Current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids.

Camelids have many unique reproductive features that considerably differ from those of other domestic species. Females are induced ovulators with subsequent development of a corpus luteum (CL) with a short lifespan. Plasma progesterone concentration starts to increase on day 4, peaks on day 8-9 and, in non-pregnant animals, basal concentration is reached around day 10-11 post-induction of ovulation. Luteolytic pulses of prostaglandin F2α (PGF2α ) are firstly detected on day 7 or 8 (approximately on day 5-6 after ovulation), with maximal luteolytic peaks observed between days 9 and 11 post-mating, in coincidence with a high endometrial expression of cyclooxygenase 2, a limiting enzyme in prostaglandins synthesis. Unlike other species, oxytocin seems not to be involved in the luteolytic process in these species. The CL is the main source of progesterone secretion, and its function is required to support pregnancy. Despite constant research efforts, aspects of reproduction and maternal recognition of pregnancy in camelids remain not fully understood. A transient decrease and subsequent recovery in plasma progesterone concentration are observed after day 9 post-mating in pregnant animals in association with a pulsatile release of PGF2α and a transitory decrease in CL vascularization. Thus, embryo recognition should occur between days 8 and 12 post-mating. In camels, conceptus tissues exhibit aromatizing activity with the capacity to synthesize large amounts of oestradiol. Similarly, llama blastocysts secrete oestradiol-17ß during the preimplantation stage, with a higher production during the elongation period. An increase in the endometrial expression of oestrogen receptor α is also observed on day 12 post-mating. All these evidences suggest that oestrogen could be the signal released by the embryo at the time of its recognition in camelids. Besides, nearly 98% of pregnancies are carried out in the left horn. A decrease in the endometrial expression of mucin 1 and 16 genes has been reported, suggesting that these changes are crucial for successful embryo implantation; however, no differences have been observed between horns. Thus, maternal recognition of pregnancy in camelids is a particularly complex process that must occur in a concise time to allow the rescue of the CL and embryo survival.

TNAP and P2X7R: New Plasma Biomarkers for Alzheimer's Disease.

Over the last few years, intense research efforts have been made to anticipate or improve the diagnosis of Alzheimer's disease by detecting blood biomarkers. However, the most promising blood biomarkers identified to date have some limitations, most of them related to the techniques required for their detection. Hence, new blood biomarkers should be identified to improve the diagnosis of AD, better discriminate between AD and mild cognitive impairment (MCI) and identify cognitively unimpaired (CU) older individuals at risk for progression to AD. Our previous studies demonstrated that both the purinergic receptor P2X7 and the tissue-nonspecific alkaline phosphatase ectoenzyme (TNAP) are upregulated in the brains of AD patients. Since both proteins are also present in plasma, we investigated whether plasma P2X7R and TNAP are altered in MCI and AD patients and, if so, their potential role as AD biomarkers. We found that AD but not MCI patients present increased plasma P2X7R levels. Nevertheless, TNAP plasma activity was increased in MCI patients and decreased in the AD group. ROC curve analysis indicated that measuring both parameters has a reasonable discriminating capability to diagnose MCI and AD conditions. In addition to confirming that individuals progressing to MCI have increased TNAP activity in plasma, longitudinal studies also revealed that CU individuals have lower plasma TNAP activity than stable controls. Thus, we propose that P2X7 and TNAP could serve as new plasma biomarkers for MCI and AD.

TNAP upregulation is a critical factor in Tauopathies and its blockade ameliorates neurotoxicity and increases life-expectancy.

