[Comments on the 2010 guidelines on cardiopulmonary resuscitation of the European Resuscitation Council]. / Kommentar zu den Leitlinien 2010 zur kardiopulmonalen Reanimation des European Resuscitation Council.

ADULTS: Administer chest compressions (minimum 100/min, minimum 5 cm depth) at a ratio of 30:2 with ventilation (tidal volume 500-600 ml, inspiration time 1 s, F(I)O2 if possible 1.0). Avoid any interruptions in chest compressions. After every single defibrillation attempt (initially biphasic 120-200 J, monophasic 360 J, subsequently with the respective highest energy), chest compressions are initiated again immediately for 2 min independent of the ECG rhythm. Tracheal intubation is the optimal method for securing the airway during resuscitation but should be performed only by experienced airway management providers. Laryngoscopy is performed during ongoing chest compressions; interruption of chest compressions for a maximum of 10 s to pass the tube through the vocal cords. Supraglottic airway devices are alternatives to tracheal intubation. Drug administration routes for adults and children: first choice i.v., second choice intraosseous (i.o.). Vasopressors: 1 mg epinephrine every 3-5 min i.v. After the third unsuccessful defibrillation amiodarone (300 mg i.v.), repetition (150 mg) possible. Sodium bicarbonate (50 ml 8.4%) only for excessive hyperkaliemia, metabolic acidosis, or intoxication with tricyclic antidepressants. Consider aminophylline (5 mg/kgBW). Thrombolysis during spontaneous circulation only for myocardial infarction or massive pulmonary embolism; during on-going cardiopulmonary resuscitation (CPR) only when indications of massive pulmonary embolism. Active compression-decompression (ACD-CPR) and inspiratory threshold valve (ITV-CPR) are not superior to good standard CPR. CHILDREN: Most effective improvement of outcome by prevention of full cardiorespiratory arrest. Basic life support: initially five rescue breaths, followed by chest compressions (100-120/min depth about one third of chest diameter), compression-ventilation ratio 15:2. Foreign body airway obstruction with insufficient cough: alternate back blows and chest compressions (infants), or abdominal compressions (children >1 year). Treatment of potentially reversible causes: ("4 Hs and 4 Ts") hypoxia and hypovolaemia, hypokalaemia and hyperkalaemia, hypothermia, and tension pneumothorax, tamponade, toxic/therapeutic disturbances, thrombosis (coronary/pulmonary). Advanced life support: adrenaline (epinephrine) 10 µg/kgBW i.v. or i.o. every 3-5 min. Defibrillation (4 J/kgBW; monophasic or biphasic) followed by 2 min CPR, then ECG and pulse check. NEWBORNS: Initially inflate the lungs with bag-valve mask ventilation (p(AW) 20-40 cmH2O). If heart rate remains <60/min, start chest compressions (120 chest compressions/min) and ventilation with a ratio 3:1. Maintain normothermia in preterm babies by covering them with foodgrade plastic wrap or similar. POSTRESUSCITATION PHASE: Early protocol-based intensive care stabilization; initiate mild hypothermia early regardless of initial cardiac rhythm [32-34°C for 12-24 h (adults) or 24 h (children); slow rewarming (<0.5°C/h)]. Consider percutaneous coronary intervention (PCI) in patients with presumed cardiac ischemia. Prediction of CPR outcome is not possible at the scene, determine neurological outcome <72 h after cardiac arrest with somatosensory evoked potentials, biochemical tests and neurological examination. ACUTE CORONARY SYNDROME: Even if only a weak suspicion of an acute coronary syndrome is present, record a prehospital 12-lead ECG. In parallel to pain therapy, administer aspirin (160-325 mg p.o. or i.v.) and clopidogrel (75-600 mg depending on strategy); in ST-elevation myocardial infarction (STEMI) and planned PCI also prasugrel (60 mg p.o.). Antithrombins, such as heparin (60 IU/kgBW, max. 4000 IU), enoxaparin, bivalirudin or fondaparinux depending on the diagnosis (STEMI or non-STEMI-ACS) and the planned therapeutic strategy. In STEMI define reperfusion strategy depending on duration of symptoms until PCI, age and location of infarction. TRAUMA: In severe hemorrhagic shock, definitive control of bleeding is the most important goal. For successful CPR of trauma patients a minimal intravascular volume status and management of hypoxia are essential. Aggressive fluid resuscitation, hyperventilation and excessive ventilation pressure may impair outcome in patients with severe hemorrhagic shock. TRAINING: Any CPR training is better than nothing; simplification of contents and processes is the main aim.

Influence of airway management strategy on "no-flow-time" in a standardized single rescuer manikin scenario (a comparison between LTS-D and I-gel).

