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During the previous decades, the indiscriminate use of anthelmintics for the control of gastrointestinal nematodes has generated anthelmintic resistance in different parts of the world. It is neces...

Sheep haemonchosis is a disease that causes anemia, diarrhea, weight loss, low production, and even death of young animals. This represents a negative economic impact on the livestock sector. For d...

This study was aimed at evaluating thein vitroeffect of the edible mushroom (EM)Pleurotus eryngiiagainst the eggs and larvae (L3) ofHaemonchus contortus. The evaluation included acetone (AE) and hydroalcoholic (HA) extracts of the following strains: ECS-1138, ECS-1156, ECS-1255, ECS-1258, ECS-1261, ECS-1282, and ECS-1292. The HA extract of the ECS-1255 strain showed the highest effect on mortality rates of L3 (18.83%) at 20 μg/mL. After subjecting this HA extract to a normal phase chromatography column, five fractions were obtained; fraction F5 (100% MeOH) was the most effective against eggs, with hatching inhibition percentages of 88.77 and 91.87% at 20 and 40 mg/mL, respectively. Gas chromatography-mass spectrometry (GC-MS) subjected this fraction to an acetylation reaction to determine the content of the secondary metabolites. The GC-MS analysis showed that the F5 fraction was composed of trehalose CAS: 6138-23-4, polyols (L-iditol CAS: 488-45-9, galactitol CAS: 608-66-2, D-mannitol CAS: 69-65-8, D-glucitol CAS: 50-70-4, and myoinositol CAS: 87-89-8), adipic acid CAS: 124-04-9, stearic acid CAS: 57-11-4, squalene CAS: 111-02-4, andβ-sitosterol CAS: 83-46-5.

Recently, the root-knot nematode Meloidogyne enterolobii have generated important economic losses (65%) in worldwide agriculture. In the present study, the nematocidal activity of the PdR-2 fraction of Pleurotus djamor was evaluated against the second instar juvenile (J2) of M. enterolobii. Different concentrations of the PdR-2 fraction (0.039, 0.078, 0.156, 0.132, 0.625, and 1.25 mg mL-1) were evaluated, as well as the respective control groups (water and Levamisole, 5 mg mL-1) in a volume of 100 µL (n=4). J2s were exposed for 24 h and subsequently quantified, and the percentage of mortality was estimated. Data were analyzed by analysis of variance with the general linear model and a comparison of means with Tukey’s test (p < 0.05). The PdR-2 fraction at concentrations of 0.132, 0.625, and 1.25 mg mL-1 were significantly equal with respect to Levamisole application, showing a mortality of 87.6, 84.5, and 86.3%, respectively. At the lowest concentration (0.039 mg mL-1), 40.3% mortality was recorded, whereas 86% was recorded at the highest concentration evaluated (1.25 mg mL-1). The PdR-2 fraction of P. djamor had nematocidal activity against J2s of M. enterolobii.

To explore the capability of soil mycobiota to degrade avocado peel waste and identify relevant successions and trophic guild shifts, fungal communities from three environments with different land uses were evaluated in a solid-state process. Soil samples used as inoculum were collected from a pristine mature tropical forest, a traditionally managed Mayan land, and an intensively managed monospecific avocado plantation. Soil-substrate mixes were evaluated for 52 weeks to evaluate organic matter decay and the carbon-to-nitrogen ratio. Amplicon-based high-throughput sequencing from internally transcribed spacer (ITS) analysis revealed significant differences in fungal communities widely dominated by Fusarium sp. and Clonostachys sp.; however, less represented taxa showed relevant shifts concomitantly with organic matter content drops. Trophic guild assignment revealed different behaviors in fungal communities between treatments over the 52 weeks, suggesting distinct preconditioning of fungal communities in these environments. Overall, the results lead to the identification of promising degradation moments and inoculum sources for further consortia enrichment or bioprospecting efforts.

The objective of this study was to evaluate and compare the in vitro lethal effect of the hydroalcoholic extract of the spent substrate of Pleurotus djamor ECS-123, obtained at 15 days of colonization (SPS) and at the first (SPS1) and second (SPS2) harvests, against infective larvae L3 of Haemonchus contortus. The in vitro lethal effect was evaluated by the L3 larval mortality test (LM) using six concentrations: 1.25, 2.5, 5, 10, 20, and 40 mg/mL, with ivermectin and thiabendazole (5 mg/mL) as controls. The first harvest extract (SPS1) of strain ECS-123 was subjected to liquid-liquid bipartition, which resulted in two fractions: aqueous (PdAcO) and ethyl acetate (PdAct). The chemical fractionation of PdAct with the highest mortality rate (80.11 %) was carried out with open-column chromatography, giving a total of 13 fractions, which were analyzed by thin-layer chromatography (TLC) and grouped into 5 mixtures (R1;1–3, R2;4–7, R3;8–9, R4;10–11 and R5;12–13). Subsequently, the mixtures were evaluated against H. contortus L3 larvae. Finally, the components of the mixtures with the highest nematocidal effects were evaluated by gas chromatography coupled to mass spectrometry (GC–MS). The data were analyzed with a completely randomized design through ANOVA using the generalized linear model (GLM) with the "R" program. The purification and characterization of R4 and R5 by GC–MS revealed the presence of the following compounds: veratryl alcohol, 4-hydroxy-3,5,5 trimethyl-4-[3-oxo-1-butenyl]-2- cyclohexen-1-one, caffeine and 5,6-dimethoxy-1(3 H) isobenzofuranone. This information allowed for the identification of nematocidal compounds in the degraded substrate of P. djamor, an activity that had not been reported previously.

Antecedentes: En los últimos años se ha presentado un marcado interés por la producción comercial de Pleurotus spp., lo cual ha ocasionado la búsqueda de sustratos o mezclas que aseguren una alta productividad. Objetivos: Determinar el efecto de los principales componentes químicos del sustrato en la producción de basidiomas de dos cepas del género Pleurotus. Métodos: A partir de olote, pasto y pulpa de café se prepararon 10 tratamientos, que fueron analizados bromatológicamente. Se cultivaron dos cepas de Pleurotus spp. y se evaluó la eficiencia biológica (EB), la tasa de producción (TP) y el rendimiento (R). Resultados y conclusiones: En ambas cepas cultivadas, ECS-123 y ECS-1123, el tratamiento 7 (100 % pasto) obtuvo los valores más altos de EB (38.2 % y 48.8 %, respectivamente), TP (0.5 % y 0.6 %, respectivamente) y R (14.7 y 18.8 %, respectivamente). Estos valores no fueron estadísticamente diferentes de otros tratamientos con pasto, pero sí de la pulpa de café (con mayor contenido de nitrógeno). Se concluye que es suficiente una concentración de nitrógeno de 0.5 % y una relación C/N de 96.75 para obtener una buena producción de basidiomas. Se resalta la incidencia de otros factores en la productividad de las cepas.

Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown.

The present paper reviewed publications on the nematocidal activity of edible mushrooms (EM) and their potential use as sustainable tools for the control of parasitic nematodes affecting agriculture and livestock industry. Nematodes are organisms living in the soil and animals’ guts where they may live as parasites severely affecting economically important crops and farm animals, thus causing economic losses to worldwide agriculture. Traditionally, parasitic nematodes have been controlled using commercial pesticides and anthelmintic (AH) drugs. Over the years, nematodes developed resistance to the AH drugs, reducing the usefulness of many commercial drugs. Also, the use of pesticides/anthelmintic drugs to control nematodes can have important negative impacts on the environment. Different EM have been not only used as food but also studied as alternative methods for controlling several diseases including parasitic nematodes. The present paper reviewed publications from the last decades about the nematocidal activity of EM and assessed their potential use as sustainable tools for the control of nematodes affecting agriculture and livestock industry. A reduced number of reports on the effect of EM against nematodes were found, and an even smaller number of reports regarding the potential AH activity of chemical compounds isolated from EM products were found. However, those studies have produced promising results that certainly deserve further investigation. It is concluded that EM, their fractions and extracts, and some compounds contained in them may have biotechnological application for the control of animal and plant parasitic nematodes.

Haemonchus contortus is a haematophagous gastrointestinal nematode (GIN) that causes severe anaemia and even death in small ruminants, negatively impacting the economic viability of farms. Traditionally, this parasite has been controlled with chemical compounds; however, inadequate use of these types of products has favoured the emergence of anthelmintic resistance. Therefore, it is necessary to search for alternatives for GIN control. Previous studies have reported the anthelmintic activity of edible mushroom extracts against H. contortus. A recent study reported that a fraction constituted of different fatty acids and β-sitosterol isolated from the basidiomata of the edible mushroom Pleurotus djamor ECS-123 has ovicidal and larvicidal activity against H. contortus. Thus, this study aimed to assess the anthelmintic activity of the pure molecules: pentadecanoic acid, palmitic acid, β-sitosterol, stearic acid and linoleic acid. For this purpose, an egg-hatching inhibition test was carried out in which the compounds were evaluated individually and in combination at a final concentration of 20 mg mL−1. Furthermore, larval mortality was assessed using a combination of the five commercial compounds previously mentioned at different concentrations (1.25–20 mg mL−1). Palmitic acid and stearic acid, in some combinations, inhibited H. contortus egg hatching by 100%. On the other hand, in the larval mortality test, the combination of the five compounds showed dose-dependent behaviour, and 100% mortality was obtained 24 h post-incubation. Pure molecules and their combinations have anthelmintic-like activity against the eggs and larvae of H. contortus.

In this work, the white-rot fungus Pleurotus djamor was used for the first time to determine the degradation kinetics of the nonsteroidal anti-inflammatory drugs diclofenac, naproxen and, ketoprofen, either individually or in mixtures, in submerged cultures. Removal of 93% individual diclofenac and 99% diclofenac in mixtures with naproxen and ketoprofen at 6 h of incubation with the fungus was achieved. The elimination levels of naproxen and ketoprofen individually were 90% and 87%, respectively, after 48 h of incubation. However, the removal levels of these compounds in mixtures were 85% and 83%, respectively. On the other hand, during the degradation kinetics analysis, the enzymatic activities of laccases, manganese peroxidases, and lignin peroxidases were evaluated, yielding values of 3700, 270 and 31 U/L, respectively. Additionally, it was demonstrated that during degradation of diclofenac or the three drugs mixed in the submerged cultures, the enzymatic activity of extracellular laccases expressed by P. djamor increased by 200% and 300%, respectively. The activity of manganese peroxides increased by 126% with diclofenac and 138% when the mixture of drugs was added to the cultures. On the other hand, lignin peroxidase only increased activity by 123% with the drug mixture.

Background: Edible mushrooms have nutraceutical properties, mainly in humans, animals and plants. Pleurotus species represent an alternative approach to parasite control because they have nematocidal activity against different taxonomic genera of nematodes of importance for livestock (e.g., eggs and infective and histotrophic larvae of Haemonchus contortus), as well as against other parasites of importance in animal health. Objective: To present a general overview of fungi of the genus Pleurotus and their characteristics as a sustainable method of biocontrol for parasitic nematodes of animals with importance in public health. Methods: This bibliographic review was carried out by consulting published studies on edible fungi of the genus Pleurotus and analyzing their properties related to nematocidal activity. The works were grouped according to their research focus; works related to keywords and topics in livestock were identified in indexed journals and on digital platforms. Results and conclusions: Pleurotus spp. produce different secondary metabolites with antiparasitic activities, mainly anthelmintic and particularly against gastrointestinal nematodes and their larval stages. The characteristics of these fungi are excellent for biotechnological applications since they produce a wide range of secondary metabolites in different parts, such as in the basidiomas, mycelia and degraded substrates.

This study evaluated in vitro the morphology of ten strains of edible mushroom: Pleurotus ostreatus (strains 1123 and 0152), P. eryngii (strains 1290 and 1291), P. cornucopiae (strains 1328 and 1330), Coprinus comatus (strain 1103), Panus sp. (strain 801), Lentinula edodes (strain 401) and L. boryanus (strain 402) on different growing media. In addition, the in vitro anthelmintic activity (AH) of the mycelium of the fungi against Haemonchus contortus infective larvae was evaluated in Petri dishes. Finally, organic extracts of six of the fungi were synthesized and evaluated against H. contortus larvae. The highest mycelial growth rate was achieved by P. ostreatus (0152), Panus sp. (8 0 1), and P. ostreatus (1123) in wheat flour with agar (HIT). The highest biomass (1.43 g/Petri dish) was produced by Panus sp. 801 in the HIT medium. The highest percentages of mycelial AH mortality were caused by P. ostreatus 0152, P. cornucopiae 1328, P. ostreatus 1123, P. eryngii 1292 and L. edodes 401, at 88, 88.5, 91, 93 and 93.93% respectively. The biological activity of the fungi could be due to toxins, which inhibit the movement of nematodes and subsequently degrade them. The extracts that caused the highest mortality rates were: P. ostreatus 0152 (81.6%) and P. eryngii 1292 (90.8%) at 72 h following exposure to 200 mg/mL of extract. The results of the present study show that these fungi are candidates for future in vivo tests against H. contortus.