Tauopathies are a family of neurodegenerative diseases characterized by the presence of abnormally hyperphosphorylated Tau protein. Several studies have proposed that increased extracellular Tau (eTau) leads to the spread of cerebral tauopathy. However, the molecular mechanisms underlying eTau-induced neurotoxicity remain unclear. Previous in vitro studies reported that the ecto-enzyme tissue-nonspecific alkaline phosphatase (TNAP) dephosphorylate eTau at different sites increasing its neurotoxicity. Here, we confirm TNAP protein upregulation in the brains of Alzheimer's patients and found a similar TNAP increase in Pick's disease patients and P301S mice, a well-characterized mouse model of tauopathies. Interestingly, the conditional overexpression of TNAP causes intracellular Tau hyperphosphorylation and aggregation in cells neighbouring those overexpressing the ectoenzyme. Conversely, the genetic disruption of TNAP reduced the dephosphorylation of eTau and decreased neuronal hyperactivity, brain atrophy, and hippocampal neuronal death in P301S mice. TNAP haploinsufficiency in P301S mice prevents the decreased anxiety-like behaviour, motor deficiency, and increased memory capacity and life expectancy. Similar results were observed by the in vivo pharmacological blunting of TNAP activity. This study provides the first in vivo evidence demonstrating that raised TNAP activity is critical for Tau-induced neurotoxicity and suggest that TNAP blockade may be a novel and efficient therapy to treat tauopathies.

Effect of Dietary Oregano (Lippia origanoides) and Clover (Eugenia caryophillata) Essential Oils' Formulations on Intestinal Health and Performance of Pigs.

The incorporation of natural essential oils to the pigs' diet in intensive production systems is a potential tool to improve gut health and prevent infections without using antibiotics. Nevertheless, different products, even containing the same compounds, coming from the same botanical species, may exert dissimilar biological effects due differences in the technological processes by which they are produced and preserved. For this reason, suitability of a given product based on natural extracts, intended for swine production must be thoroughly evaluated. In the present study, we assessed the effects of three additives containing oregano (Lippia origanoides) essential oil, alone or in combination with clover (Eugenia caryophillata) essential oil, with or without being microencapsulated, on gastrointestinal health and on some performance parameters in a commercial pig production farm. Recently weaned piglets were randomly divided in four groups, and basal diet or essential oil-supplemented diet (OCE; MOCE; MOE) was randomly assigned to each of the groups from weaning to finishing. Blood samples were collected at pre-established days after weaning. Intestinal sampling took place at 42 and 72 days of age. Pigs consuming the supplemented diets showed higher intestinal metabolic activity during the post-weaning period, decreasing the impact of weaning stress on enterocytes' metabolism. Intestinal barrier function was not affected in pigs consuming microencapsulated products. All treated groups showed improved intestinal architecture, increased digestive enzymes activity and caecal VFA concentrations. The incorporation of the dietary essential oils products brought beneficial effects on gastrointestinal health that were reflected in improved performance parameters.

Effect of short-term artificial light and transvaginal progesterone device on first ovulation in late transitional mares.

In study I, plasma progesterone concentrations were evaluated in anoestrous mares that received an intravaginal progesterone release device (IPRD) for 10 days. Mares were divided into 3 groups based on the dosage of progesterone (0 g, n=3; 1.38 g, n=5; and 1.9 g, n=5). No statistical differences were found in plasma progesterone concentrations between the two doses tested. In study II, the effects of a protocol based on a short program of artificial light combined with an IPRD containing 1.38 g of progesterone on oestrous behaviour and onset of ovulation were evaluated. IPRDs were inserted into 31 late transitional mares (10 days of treatment). The mares were divided into a control group (n=9, IPRD with 0 g of progesterone) and two treatment groups (T1, n=10, IPRD with 0 g of progesterone and artificial light; T2, n=12, IPRD with 1.38 g of progesterone and artificial light). The percentages of mares in heat within the first 14 days after treatment were 100%, 70%, and 100% in the control, T1, and T2 groups, respectively (P=0.097), and their ovulation rates were 44%, 60%, and 100%, respectively (P≤0.01). In conclusion, a protocol based on artificial light and an IPRD containing 1.38 g of progesterone for 10 days could be considered to advance the first ovulation of the year in late transitional mares, as it ensures a higher rate of ovulation within the first 14 days after treatment.

