Naiara Agirregoitia

The CB2 cannabinoid receptor regulates human sperm cell motility

OBJECTIVE To analyze the expression and distribution of cannabinoid receptors in human sperm cells and evaluate the effects of activation of receptors by specific agonists and antagonists, with a special emphasis on the CB(2) receptor. DESIGN We performed expression assays for CB(1) and CB(2) by reverse transcriptase PCR, Western blot, and immunofluorescence techniques in spermatozoa and performed motility analysis after incubation of semen samples with cannabinoid agonists and CB(2) antagonist SR144528. SETTING Academic research laboratory. PATIENT(S) Semen from 50 normozoospermic, healthy human donors. INTERVENTION(S) Spermatozoa isolated from semen by two consecutive swim-ups were used for all techniques. MAIN OUTCOME MEASURE(S) Reverse transcriptase PCR amplification gels, immunoblots, indirect immunofluorescence antibody assays, and percentage of motile sperm. RESULT(S) We have verified the presence of CB(1) and CB(2) receptors in human spermatozoa. The distribution of both of these receptors was distinct. Incubation with selective cannabinoid receptor agonists induced a significant reduction in the proportion of rapidly progressive motile spermatozoa, and whereas the CB(1) agonist increased the proportion of immobile sperm cells, the CB(2) receptor agonist increased the slow/sluggish progressive sperm cell population. The effect of the CB(2) agonist was antagonized by the CB(2)-specific antagonist. CONCLUSION(S) The functional CB(2) cannabinoid receptor is present in human spermatozoa and regulates the sperm motility in a more distinct manner than CB(1).

Ontogeny of prolyl endopeptidase and pyroglutamyl peptidase I in rat tissues.

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.

Interactions among challenges of hydromineral balance, angiotensin-converting enzyme, and cystine aminopeptidase

Enzymatic cleavage of some peptides could be included among the mechanisms of water-electrolyte homeostasis. To test this hypothesis, the angiotensin-converting activity (ACE) of plasma and the L-cystine-di-beta-naphthylamidase activity (CAP) of plasma and of soluble and particulate fractions from different areas of the central nervous system (CNS) were investigated in rats submitted to treatments eliciting hydromineral imbalance. CAP in the CNS was unchanged by hydromineral challenges. The correlations observed between plasma osmolality and CAP, and plasma CAP and ACE suggested a contribution of these activities to the restoration of basal water-electrolyte and blood pressure conditions through the hydrolysis of vasopressin, oxytocin, angiotensin I and bradykinin.

Expression and localization of cannabinoid receptors in human immature oocytes and unfertilized metaphase-II oocytes

Endocannabinoid anandamide and cannabinoid receptors have been described in some organs of the female reproductive system, but little is known about the expression of these receptors in human oocytes. The aim of the study was to describe the expression of cannabinoid receptors in human oocytes and to investigate their differential distribution at various stages of meiotic resumption in human oocytes. A total of 750 human oocytes from 214 patients were analysed by Western blot, immunocytochemistry and PCR. For this study, oocytes that were not suitable for intracytoplasmic sperm injection (ICSI) (germinal-vesicle and metaphase-I stages), as well as metaphase-II oocytes that had not developed into an embryo after ICSI were used. Western blot analysis revealed the presence of CB1 and CB2 receptor proteins in human oocytes. CB1 and CB2 receptor immunostaining patterns changed during the various stages of meiotic resumption. Localization of CB1 receptor was peripheral at germinal-vesicle stage, homogeneous over the entire oocyte at metaphase I and peripheral at mature metaphase II. CB2 receptor localization was peripheral at germinal-vesicle and metaphase-I stages but homogeneous over the entire cell at metaphase II. This finding suggests a possible role for endocannabinoids, acting via receptors, in the maturation of female gametes and fertilization. The number of couples with sterility problems attending fertility programmes is rising but treatment is not always successful. Important problems associated with failure to conceive remain unresolved because many physiological aspects of human reproduction are still unknown. Endocannabinoids are endogenous chemical compounds that mimic the action of the main psychoactive component of marijuana, delta-9-tetrahydrocannabinol. An endogenous cannabinoid named anandamide has been found in human follicular fluid. Thus, in order to develop knowledge in this field, in the present study we have described the presence of the cannabinoid receptors CB1 and CB2 (the proteins required to mediate the action of the cannabinoids) in the early stages of meiotic resumption of oocytes (the stages before ovulation) and we could postulate that the endocannabinoids could act in the regulation of maturation of oocytes. Our study, together with other studies, indicates that the endocannabinoid system may play a role in human reproduction.

Ontogeny of Soluble and Particulate Prolyl Endopeptidase Activity in Several Areas of the Rat Brain and in the Pituitary Gland

We have analyzed the activity of prolyl endopeptidase (PEP) in several areas of the rat brain (brain cortex, striatum, brain stem, cerebellum and hypothalamus) and in the pituitary gland during ontogeny. In all of these areas, we observed a reduction in PEP activity during development. However, the temporal profile of these alterations was found to be area specific and differences in the ontogeny of the soluble and particulate forms of PEP were observed. Thus, by postnatal day 20 (PD20), soluble PEP activity had began to decrease in the brain cortex and striatum, whereas decreased soluble PEP activity was observed earlier, at PD15, in the brain stem and cerebellum. Changes in the particulate fraction were even more pronounced. Senescence was associated with decreased soluble PEP activity in the striatum, but in contrast, particulate PEP activity was found to be increased in the senescent brain stem. The present results indicate that alterations in the levels of activity of PEP may represent an important event in the development and aging of the central nervous system.