The aim of this study was to evaluate a self-heating pasteurization technique in preparing substrates for mushroom production. Seven species were used: Agrocybe aegerita, Auricularia fuscosuccinea, Pleurotus djamor, P. eryngii, P. ostreatus, Lentinula edodes, and Ganoderma lucidum. They were cultivated on grass, corncob, wood shavings, and a mixture thereof. The self-heating technique allowed for pasteurization of 3 of the substrates (grass, corncob, and the mixture). The preheating chamber comprised a drawer placed under the pasteurization crate. With this chamber, it was possible to increase inlet air temperatures by 4--5°C. The evaluated mushroom species responded in different ways to the pasteurization process. P. ostreatus (control) and P. djamor produced basidiomes when cultivated in all pasteurization substrates. A. aegerita and P. eryngii fruited only on corncob and the mixture, whereas A. fuscosuccinea fruited only on the pasteurized corncob. G. lucidum and L. edodes did not fructify on the pasteurized substrates.

Fifty-four macromycetes, isolated from southeastern Mexico, were used in order to evaluate their capacity for degradation and tolerance to the herbicide paraquat. Ten of these strains were capable of growing in a solid culture medium in the presence of 200 ppm paraquat. Subsequently, assays to evaluate the degradation of the xenobiotic in a liquid medium were carried out. Of the ten strains evaluated, three presented the highest levels of degradation of the compound, which were Trametes pavonia (54.2%), Trametes versicolor (54.1%) and Hypholoma dispersum. They presented the highest overall degradation percentage (70.7%) after 12 days culture. The presence of ligninolytic enzymes in these strains was evaluated. H. dispersum only presented aryl alcohol oxidase activity; however, with the data obtained, it was not possible to conclude whether this specific enzyme is responsible for paraquat degradation. The level of degradation obtained is above the one reported for Pseudomonas putida, one of the few reports on paraquat degradation. This is the first report on the contaminant degradation capacity of H. dispersum.

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57°C and pH 5.5 and the other, a temperature of 61°C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24h until 144h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

Background: Certain microorganisms during the preparation of substrate for growing Agaricus bisporus are of great interest for the conversion of organic material and for the excretion of a wide range of metabolites with growth regulation activities that may affect the mycelial growth processes and mushroom yield. Adding beneficial microorganisms in the substrate may be a biotechnological alternative to optimize A. bisporus cultivation. Objective: Isolate and evaluate thermophilic microorganisms from self-heating pasteurized substrate with growth-promoting effects on A. bisporus cultivation. Methods: Different microorganisms were isolated and selected at 45 and 55 °C. They were tested for siderophore production, 1-octen-3-ol consumption, and phosphate solubilization in coculture with A. bisporus to determine their growth effects on potato dextrose agar (PDA) and on sterile pangola grass (Digitaria eriantha). Results: Of the 106 microorganisms isolated, 88 % were able to grow in the presence of 1-octen-3-ol, while 1 % had the capacity to produce siderophores, and 55 % had the ability to solubilize phosphate. The strains Bacillus hisashii ECS-B-65, B. licheniformis ECS-B-78, Rhizomucor pusillus ECS-710 and ECS-712, Aspergillus fumigatus ECS-709, and Thermomyces sp. ECS-711 were found to have a positive effect on A. bisporus mycelial growth.

Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown.

Lignocellulosic materials are used as substrate for the cultivation of the edible mushroom Pleurotus pulmonarius. After two or three flushes of mushrooms, the spent substrate is discarded although it still has an important enzymatic activity that can be used for several purposes. In this study, we sought to determine the technical feasibility of using spent substrate from P. pulmonarius to degrade chlorothalonil. Reaction mixture was prepared with 6 ml of pesticide aqueous solution (2 mg active ingredient/l) and 3 ml of enzymatic extract obtained from spent P. pulmonarius substrate. The enzymatic reaction (27 °C, pH 7.4) was conducted for 1 h with sampling at 15 min intervals. The effect of storage time and temperature (freezing or refrigerating) of spent substrate and enzymatic extract, respectively, on the activity over chlorothalonil was determined. Freshly obtained spent substrate extract was able to reduce 100% of the initial concentration of chlorothalonil (2 mg/l) after 45 min of reaction. Storage time had a negative effect on the stability of the enzymatic activity: with spent substrate stored for a week, chlorothalonil concentration was reduced in 49.5% after 1 h reaction and with substrate stored for two and three weeks, the degradation efficiency decreased to 9.15% and 0%, respectively. Cooling and freezing the spent substrate extract also had a negative effect on chlorothalonil degradation.

Rat ovaries stimulated with human follicle-stimulating hormone (hFSH) overexpress a factor that attenuates the LH surge in the rat: the putative gonadotropin surge-attenuating factor (GnSAF). A reduced gondadotrope progesterone receptor (PR) phosphorylation/activation is likely to be the main causative factor involved in GnSAF bioactivity on LH release. Besides, GnSAF reduces LH synthesis as well as LH secretion, and it is not known whether PR is involved in the inhibitory action of GnSAF on LH synthesis. Thus, the purpose of the present work was to evaluate the involvement of PR in the inhibitory effects of GnSAF on LH synthesis in cycling rats. To this end we used a specific radioimmunoassay and reverse transcription-polymerase chain reaction (RT-PCR) to study the effect on LH pituitary content and LHβ mRNA expression of PR occupancy with P (3 mg/0.2 ml oil in diestrus) on the inhibitory effects of hFSH (0, 0.1, 1, and 10 IU) in metestrus (day 2) and diestrus (day 3) on LH synthesis on proestrus in intact and on day 4 in day 2 ovariectomized (OVX) rats injected with 5 and 10 μg of estradiol benzoate (EB) on days 2 and 3, respectively. Results showed that (1) hFSH decreased pituitary LH content in intact, but not in OVX rats injected with EB, without affecting LHβ mRNA levels, and (2) PR occupancy with P annulled the inhibitory action of hFSH on pituitary LH content. These results indicate that PR is involved in ovarian GnSAF effect on LH content probably at a post-transcriptional level.