Corpus luteum vascularization during the maternal recognition of pregnancy in llamas (Lama glama).

The aim of this study was to characterize corpus luteum vascularization and its association with plasma progesterone concentration in early stages of pregnancy, when maternal recognition of pregnancy is expected to occur. In all animals, both plasma progesterone concentration and corpus luteum vascularization increased from Day 6 to Day 8 post-mating and afterwards in non-pregnant llamas they started to decrease to reach basal levels around Days 12 to 14 post-mating, while in pregnant animals, both variables remained elevated until the end of the study. A lineal positive relationship between corpus luteum vascularization and plasma progesterone concentration was observed in pregnant (r2  = .46, p < .0001) and non-pregnant llamas (r2  = .66, p < .0001). Pregnant animals showed higher plasma progesterone concentration and corpus luteum vascularization than the non-pregnant ones from Day 12 post-mating until the end of the study (p Ë‚ .05 and p Ë‚ .01, respectively). These results suggest that maternal recognition of pregnancy should occur before Day 12 post-mating in order to expand luteal lifespan, maintaining corpus luteum vascularization and progesterone production. Also, the assessment of CL vascularization area could be a useful and non-invasive method for early pregnancy diagnosis due to its association with plasma progesterone concentration.

Administration of progesterone BioRelease LA inhibits follicular growth in llamas (Lama glama) regardless of follicle diameter at the start of treatment.

The aims of the study were twofold: first, the comparison of the pharmacokinetics parameters of two doses of Progesterone BioRelease® LA, (BioRelease Technologies, Lexington, KY, USA) one of 300 mg and other of 150 mg and their effects on ovarian dynamics in llamas. Based on the results from the first study, the aim of the second study was to evaluate the effect of the doses of 150 mg of progesterone on follicular activity considering the stage of the largest follicle at the beginning of treatment. The results in Study 1 showed that both doses of the formulation induced plasma progesterone concentrations higher than 1 ng/ml during the first 6 days of treatment in all females, progesterone concentrations steadily decline until Day 5 following by a slowly decrease. The total amount of progesterone released during treatment was higher in Group 300 than in Group 150 (p = 0.045). Mean maximum concentrations were 14.9 ± 2.24 and 14.3 ± 2.16 ng/ml for Group A versus Group B (p = 0.58), and they were registered on Day 1.5 ± 0.22 and 1.7 ± 0.34 days, respectively (p = 0.10). None of the animals of Group A showed progesterone concentration below 1 ng/ml during all studied period. The treatment applied in Study 2 was efficient in inhibiting the ovarian follicular dynamics and to start a superestimulatory treatment. The use of progesterone Biorelease® LA of 150 mg in comparison with the dose of 300 mg could be more effective in the use of synchronization protocols in llamas for AI or prior to the application of an ovarian superstimulatory treatment.

Oncogenic p95HER2 regulates Na+-HCO3- cotransporter NBCn1 mRNA stability in breast cancer cells via 3'UTR-dependent processes.