Expression and localization of opioid receptors during the maturation of human oocytes

The endogenous opioid system has been characterized in some female reproductive organs, but little is known about the expression of these receptors in human oocytes. This study investigated the presence and differential distribution of the opioid receptors during the maturation of human oocytes. A total of 821 human oocytes from an intracytoplasmic sperm injection (ICSI) programme were studied including 213 at germinal-vesicle (GV) stage and 164 at metaphase-I (MI) stage and 444 failed fertilization metaphase-II (MII) oocytes. Additionally 31 MII oocytes corresponding to cases where ICSI was not attempted and 50 failed fertilization MII oocytes from the IVF programme were included. Western blot analysis revealed the presence of the delta (OPRD1), kappa (OPRK1) and mu (OPRM1) opioid receptors in human oocytes. The OPRK1 and OPRM1 immunostaining patterns changed during the maturation of the oocyte, while the OPRD1 pattern was the same throughout. In particular, OPRD1 were detected in peripheral tissue from the GV to the MII stage. OPRK1 were found peripherally at the GV stage, more internally at MI and homogeneously at MII. Finally, OPRM1 were located peripherally at the GV stage and homogeneously in MI and MII oocytes. Opioids may have a role in oocyte maturation, acting via receptors. The opioid system has been well characterized in the central nervous system, but it is now known that opioids also act in reproductive organs. However, little is known about the presence and function of this system in human oocytes and its role in their maturation. In this study, we investigated the presence and differential distribution of three (delta, kappa and mu) opioid receptors (proteins which bind the opioids) during the maturation of human oocytes. A total of 821 human oocytes (from 253 patients) not suitable for intracytoplasmic sperm injection (ICSI) or which did not develop into an embryo after ICSI were studied. Thus, we have verified the presence of the delta, kappa and mu opioid receptors in human oocytes. The kappa and mu localization changed during the maturation of the oocyte, while the Delta localization was the same throughout. In particular, the delta receptor was detected in the periphery of the oocyte. On the other hand, the kappa receptor was found peripherally at the beginning, more internally during maturation and homogeneously at the end of maturation. Finally, the Mu receptor was located peripherally at the beginning of maturation and homogeneously in the rest of the maturation stages. This finding suggests a possible role for opioids, acting via receptors, in the maturation of the oocyte.

Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain

We evaluated the subcellular distribution of four membrane-bound aminopeptidases in the human and rat brain cortex. The particulate enzymes under study--puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), pyroglutamyl-peptidase I (PG I) and aspartyl-aminopeptidase (Asp-AP)--were fluorometrically measured using beta-naphthylamide derivatives. Membrane-bound aminopeptidase activity was found in all the studied subcellular fractions (myelinic, synaptosomal, mitochondrial, microsomal and nuclear fractions), although not homogenously. Human PSA showed highest activity in the microsomal fraction. APN was significantly higher in the nuclear fraction of both species, while PG I showed highest activity in the synaptosomal and myelinic fractions of the human and rat brain. The present results suggest that in addition to inactivating neuropeptides at the synaptic cleft, these enzymes may participate in other physiological processes. Moreover, these peptidases may play specific roles depending on their activity levels at the different subcellular structures where they are localized.

Effect of the disruption of body fluid balance on pyroglutamyl aminopeptidase (type-1) in rat brain structures

The activity of soluble and membrane-bound pyroglutamyl aminopeptidase Type-1 (PAP I) was evaluated in the hypothalamus, hippocampus, thalamus, brain cortex, and pituitary gland of rats after applying certain hydromineral challenges. Compared to euhydrated rats, decreased enzyme activity was found in the hypophysis of rats deprived of water for 48 h, or rats drinking ad libitum hypertonic sodium chloride solution (2%) for 6 days or distilled water for 6 days and then submitted to acute water overload. PAP I cleaves the pGlu-amino acid bond of neuropeptides such as thyroliberin, luliberin, neurotensin, and bombesin. The decay of particulate PAP I activity may cause an increase of these pyroglutamate peptides in the whole pituitary. Although the deleterious or pro-homeostatic influence of this decay remains to be elucidated, the present data provide evidence for the involvement of this enzyme activity at this anatomical location in the water-electrolyte imbalance.

Dynamic of expression and localization of cannabinoid-degrading enzymes FAAH and MGLL in relation to CB1 during meiotic maturation of human oocytes.

The endogenous cannabinoid system has been characterized in some female reproductive organs but little is known about the expression and localization pattern of cannabinoid-degrading enzymes in relation to the CB1 cannabinoid receptor in human oocytes. In this study, we focus on the investigation of the presence and differential distribution of fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in relation to CB1 during the maturation of human oocytes. We used a total of 290 human oocytes not suitable for in vitro fertilization/intracytoplasmic sperm injection (ICSI): germinal-vesicle (GV) and metaphase-I (MI) stages and metaphase-II (MII) oocytes that had not developed into an embryo after ICSI. Cannabinoid-degrading enzymes and the cannabinoid CB1 receptor were present in human oocytes. Specifically, FAAH was detected in the periphery of the oocyte from the GV to MI stage and co-localized with CB1. Later, by the MII stage, FAAH was spread within the oocyte, whereas MGLL immunostaining was homogeneous across the oocyte at all stages of maturation and only overlapped with CB1 at the GV stage. This coordinated redistribution of cannabinoid system proteins suggests a role for this system in the maturation of the female gamete.