These experiments investigated the involvement of gonadotrope progesterone receptor (PR) in the effects of the putative gonadotropin surge-attenuating factor (GnSAF) on gonadotropin (LH and FSH) secretion. Human follicular fluids (hFF) used in this study were aspirated from follicles in gonadotropin-treated women for in vitro fertilization. Samples were subjected to two-fold charcoal extraction of steroid hormones and two-fold inhibin immunoprecipitation. Gonadotropin secretion parameters were assessed by specific radioimmunoassays. In the first experiment, the effects of hFF on both basal and GnRH-stimulated gonadotropin secretion and GnRH self-priming were studied in incubated hemipituitaries from rats on each day of the 4-day estrous cycle. hFF inhibited only GnRH self-priming in pituitaries from rats in diestrus. In the second experiment, immunohistochemical PR expression and action were evaluated in pituitaries from rats in diestrus. PR-positive (PR10A9 antibody) gonadotropes were detected (4–5/field 40x), and antiprogestins added to the incubation media blocked the ligand-independent (GnRH) activation of PR effects on GnRH self-priming. Finally, the third experiment evaluated the effects of hFF on P-induced potentiation of GnRH-stimulated LH secretion. GnSAF bioactivity, as evidenced by inhibition of PR-induced potentiation of GnRH-stimulated LH secretion, was found in diestrous pituitaries incubated with hFF. The results indicate that GnSAF attenuated GnRH-dependent LH secretion in diestrus through the inhibition of PR-dependent GnRH self-priming.

Passive immunization against inhibin with an anti-inhibin serum (AIS) during the diestrous phase in cycling rats increased follicle-stimulating hormone secretion, stimulated the ovaries and reduced the magnitude of the luteinizing hormone (LH) surge in the afternoon of proestrus. The involvement of gonadotrope progesterone receptor (PR) expression/action in the inhibitory effects of the follicle-stimulating hormone-dependent putative ovarian factor gonadotropin surge-attenuating factor on preovulatory LH secretion was studied in the absence of circulating free inhibin. Proestrous pituitaries from rats injected with AIS or a non-immune serum (NIS) were studied for determination of PR-AB and PR-B mRNAs by RT-PCR and PR-B and PR-A isoform proteins by Western blot. In addition, pituitaries from AIS- and NIS-injected rats were incubated and studied for PR-dependent LH secretion parameters: LH-releasing hormone (LHRH)-stimulated LH secretion, progesterone-potentiated LHRH-stimulated LH secretion and LHRH self-priming. Also, the effects of the antiprogestagen RU486 on these LH secretion parameters were evaluated and compared with those of AIS. Finally, gonadotrope PR phosphorylation was evaluated by immunohistochemistry. Results showed that the hyperstimulated ovaries of AIS-injected rats produce a factor, different from inhibin, that blocked LHRH self-priming and P-potentiation of LHRH-stimulated LH secretion. These effects were not due to decreased pituitary PR mRNAs, PR protein expression or PR protein B/A ratio. The inhibitory effect of AIS on PR-dependent LH secretion seemed to be due to gonadotrope PR dephosphorylation. Taken together, the findings indicated that the putative gonadotropin surge-attenuating factor affected LH surge through an inhibition of PR phosphorylation/action but not PR expression.

Administration of human follicle-stimulating hormone (hFSH) to female rats during diestrus phase attenuates the spontaneous luteinizing hormone (LH) surge in proestrous afternoon. The inhibition of LH secretion is associated with a decreased pituitary LH content in intact, but not in ovariectomized rats injected with 25 μg estradiol benzoate (EB). This suggests that the mechanism of action of the putative non-steroidal ovarian bioactive FSH-dependent gonadodotropin surge attenuating factor (GnSAF) might, in addition, involve a reduction in LH synthesis. The present experiments studied, in proestrous pituitaries, the effects of different doses of hFSH, with or without EB on: (i) basal and GnRH-stimulated LH release and GnRH self-priming, (ii) LHβ mRNA values, and (iii) LH content. Results showed that bioactive GnSAF reduced mainly GnRH self-priming, but also GnRH-stimulated LH secretion and pituitary LH content in a dose-dependent manner. GnSAF had no effect on basal LH secretion or pituitary LHβ mRNA values. EB increased pituitary sensitivity to GnRH in controls, and overcame the inhibitory effects of GnSAF after low doses of hFSH but not after 10 IU of hFSH. In contrast with the sensitizing action of EB on LH secretion, EB had no effect on pituitary LHβ mRNA content or LH protein. It is concluded that the putative GnSAF blunted the LH surge by reducing LH synthesis at post-transcriptional level and antagonizing the GnRH-dependent LH secretion and the sensitizing effect of estradiol to GnRH.

Kisspeptins, the products of the KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54), have recently emerged as pivotal signals in the hypothalamic network triggering the preovulatory surge of gonadotropins and, hence, ovulation. Additional actions of kisspeptins at other levels of the hypothalamic-pituitary-ovarian axis have been suggested but remain to date scarcely studied. We report herein the pattern of expression of KiSS-1 and GPR54 in the human and nonhuman primate ovary and evaluate changes in ovarian KiSS-1 expression in a rat model of ovulatory dysfunction. KiSS-1 and GPR54 mRNAs were detected in human ovarian tissue and cultured granulosa-lutein cells. In good agreement, kisspeptin immunoreactivity was observed in cyclic human and marmoset ovaries, with prominent signals in the theca layer of growing follicles, corpora lutea, interstitial gland, and ovarian surface epithelium. GPR54 immunoreactivity was also found in human theca and luteal cells. Administration of indomethacin to cyclic female rats disturbed ovulation and resulted in a dramatic drop in ovarian KiSS-1, but not GPR54, cyclooxygenase-2 (COX-2), or progesterone receptor, mRNA levels at the time of ovulation; an effect mimicked by the selective COX-2 inhibitor NS398 and rescued by coadministration of PGE2. Likewise, the stimulatory effect of human choriogonadotropin on ovarian KiSS-1 expression was partially blunted by indomethacin. In contrast, KiSS-1 mRNA levels remained unaltered in another model of ovulatory failure, i.e., the RU486-treated rat. In summary, we document for the first time the expression of KiSS-1/kisspeptin and GPR54 in the human and nonhuman primate ovary. In addition, we provide evidence for the ability of inhibitors of COX-2, known to disturb follicular rupture and ovulation, to selectively alter the expression of KiSS-1 gene in rat ovary. Altogether, our results are suggestive of a conserved role of local KiSS-1 in the direct control of ovarian functions in mammals.