The Na+-HCO3- cotransporter NBCn1 (SLC4A7) is up-regulated in breast cancer, important for tumor growth, and a single nucleotide polymorphism (SNP), rs4973768, in its 3' untranslated region (3'UTR) correlates with increased breast cancer risk. We previously demonstrated that NBCn1 expression and promoter activity are strongly increased in breast cancer cells expressing a constitutively active oncogenic human epidermal growth factor receptor 2 (HER2) (p95HER2). Here, we address the roles of p95HER2 in regulating NBCn1 expression via post-transcriptional mechanisms. p95HER2 expression in MCF-7 cells reduced the rate of NBCn1 mRNA degradation. The NBCn1 3'UTR down-regulated luciferase reporter expression in control cells, and this was reversed by p95HER2, suggesting that p95HER2 counteracts 3'UTR-mediated suppression of NBCn1 expression. Truncation analyses identified three NBCn1 3'UTR regions of regulatory importance. Mutation of putative miRNA-binding sites (miR-374a/b, miR-200b/c, miR-29a/b/c, miR-488) in these regions did not have significant impact on 3'UTR activity. The NBCn1 3'UTR interacted directly with the RNA-binding protein human antigen R (HuR), and HuR knockdown reduced NBCn1 expression. Conversely, ablation of a distal AU-rich element increased 3'UTR-driven reporter activity, suggesting complex regulatory roles of these sites. The cancer-associated SNP variant decreased reporter expression in T-47D breast cancer cells, yet not in MCF-7, MDA-MB-231 and SK-BR-3 cells, arguing against a general role in regulating NBCn1 expression. Finally, p95HER2 expression increased total and plasma membrane NBCn1 protein levels and decreased the rate of NBCn1 protein degradation. Collectively, this is the first work to demonstrate 3'UTR-mediated NBCn1 regulation, shows that p95HER2 regulates NBCn1 expression at multiple levels, and substantiates the central position of p95HER2-NBCn1 signaling in breast cancer.

Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep.

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Effect of early administration of progesterone on the function of the corpus luteum of llamas.

The aim of the present study was to evaluate if early administration of progesterone immediately after ovulation affects corpus luteum lifespan in llamas. Female llamas (n = 16) were induced to ovulate by Buserelin injection in the presence of an ovulatory follicle (Day 0). On Day 2, ovulation was confirmed and animals were randomly divided into two groups: treated animals (n = 8) received an intravaginal device containing 0.3 g of progesterone from Day 2 to Day 6 post-induction of ovulation and control group (n = 8) received a device with 0 g of progesterone. Blood samples were collected daily to determine plasma progesterone concentration and transrectal ultrasonographies were performed from Day 7 to Day 12 post-induction of ovulation. Mean maximum diameter of the corpus luteum was significantly lower and was reached before in the treated group than in the control group. The mean highest plasma progesterone concentration and the day that concentration was achieved were similar between groups. However, mean plasma progesterone concentration was significantly higher in the treated group than in the control group on Days 3 and 4 and lower on Days 8 and 9 post-induction of ovulation. The day that plasma progesterone concentration returns to 1 ng/ml differed between groups, occurring earlier in the treated group. In conclusion, the early increase of plasma progesterone concentration during the luteal phase, promoted the premature activation of the luteolytic process affecting corpus luteum function in llamas as it was previously reported in other species.

P2X7 receptor inhibition ameliorates ubiquitin-proteasome system dysfunction associated with Alzheimer's disease.

BACKGROUND: Over recent years, increasing evidence suggests a causal relationship between neurofibrillary tangles (NFTs) formation, the main histopathological hallmark of tauopathies, including Alzheimer's disease (AD), and the ubiquitin-proteasome system (UPS) dysfunction detected in these patients. Nevertheless, the mechanisms underlying UPS failure and the factors involved remain poorly understood. Given that AD and tauopathies are associated with chronic neuroinflammation, here, we explore if ATP, one of the danger-associated molecules patterns (DAMPs) associated with neuroinflammation, impacts on AD-associated UPS dysfunction. METHODS: To evaluate if ATP may modulate the UPS via its selective P2X7 receptor, we combined in vitro and in vivo approaches using both pharmacological and genetic tools. We analyze postmortem samples from human AD patients and P301S mice, a mouse model that mimics pathology observed in AD patients, and those from the new transgenic mouse lines generated, such as P301S mice expressing the UPS reporter UbG76V-YFP or P301S deficient of P2X7R. RESULTS: We describe for the first time that extracellular ATP-induced activation of the purinergic P2X7 receptor (P2X7R) downregulates the transcription of ß5 and ß1 proteasomal catalytic subunits via the PI3K/Akt/GSK3/Nfr2 pathway, leading to their deficient assembly into the 20S core proteasomal complex, resulting in a reduced proteasomal chymotrypsin-like and postglutamyl-like activities. Using UPS-reported mice (UbGFP mice), we identified neurons and microglial cells as the most sensitive cell linages to a P2X7R-mediated UPS regulation. In vivo pharmacological or genetic P2X7R blockade reverted the proteasomal impairment developed by P301S mice, which mimics that were detected in AD patients. Finally, the generation of P301S;UbGFP mice allowed us to identify those hippocampal cells more sensitive to UPS impairment and demonstrate that the pharmacological or genetic blockade of P2X7R promotes their survival. CONCLUSIONS: Our work demonstrates the sustained and aberrant activation of P2X7R caused by Tau-induced neuroinflammation contributes to the UPS dysfunction and subsequent neuronal death associated with AD, especially in the hippocampus.

