahydrocannabinol Modulates in Vitro Maturation of Oocytes and Improves the Blastocyst Rates after in Vitro Fertilization.

BACKGROUND/AIMS: Among the assisted reproductive techniques, the in vitro maturation of oocytes (IVM) is less developed than other techniques, but its implementation would entail a qualitative advance. This technique consists in the extraction of immature oocytes from antral ovarian follicles with the patient under low hormone stimulation or without hormone to mature exogenously in culture media supplemented with different molecules to promote maturation. In this sense, we are interested in the role that cannabinoids could have as IVM promoters because cannabinoid's molecular pathway is similar to the one by which oocyte's meiosis resumption is activated. With the intention of advancing in the possible use of cannabinoids as supplements for the media for in vitro maturation of oocytes, we intend to deepen the study of the function of the phytocannabinoid Δ-9-tetrahydrocannabinol (THC) in the IVM process. METHODS: By immunocytochemistry, we detected the location pattern of cannabinoid receptor type 1 (CB1) and type 2 (CB2) during oocyte maturation in presence or absence of THC, as well as, the staining pattern of p-AKT and p-ERK. We used a genetic/ pharmacological approach generating knockout oocytes for CB1 and/or CB2 and they were incubated with THC during the oocyte maturation to visualize the physiological effects of THC, observing the rate of blastocyst achieved by oocyte. RESULTS: This study confirms that the incubation of oocytes with THC during IVM accelerated some events of that process like the phosphorylation pattern of ERK and AKT and was able to increase the blastocyst rate in response to IVF. Moreover, it seems that both CB1 and CB2 are necessary to maintain a healthy oocyte maturation. CONCLUSION: Our data suggest that THC may be useful IVM supplements in clinic as is more feasible and reliable than any synthetic cannabinoid.

Implication of mu opioid receptor in the in vitro maturation of oocytes and its effects on subsequent fertilization and embryo development in mice.

Oocyte maturation is the process by which immature oocytes acquire all the necessary characteristics for successful fertilization. The endogenous opioid peptides have been suggested to have a role modulating this process. However, little is known about its implication and the effect of exposing oocyte maturation to opioids on the subsequent fertilization and embryo development. Hence, in the present work, we focused on elucidating the function of the mu opioid receptor (OPRM1) in the modulation of the oocyte maturation. We analyzed the expression and localization of OPRM1 in mice oocytes and granulosa cells by reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. To observe the activity of the OPRM1, immature oocytes were incubated with morphine agonist and/or naloxone antagonist and we evaluated the PI3K/Akt and MAPK pathways, as well as the effect on the subsequent fertilization and embryo development. OPRM1 was present in mice oocytes and granulosa cells, changing its expression pattern depending on the maturation stage. Moreover, morphine, modulating PI3K/Akt and MAPK pathways, helped oocytes to reach blastocyst stage, which was reverted by naloxone. These results propose the OPRM1 as a possible therapeutic target for in vitro maturation culture medium, as it could improve the blastocyst rates obtained in the actual reproduction assisted techniques.

Mu opioid receptor in the human endometrium: dynamics of its expression and localization during the menstrual cycle.

OBJECTIVE: To study the dynamics of the expression and localization of the mu opioid receptor (MOR) in human endometrium throughout the menstrual cycle. DESIGN: Analysis of human endometrial samples from different menstrual cycle phases (menstrual, early/midproliferative, late proliferative/early secretory, midsecretory, and late secretory) by reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry. SETTING: Academic research laboratory. PATIENT(S): Women from the Human Reproduction Unit of the Cruces University Hospital, fulfilling the following criteria: normal uterine vaginal ultrasound; absence of endometriosis, polycystic ovary syndrome, implantation failure, or recurrent miscarriage; and no history of opioid drug use. INTERVENTION(S): Endometrial samples of 86 women categorized into groups for the menstrual cycle phases: 12 menstrual, 21 early/midproliferative, 16 late proliferative/early secretory, 17 midsecretory, and 20 late secretory. MAIN OUTCOME MEASURE(S): MOR gene and protein expression and localization in the different compartments of the human endometrium at different stages of the menstrual cycle. RESULT(S): The expression of MOR mRNA and protein changed throughout the cycle in human endometrium. MOR expression increased during the proliferative phase and decreased during the secretory one. Lower values were found at menstruation, and maximum values around the time of ovulation. Small variations for each endometrial compartment were found. CONCLUSION(S): The presence of MOR in human endometrium and the dynamic changes during the menstrual cycle suggest a possible role for opioids in reproduction events related to the human endometrium or endometriosis.