Administration of human FSH(hFSH) to cyclic rats during the dioestrous phase attenuates progesterone receptor (PR)-dependent events of the preovulatory LH surge in pro-oestrus. The increased bioactivity of the putative ovarian gonadotropin surge inhibiting/attenuating factor induced by hFSH treatment is not associated with a decrease in PR protein expression, and the possibility of its association at a PR posttranslational effect has been raised. The present experiments aimed to analyse PR phosphorylation status in the gonadotrope of ratswith impaired LH secretion induced by in vivo hFSH injection. Two experimental approaches were used. First, incubated pro-oestrous pituitaries from hFSH-injected cycling and oestrogen-treated ovariectomized (OVX) rats were used to analyze the effect of calyculin, an inhibitor of intracellular phosphatases, on PR-dependent LH release, which was measured in the incubation medium by RIA. Second, pituitaries taken from hFSH-injected intact cycling and OVX rats and later incubated with P or GNRH1 were used to assess the phosphorylation rate of gonadotrope. The latter was analysed in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry using a MAB that recognizes the phosphorylated (p) form of PR at Ser294. Calyculin reduced the ovary-mediated inhibition of hFSH in GNRH1-stimulated LH secretion. In addition, the immunohistochemical expression of pSer294 PR was significantly reduced after ovarian stimulation with hFSH in pituitaries frompro-oestrous rats incubated with PorGNRH1.Altogether, these results suggested that the ovarian-dependent inhibitory effect of FSH injection on the preovulatory LH secretion in the rat may involve an increase in dephosphorylation of PR.

The corpus luteum (CL) is an exquisitely regulated transitory endocrine gland necessary for the onset and maintenance of pregnancy in mammals. Most of the data on the mechanisms of CL differentiation at the molecular level come from genomic studies, but direct protein data are scarce. Here we have undertaken a differential expression proteomic approach to identify, in an unbiased way, those proteins whose levels change significantly in the rat CL as it evolves from functionality during pregnancy to regression after parturition. Moreover, we have compared the regressing CL with the newly formed functional CL that coexist during lactation under the same endocrine environment. We have defined a "proteomic signature" of CL functionality, which is constituted by a set of 24 proteins with a few differences between pregnancy and lactation. Most of these markers are new and are involved in microtubule assembly, retinoic acid transport, and Raf kinase signaling cascade; 10 are enzymes that define a ketogenic metabolic landscape, demonstrating, for the first time, the prevalence of de novo cholesterol synthesis in luteal cells. The "proteomic signature of regression," on the other hand, is composed of nine proteins, one of which is 20alpha-hydroxysteroid dehydrogenase and two, ferritin and gamma-actin, are new. The discovery of unpredictable new actors in the differentiation process of CL reported here will contribute to new hypotheses that explain the complex female reproductive function at the protein level. It will also open new doors to research on each identified protein by relating them to cellular differentiation.

La subregión turística del Litoral Norte de Holguín, y en particular el sector entre las bahías de Vita y Samá, es uno de los destinos más importantes para el desarrollo del turismo internacional en Cuba. En el presente artículo se hace un análisis de los principales peligros naturales y antrópicos que inciden en la actividad turística del sector de estudio, los cuales fueron caracterizados y sintetizados utilizando como herramienta los Sistemas de Información Geográfica (SiG).Se obtiene como resultado el mapa de superposición de peligros a los que se encuentra expuesta la actividad.

The use of fungi in biodegradation processes has increased in recent years, mainly because they are relatively easy to cultivate and they possess an interesting metabolism process with a powerful enzymatic system. In this study, the degradation of pesticides used for agricultural purposes is reviewed. Three compounds are taken as models of degradation: endosulfan (insecticide), chlorothalonil (fungicide), and paraquat (herbicide). These pesticides pose a significant health risk, and their indiscriminate use in agriculture can cause long-term problems such as birth defects, chromosome abnormalities, blood disorders, cancer (confirmed in animals and probable in human), and even death. Several genera of mushrooms have been studied for biodegradation of agro-pesticides, of which Pleurotus is considered to be one of the most promising due to its ligninolytic capabilities. Strains of this genus are able to degrade endosulfan and chlorothalonil. There are few reports on paraquat degradation; however, a study on using native strains of tropical mushrooms is currently in progress, with promising results. All the studied taxa have shown the ability to degrade some of these compounds, with up to 95% degradation in certain cases, thus making them excellent candidates for bioremediation strategies.

Hyperstimulation of ovarian function with human FSH (hFSH) attenuates the preovulatory surge of LH. These experiments aimed at investigating the mechanism of ovarian-mediated FSH suppression of the progesterone (P4) receptor (PR)-dependent LH surge in the rat. Four-day cycling rats were injected with hFSH, oestradiol benzoate (EB) or vehicle during the dioestrous phase. On pro-oestrus, their pituitaries were studied for PR mRNA and protein expression. Additionally, pro-oestrous pituitaries were incubated in the presence of oestradiol-17β (E2), and primed with P4 and LH-releasing hormone (LHRH), with or without the antiprogestin RU486. After 1 h of incubation, pituitaries were either challenged or not challenged with LHRH. Measured basal and LHRH-stimulated LH secretions and LHRH self-priming were compared with those exhibited by incubated pituitaries on day 4 from ovariectomized (OVX) rats in metoestrus (day 2) injected with hFSH and/or EB on days 2 and 3. The results showed that: i) hFSH lowered the spontaneous LH surge without affecting basal LH and E2 levels, gonadotroph PR-A/PR-B mRNA ratio or immunohistochemical protein expression; ii) incubated pro-oestrous pituitaries from hFSH-treated rats did not respond to P4 or LHRH, and lacked E2-augmenting and LHRH self-priming effects and iii) OVX reversed the inhibitory effects of hFSH on LH secretion. It is concluded that under the influence of hFSH, the ovaries produce a non-steroidal factor which suppresses all PR-dependent events of the LH surge elicited by E2. The action of such a factor seemed to be due to a blockade of gonadotroph PR action rather than to an inhibition of PR expression.