Effect of hCG administration on Day 7 post-mating on accessory corpus luteum development and progesterone concentration in llamas.

A high embryo loss rate has been reported in llamas. As strategies that lead to an increase in plasma progesterone (P4) concentration might improve fertility, the aim of the present study was to evaluate if the administration of hCG on Day 7 post-mating is useful to develop an accessory corpus luteum (CL), increasing plasma P4 concentration. Twenty (n = 20) female llamas, ranging between 5 and 10 years of age and four (n = 4) males of proven fertility, ranging between 8 and 10 years of age were included in the study. Accessory CL developed in all treated llamas after hCG administration and plasma P4 concentration was significantly greater in treated than in control females (P˂0.0001). The diameter and vascularization of the original CL were not affected by treatment in pregnant llamas. However, in treated non-pregnant llamas, corpus luteum diameter was greater than in the control group from Day 14 post-mating until the end of the study (P˂0.001). In treated llamas, the accessory CL was detected throughout the study in pregnant and non-pregnant females, but its vascularization started to decrease around Day 16 post-mating in non-pregnant animals (P˂0.05). In conclusion, hCG treatment on Day 7 post mating was useful to induce an accessory CL and increase plasma P4 concentration in llamas. Thus, this treatment could be considered as a useful strategy to improve pregnancy rates in llama herds.

A feasible method for llama embryo paraffin embedding.

Maternal recognition of pregnancy in llamas would be related to a still-unknown signal secreted by the embryo to extend the corpus luteum lifespan. In order to study llamas reproductive physiology and early pregnancy aspects, research on embryos between 8 and 12 days post-mating is essential. However, there is a lack of information on llama embryo paraffin embedding, a procedure that exhibits difficulties, mostly for embryo manipulation. This work presents an accessible, easy and rapid protocol for embedding llama blastocysts. Uterine flushings from four superovulated llamas were performed to recover embryos 8 days post-mating. Each embryo was fixed in 1 ml of 10 % neutral buffered formalin for 24 h, at 4 °C, and transferred to 70 % ethanol. Later, they were submitted to a series of baths: 500 µl PBS with 5 % bovine foetal serum solution (PBS+BFS) for 1 min; zona pellucida permeabilization with 500 µl PBS with 0.2 % Triton X-100 for 20 min; 500 µl PBS+BFS for 1 min; dehydration on baths with ascendent grades of ethanol (70 %, 96 %, and 100 % twice) for 10 min each; and bath of butanol for 5 min. Finally, embryos were embedded in a pellet of paraffin and afterwards in a paraffin block. Complete sections from 8 of 13 grade I and II embryos were obtained. In conclusion, by applying this method, good quality sections with preserved embryo morphology suitable for histological, histochemical or immunohistochemical approaches would be possible to acquire.

Studying the Role of P2X7 Receptor in Axonal Growth Using In Utero Electroporation Technique.