El presente estudio tuvo como objetivos contribuir al conocimiento de los macromicetos que crecen en el Parque Educativo Laguna Bélgica (PELB), en Ocozocoautla de Espinosa, Chiapas y determinar el índice de diversidad de Simpson y similitud de Sorensen de la micobiota existente en los diferentes tipos de vegetación. Se realizaron 24 exploraciones micológicas durante un año, encontrándose 144 especies (24 Ascomycota y 120 Basidiomycota). El índice de diversidad, mostró que la vegetación con la micobiota más diversa fue la del bosque de Quercus elliptica (D= 0.9678) la cual presentó mayor similitud con el bosque de Liquidambar stracyflua (Is= 83 %). El sustrato más frecuente fue la madera en descomposición (57.63 %). Se determinaron las especies de macromicetos potencialmente utilizables para el consumo humano (22 especies), lo que determinó el valor micológico del área de estudio en 15.27 %. Del total de especies determinadas 22 son nuevos registros para el estado de Chiapas; 4 Ascomicetos Scutellinia scutellata, Xylaria amphitele, X. persicaria, Chlorociboria aeruginosa, y 18 Basidiomicetos Amanita pantherina, Geastrum striatum, Hydnum repandum, Hygrocybe miniata, Scleroderma verrucosum, Cotylidia diaphana, Lactarius indigo, Phlogiotis helvelloides, Hydnochaete olivaceae, Phellinus ferruginosus, P. contiguus, P.rufitinctus, Thelephora terrestris, T. cervicornis, Perenniporia ohiensis, Diplomitoporus lenis, Schizopora paradoxa y Tremella fuciformis.The objectives of this study were to contribute to the knowledge of macro fungi growing in the "Parque Educativo Laguna Belgica" (PELB), in Chiapas, Mexico and to determine the diversity and similarity indexes for its mycobiota in each type of vegetation. Twenty four mycological explorations were made during a year, resulting in the identification of 144 species (24 Ascomycota and 120 Basidiomycota). The diversity of fungal species was determined through the index of Simpson and the similarity among the existing mycobiota inside the different types of vegetation was determined by means of the index of Sorensen. The index of diversity pointed out that the vegetation with the most diverse mycobiota was Quercus elliptica forest (D= 0.9678) which presented greater similarity than the forest of Liquidambar styraciflua (Is= 83%). Rot wood was found to be the most frequently used substratum for the macromycetes (57.63%). Twenty two species of potential use for human consumption were found, determining a mycological value of 15.27 % for the area. Twenty two species were considered as new records for the state of Chiapas; 4 Ascomycetes Scutellinia scutellata, Xylaria amphitele, Chlorociboria aeruginosa, X. persicaria and 18 Basidiomycetes (Amanita pantherina, Geastrum striatum, Hydnum repandum, Hygrocybe miniata, Scleroderma verrucosum, Cotylidia diaphana, Lactarius indigo, Phlogiotis helvelloides, Hydnochaete olivaceae, Phellinus ferruginosus, P. contiguus, P.rufitinctus, Thelephora terrestris, T. cervicornis, Perenniporia ohiensis, Diplomitoporus lenis, Schizopora paradoxa and Tremella fuciformis.

Ovarian tissue homeostasis is maintained by highly regulated cyclic phases of cell proliferation/differentiation and programmed cell death. Compelling evidence indicates that both apoptotic and autophagic types of programmed cell death are involved in the regression of the corpus luteum (CL) in primate species. Beclin 1 is an autophagy-related protein that is involved in the inter-relationships between apoptosis and autophagy, through interaction with the anti-apoptotic protein bcl-2. We studied the presence and expression pattern of beclin 1 in the adult human ovary. In ovarian follicles, beclin 1 immunostaining was found in the theca layer, whereas granulosa cells were negative. After ovulation, beclin 1 immunostaining was present in both theca-lutein and granulosa-lutein areas. The expression of beclin 1 in granulosa-lutein cells was related to the functional and structural status of the CL, being strong at the early and mid luteal phases, barely detectable at the late luteal phase, and absent in granulosa-lutein cells in subsequent cycles. Our results indicated that beclin 1 expression was related to luteal cell survival rather than to cell death. Accordingly, persistent beclin 1 expression was found in granulosa-lutein cells under either physiological (i.e., CL of pregnancy) or pathological (irregularly regressing CL in climacteric women) conditions involving prolonged CL life span. Strong beclin 1 immunostaining was also found in ovarian androgen-producing cells (i.e., secondary interstitial and hilus cells). Our data thus suggest that beclin 1 plays important roles in the regulation of the life span of human CL and ovarian androgen-secreting cells, by maintaining autophagy at levels promoting cell survival rather than cell death.

Experiments were performed to determine the effect of adding nutrient supplements to colonized mushroom compost (MC) for the production of a second crop of mushrooms. Mushrooms were harvested for 1, 2 or 3 flushes, the casing removed and the MC then was fragmented and re-supplemented with delayed release supplements treated or non-treated with fungicide (thiophanate-methyl; Topsin M 70WP) and re-cased. Overall double-crop yields were higher when MC was re-supplemented after 1st flush (1st flush MC) as compared to re-supplementation after the 2nd or 3rd flushes. Mean double-crop BEs were 128, 119 and 109% when 1st-, 2nd- and 3rd-flush MCs were used, respectively. Treatment of delayed release supplement with thiophanate-methyl fungicide did not affect mushroom yields. Soluble salts and potassium concentrations increased 350 and 900%, respectively, in the casing overlay through three flushes suggesting that removal of the casing would help to alleviate the build up of these potential growth-limiting materials. Re-supplementing and re-casing of MC represents a potential opportunity for growers to increase revenues and reduce costs associated with preparation and disposal of compost. The ability to double-crop mushroom compost would provide growers a chance to increase yields by 40% or more, depending on whether they re-supplement and re-case after 1st, 2nd or 3rd flush.

The variability of antioxidant capacity of 14 strains of the edible oyster mushroom Pleurotus spp. was determined, and the effect of selected mushroom supplements on the longevity of the Mexican fruit fly, Anastrepha ludens, was evaluated. The antioxidant capacity of the fruiting bodies was determined by three different methods, measuring the free radical scavenging activity of methanolic extracts, the OH radical scavenging capacity, and the total phenol content. The inhibition percentage of the DPPH radical varied between 32.6 and 85.7 % and total phenols varied between 30.6 and 143.3 mg/g. The strains with the highest (Pleurotus djamor ECS-0142) and lowest (Pleurotus ostreatus ECS-1123) antioxidant capacity were selected to study their effect on the survival, life expectancy, and mortality of the Mexican fruit fly A. ludens. The results demonstrated differing responses between male and female flies. High concentrations of mushrooms (5 and 20 %) in the diet resulted in a decrease in life expectancy. However, flies on the diet with 1 % P. djamor ECS-0142 showed slightly but significantly greater survival than those on the control diet. The possible adverse effect of protein content in mushroom extracts is discussed.

Administration of human FSH (hFSH) during the diestrus phase in cyclic rats is followed by a reduction in the preovulatory LH surge. This inhibitory action of FSH involves a decrease in the stimulatory effect of gonadotrope progesterone receptor (PR) activation, in a ligand-dependent (progesterone) and -independent (GNRH) manner. PR activation and action are mandatory for LH surge, and are dependent on the phosphorylation of serine (Ser) residues. Together with this post-translational modification, PR is marked for downregulation by proteasome machinery. These experiments used the western blotting technique to measure pituitary expression of PR-A and PR-B isoforms and phosphorylation levels of Ser294 and Ser400 PR-B in rats bearing i) hFSH treatment or ii) PR downregulation. Treatment with hFSH reduced LH secretion and increased that of estradiol in proestrus afternoon. hFSH injections, without altering PR-A and PR-B content or ratio, caused a reduction in phosphorylation of Ser294 and Ser400 but only when pituitaries were previously challenged with progesterone or GNRH for 2 h. However, while pSer294 levels increased after 2 h of pituitary incubation with progesterone or GNRH, those of pSer400 were not modified by thesein vitrotreatments. Finally, progesterone had a biphasic effect: in 2-h incubations increased pituitary PR-A and PR-B content, but after 8 h caused downregulation and altered PR-A:PR-B ratio. The results provide a potential mechanism through which LH levels are decreased by hFSH administration and better understanding of the control of PR expression and phosphorylation in rat pituitaries.

Summary Endosulfan is an insecticide used on many vegetable crops. In mushroom cultivation, vegetable materials used as a growth substrate may contain residues of endosulfan that may accumulate in the final mushroom biomass. After preparing the substrate, it is subjected to pasteurization and/or composting and then inoculated with the desired fungus. The purpose of this research was to determine the rate and extent of endosulfan reduction from a grass substrate that was either composted or sterilized by autoclaving. In addition, the rate and extent of removal of endosulfan from substrate colonized with Pleurotus pulmonarius was determined. The degradation of 65 mg/kg endosulfan was analyzed on both, the substrate preparation and the culture of P. pulmonarius on the grass Digitaria decumbens. During composting in presence of Ca(OH)2 for 120 h, the concentrations of a and b endosulfan were reduced by 61.4 and 49.5% respectively, significantly higher compared with the control (without Ca(OH)2,) in which the reduction was 38.5%. After sterilization the concentration of a and b endosulfan was reduced by 84.8 and 87.5% respectively. After the colonization of substrate by P. pulmonarius (15 days after spawning) a and b endosulfan were reduced by 96% and at the end of cultivation (35 days after spawning) were reduced by 99%. When carpophores were analyzed, residues of a and b endosulfan were observed between 0.019–0.084 mg/kg. The results showed that a and b endosulfan were partially removed during the preparation of substrate and entirely eliminated during fungal colonization on the substrate.

The selective oestrogen receptor modulator (SERM) tamoxifen (TX) has agonist/antagonist actions on LH secretion in the rat. Whereas in the absence of oestrogens TX elicits progesterone receptor (PR)-dependent GnRH self-priming, it antagonizes oestrogen-stimulatory action on LH secretion. The aim of these experiments was to explore whether TX treatment-induced differential expression of oestrogen receptor (ER)alpha and ERbeta in the gonadotrope may determine its agonist effect on LH secretion. In the first experiment, basal LH secretion, GnRH-stimulated LH secretion and PR-dependent GnRH self-priming were determined in incubated pituitaries from ovariectomized (OVX) rats treated with oestradiol benzoate (EB), TX or raloxifene (RX). Cycling rats in metoestrus or pro-oestrus were used as basic controls. As in pro-oestrus, pituitaries from OVX rats treated with EB exhibited GnRH-stimulated LH secretion, immunohistochemical PR expression and GnRH self-priming. While RX had no effect on these parameters, TX induced PR expression and GnRH self-priming. GnRH self-priming was absent in pituitaries incubated with the antiprogestin ZK299. In the second experiment, we evaluated the immunohistochemical expression of ERalpha and ERbeta in gonadotropes of cycling rats and OVX rats treated with EB, TX or RX. We found that while ERalpha expression was similar in all six groups, ERalpha expression was oestrous cycle dependent. Moreover, ERalpha expression in gonadotropes of TX-treated rats was as high as that found in pro-oestrus, while ERalpha expression in the gonadotropes of RX-treated rats was lower than in metoestrous or pro-oestrous pituitaries. These results suggest that, in the absence of the cognate ligand, TX, unlike RX, may regulate LH secretion through the ERalpha subtype in gonadotropes.