The nervous system is formed by a complex network of neuronal connections. During development, neurons elongate their axons through highly stereotyped anatomical pathways to form precise connections. Defects in these mechanisms are related with neurological disorders. Previous studies have reported that inhibition of the P2X7 receptor, an ionotropic purinergic receptor, promotes axonal growth and branching in cultured neurons. However, little is known about the in vivo mechanism of axonal elongation regulated by P2X7. Here, we detailed a step-by-step method to perform in utero cortical electroporation and quantified the electroporated axons employing accessible and open-source image processing software. This effective surgical procedure manipulates in vivo the gene expression in a discrete population of callosal projection neuron. Thus, a better understanding of the involvement of P2X7 in the in vivo establishment of neuronal circuits might help to clarify the basic biology of several neurodevelopmental disorders and axonal regenerative processes.

P2X7 receptor blockade reduces tau induced toxicity, therapeutic implications in tauopathies.

Tauopathies are neurodegenerative diseases characterized by the presence of aberrant intraneuronal aggregates of hyperphosphorylated Tau protein. Recent studies suggest that associated chronic neuroinflammation may contribute to the pathological Tau dissemination. However, the underlying molecular mechanisms remain unknown. Since purinergic P2X7 receptors (P2X7) can sense the rise of extracellular ATP levels associated with neuroinflammation, its involvement in neurodegeneration-associated inflammation was suggested. We found a P2X7 upregulation in patients diagnosed with different tauopathies and in a tauopathy mouse model, P301S mice. In vivo pharmacological or genetic blockade of P2X7 reverted microglial activation in P301S mice leading to a reduction in microglial migratory, secretory, and proliferative capacities, and promoting phagocytic function. Furthermore, it reduced the intraneuronal phosphorylated Tau levels in a GSK3-dependent way and increased extracellular phosphorylated Tau levels by reducing the expression of ectoenzyme TNAP. Accordingly, pharmacological or genetic blockade of P2X7 improved the cellular survival, motor and memory deficits and anxiolytic profile in P301S mice. Contrary, P2X7 overexpression caused a significant worsening of Tau-induced toxicity and aggravated the deteriorated motor and memory deficits in P301S mice. Our results indicate that P2X7 plays a deleterious role in tauopathies and suggest that its blockade may be a promising approach to treat Tauopathies.

Associations between brain-derived neurotrophic factor plasma levels and severity of the illness, recurrence and symptoms in depressed patients.

BACKGROUND: There is increasing evidence that the brain-derived neurotrophic factor (BDNF) is involved in the pathophysiology of mood disorders and that its peripheral levels represent a reliable mirror of its concentration in the brain. The aim of the present study was to measure BDNF plasma levels in patients affected by major depression and to explore the possible relationship between the biological parameter and characteristics of the illness. METHOD: BDNF plasma levels were evaluated in 30 inpatients suffering from major depression, according to DSM-IV criteria, by means of a commonly employed ELISA method. The clinical characteristics were assessed by the Hamilton Rating Scale for Depression (HRSD) and the Clinical Global Impression Scale. RESULTS: BDNF plasma levels were significantly lower in the patients with the severest illness compared with the others, and the same was true for patients with dissociative symptoms, severe sleep disturbance and recurrent depression. A significant and negative correlation was observed between the biological parameter and the retardation factor score of the HRSD. CONCLUSION: These findings suggest that low BDNF levels are related to both recurrence and severity of depression, as well as to symptoms typical of dysfunctions of the hypothalamic-pituitary-adrenal axis.

Plasma IGF1 and 17ß-Estradiol Concentrations During the Follicular Wave in Llamas.