The gonadotropic axis is centrally controlled by a complex regulatory network of excitatory and inhibitory signals that is activated at puberty. Recently, loss of function mutations of the gene encoding G protein-coupled receptor 54 (GPR54), the putative receptor for the KiSS-1-derived peptide metastin, have been associated with lack of puberty onset and hypogonadotropic hypogonadism. Yet the pattern of expression and functional role of the KiSS-1/GPR54 system in the rat hypothalamus remain unexplored to date. In the present work, expression analyses of KiSS-1 and GPR54 genes were conducted in different physiological and experimental settings, and the effects of central administration of KiSS-1 peptide on LH release were assessed in vivo. Persistent expression of KiSS-1 and GPR54 mRNAs was detected in rat hypothalamus throughout postnatal development, with maximum expression levels at puberty in both male and female rats. Hypothalamic expression of KiSS-1 and GPR54 genes changed throughout the estrous cycle and was significantly increased after gonadectomy, a rise that was prevented by sex steroid replacement both in males and females. Moreover, hypothalamic expression of the KiSS-1 gene was sensitive to neonatal imprinting by estrogen. From a functional standpoint, intracerebroventricular administration of KiSS-1 peptide induced a dramatic increase in serum LH levels in prepubertal male and female rats as well as in adult animals. In conclusion, we provide novel evidence of the developmental and hormonally regulated expression of KiSS-1 and GPR54 mRNAs in rat hypothalamus and the ability of KiSS-1 peptide to potently stimulate LH secretion in vivo. Our current data support the contention that the hypothalamic KiSS-1/GPR54 system is a pivotal factor in central regulation of the gonadotropic axis at puberty and in adulthood.

Net estrogen sensitivity in target tissues critically depends on the regulated expression of full-length and alternately processed estrogen receptor (ER) isoforms. However, the molecular mechanisms for the control of pituitary responsiveness to estrogen remain partially unknown. In the present communication, we report the ability of different ligands, with distinct agonistic or antagonistic properties at the ER, to modulate the expression of the transcripts encoding ERalpha and ERbeta isoforms, as well as those for the truncated ERalpha product (TERP), and the variant ERbeta2, in pituitaries from ovariectomized rats, i.e., a background devoid of endogenous estrogen. Compared with expression levels at the morning of proestrus, ovariectomy (OVX) resulted in increased pituitary expression of ERbeta and ERbeta2 mRNAs, whereas it decreased TERP-1 and -2 levels without affecting those of ERalpha. Administration of estradiol benzoate (as potent agonist for alpha and beta forms of ER) or the selective ERalpha agonist, propyl pyrazole triol, fully reversed the responses to OVX, while the ERbeta ligand, diarylpropionitrile, failed to induce any significant effect except for a partial stimulation of TERP-1 and -2 mRNA expression levels. To note, the ERbeta agonist was also ineffective in altering pituitary expression of progesterone receptor-B mRNA, i.e., a major estrogen-responsive target. In all parameters tested, tamoxifen, a selective ER modulator with mixed agonist/antagonist activity, behaved as ERalpha agonist, although the magnitude of tamoxifen effects was significantly lower than those of the ERalpha ligand, except for TERP induction. In contrast, the pure antiestrogen RU-58668 did not modify the expression of any of the targets under analysis. Overall, our results indicate that endogenous estrogen differentially regulates pituitary expression of the mRNAs encoding several ER isoforms with distinct functional properties, by a mechanism that is mostly conducted through ERalpha. Differential regulation of ER isoforms may represent a relevant system for the self-tuning of estrogen responsiveness in female pituitary.

SummarySubstrate for cultivating Pleurotus ostreatus was prepared by soaking it in water containing 0.5% lime with no further thermal treatment. Five substrates were used: grass (Digitaria decumbens), corn cob, corn straw and two mixtures of corn straw or grass with coffee pulp. Immersion time varied between 0–48 h. Mushroom yield measured as biological efficiency varied between 37.3 and 126%. No fungal contamination was found, however certain group of bacteria like pseudomonads, bacilli and coliforms were detected. This technique seems to have potential; however good control of the moisture of the substrate must be maintained to limit proliferation of substrate bacteria.

Estrogen (E) is a key regulator of the synthesis and secretion of pituitary reproductive hormones [luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL)]. Until recently, it was thought that all biological actions of E at the pituitary were manifested through a single E receptor (R). The pituitary, like many other reproductive tissues, expresses two isoforms of ER, α and β, both activated by E. The relative contribution of α and β forms in E regulatory actions is largely unknown. To this end, 2-week-old ovariectomized (OVX) rats were injected over 3 days with 25 µg estradiol benzoate (EB), 1.5 mg of propylpyrazole triol (PPT), a selective ERα agonist, 1.5 mg of the selective ERβ agonist diarylpropionitrile (DPN) or a combination of PPT and DPN. Controls were injected with 0.2 ml oil. At 10:00 h on the day after treatment, trunk blood was collected to determine serum concentration of LH, FSH and PRL, and pituitaries were processed for RT-PCR analysis of total (A+B) progesterone receptor (PR) mRNA, immunocytochemistry of PR and incubation. Pituitaries from each of the five groups were incubated in DMEM, with or without 20 nM of the antiprogestin at the receptor ZK299, for 3 h with: 10–8M 17β-estradiol, 10–6M PPT, 10–6M DPN, PPT+DPN or medium alone, respectively, to determine LH, FSH and PRL secretion, and, when challenged with two pulses of 15 min 1 h apart of 10–8M gonadotropin-releasing hormone (GnRH) (GnRH self-priming). EB, PPT and PPT+DPN treatments increased PR mRNA and the number and intensity of nuclei immunoreactive (IR) for PR in gonadotropes, and reduced the number of gonadectomy cells. Like E, PPT alone or in combination with DPN stimulated PRL secretion, increased basal and GnRH-stimulated LH and FSH secretion and induced GnRH self-priming in the absence of ZK299 in the incubation medium. DPN alone had only a significant E-like effect on gonadectomy cells and IR-PR, but not on GnRH self-priming. In addition, while DPN lacked an agonistic action on peripheral tissue and serum pituitary reproductive hormones concentration, EB, PPT and PPT+DPN induced similar uterine ballooning and vaginal cornification, and increased and decreased, respectively, serum concentrations of PRL and gonadotropins. Overall, these results indicate that most of these E actions on the pituitary are exerted through the ERα isoform. The finding that activation of ERβ with its selective DPN agonist had an estrogenic effect on IR-PR nuclei, but not on GnRH self-priming, a characteristic ERα-mediated effect of E, suggests that the biological action of E at the pituitary may involve both isoforms of ER.