The aim of this study was to characterize the temporal association between follicular waves and circulating concentrations of 17ß-estradiol (E2) and IGF1 in llamas. Follicular waves could be clearly divided in three phases: growth, plateau and regression; with a mean duration of 18.8 ± 0.32 days. All follicular waves showed overlapping, so that as one dominant follicle was regressing, another one was growing. E2 plasma concentration showed a wavelike pattern, similar to that followed by the dominant follicle; reaching its maximum concentration at the end of the growth phase and decreasing at the end of the plateau phase. IGF1 also showed variations during the follicular wave. It tended to increase during the growth phase and decreased toward Days 14 and 16. IGF1 reached its maximum concentration before E2 did (5 ± 0.8 vs. 7.2 ± 0.5 days after wave emergence) and before the maximum follicular diameter was attained (10.2 ± 0.46 days after wave emergence). Both hormones started to rise again in coincidence with the development of a new follicular wave. The observed profiles allow to suggest that IGF1 could have a role on folliculogenesis and ovarian steroideogenesis in llamas, as reported for other species.

Estradiol-17ß Injection Induces Ovulation in Llamas.

This study aimed to investigate the effect of three different doses of estradiol-17ß on ovulation and subsequent luteal development and function in llamas. Twenty-three llamas were examined daily by transrectal ultrasonography until the detection of an ovulatory follicle (≥8 mm). Thereafter, animals were divided into five groups: Control (n = 3; treated with 1.6 ml of saline solution), GnRH group (n = 6, treated with an intravenous injection of 8.4 µg Buserelin), and estradiol groups that received 0.6 mg (E1, n = 4), 1 mg (E2, n = 4), or 1.6 mg (E3, n = 6) of estradiol-17ß intravenously. Detection of ovulation was based on ultrasonographic visualization of disappearance of the largest follicle and subsequent presence of a newly formed corpus luteum (CL) and progesterone concentration exceeding 1 ng ml-1. Daily blood samples were collected to determine plasma progesterone concentration. Ovulation rate was 0% for control and E1 groups, 25% for E2 group, and 100% for GnRH and E3 groups. Differences in the mean CL diameter between GnRH and E3 groups were not statistically significant. Plasma progesterone concentration was similar between groups during the different days in ovulated animals. However, the day that the plasma progesterone concentration was above 1 ng ml-1 and the day that the highest plasma progesterone concentration was achieved differed among E3 and GnRH groups, occurring later in females treated with estradiol. In conclusion, an injection of estradiol-17ß is capable of inducing ovulation in llamas and the response depends on the dose used. Most of the animals required the highest tested dose (1.6 mg) to induce the ovulatory process. Although the CL diameter in females induced to ovulate with estradiol was similar to that in llamas induced to ovulate with a GnRH analog, the rise in plasma progesterone concentration above 1 ng ml-1 and the peak progesterone concentration were attained 1 day later in the estradiol treated females.

ATP Measurement in Cerebrospinal Fluid Using a Microplate Reader.

Imbalance in extracellular ATP levels in brain tissue has been suggested as a triggering factor for several neurological disorders. Here, we describe the most sensitive and reliable technique for monitoring the ATP levels in mice cerebrospinal samples collected by cisterna magna puncture technique and quantified using a microplate reader.

The Role of P2X7 Receptor in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease characterized by a progressive cognitive decline associated with global brain damage. Initially, intracellular paired helical filaments composed by hyperphosphorylated tau and extracellular deposits of amyloid-ß (Aß) were postulated as the causing factors of the synaptic dysfunction, neuroinflammation, oxidative stress, and neuronal death, detected in AD patients. Therefore, the vast majority of clinical trials were focused on targeting Aß and tau directly, but no effective treatment has been reported so far. Consequently, only palliative treatments are currently available for AD patients. Over recent years, several studies have suggested the involvement of the purinergic receptor P2X7 (P2X7R), a plasma membrane ionotropic ATP-gated receptor, in the AD brain pathology. In this line, altered expression levels and function of P2X7R were found both in AD patients and AD mouse models. Consequently, genetic depletion or pharmacological inhibition of P2X7R ameliorated the hallmarks and symptoms of different AD mouse models. In this review, we provide an overview of the current knowledge about the role of the P2X7R in AD.