BACKGROUND: In 2005 the European Resuscitation Council (ERC) published a revised version of the guidelines for Advanced Life Support (ALS). One of the aims was to reduce the time without chest compression in the first period of cardiac arrest (no-flow-time; NFT). We evaluated in a manikin study the influence on NFT using the single use laryngeal tube with suction option (LTS-D) compared to single use I-gel for emergency airway management. METHODS: A randomised prospective study with 200 paramedics who performed standardised simulated cardiac arrest management in a manikin. RESULTS: The use of the LTS-D did not significantly reduce NFT compared with the I-gel (104.7s vs. 105.1s; p>0.05). The LTS-D was inserted as fast as the I-gel (10.4s vs. 9.3s; p>0.05). The LTS-D was correctly positioned by 98% of the participants on the first attempt compared to 96% with the I-gel. During the cardiac arrest simulation, establishing and performing first ventilation took an average of 40.5s with the LTS-D compared to 40.9s with the I-gel. CONCLUSION: In our manikin study, NFT was comparable using the LTS-D and the I-gel. Therefore, for personnel not experienced in tracheal intubation, the LTS-D and the I-gel seem to be equal alternatives in establishing the airway during cardiac arrest. However, relevant clinical studies are appropriate because any change in guidelines in this area must be based on clinical evidence.

The use of the laryngeal tube disposable (LT-D) by paramedics during out-of-hospital resuscitation-an observational study concerning ERC guidelines 2005.

UNLABELLED: In the current guidelines of the European Resuscitation Council (ERC), tracheal intubation, as an instrument for securing the airway during resuscitation, has become less important for persons not trained in this method. For those persons, different supraglottic airway devices are recommended by the ERC. The present investigation deals with the application of the laryngeal tube disposable (LT-D) during pre-hospital resuscitation by paramedics. METHODS: During a period of 2 years (2006-2008), we registered all cardiac arrest situations in which the LT-D had been applied according to the ERC guidelines 2005. Therefore, we investigated one emergency medical system in Germany. RESULTS: During the defined period, 92 resuscitation attempts, recorded on standardised data sheets, were included. The LT-D was used in 46% of all cardiac arrest situations. Overall, the LT-D was successfully inserted in more than 90% of all cases on first attempt. In 95% of all cases, no problems concerning ventilation of the patient were described. CONCLUSION: As an alternative airway device recommended by the ERC in 2005, the LT-D may enable airway control rapidly and effectively. Additionally, by using the LT-D, a reduced "no-flow-time" and a better outcome may be possible.

Inhibition of BCL11B expression leads to apoptosis of malignant but not normal mature T cells.

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.

[Reduction in no flow time using a laryngeal tube: comparison to bag-mask ventilation]. / "No-flow-time"-Reduzierung durch Einsatz des Larynxtubus: Vergleich zur Beutel-Masken-Beatmung.

OBJECTIVE: In 2005 the European Resuscitation Council (ERC) published the new guidelines for Advanced Life Support (ALS). One of the aims was to reduce the no flow time (NFT), without chest compression in the first period of cardiac arrest. Furthermore the guidelines recommend that endotracheal intubation should only be carried out by personnel experienced in this procedure. METHODS: An attempt was made to evaluate whether the use of the laryngeal tube suction (LTS-D) for emergency airway management could contribute to reduce NFT compared to bag-mask ventilation (BMV). In a randomised prospective study 50 participants were asked to perform standardised simulated cardiac arrest management on a full-scale simulator following a one-day cardiac arrest training. Each participant was randomised into the LTS-D and the BMV group for airway management. At the end of each scenario an evaluation of the use of each ventilation procedure by the participants was made by means of a questionnaire. RESULTS: During the manikin scenario (430 s for LTS-D and 420 s for BMV) there was a significant difference in the overall NFT comparing the use of the LTS-D vs. BMV (105.8 s, range 94-124 s vs. 150.7 s, range 124-179 s; p<0.01). This corresponded during the whole scenario to a proportion of 24.6% (LTS-D) or 35.9% (BMV). Using the LTS-D all participants were able to ventilate the manikin successfully (tidal volume 500-600 ml). In a subjective evaluation of the different airway management procedures by the participants more than 90% expressed a positive opinion about the LTS-D with respect to ease of insertion and safety of ventilation. CONCLUSION: The use of the LTS-D on a manikin by emergency physicians after standardised cardiac arrest training significantly reduces the NFT in comparison to BMV. Therefore the LTS-D seems to be a good alternative to BMV during a simulated cardiac arrest scenario.

[Chest compression without ventilation during basic life support? Confirmation of the validity of the European Resuscitation Council (ERC) guidelines 2005]. / Thoraxkompression ohne Beatmung bei der Laienreanimation? Bestätigung der Gültigkeit der "European Resuscitation Council (ERC) Guidelines 2005".

Basic life support (BLS) refers to maintaining airway patency and supporting breathing and the circulation, without the use of equipment other than infection protection measures. The scientific advisory committee of the American Heart Association (AHA) published recommendations (online-first) on March 31 2008, which promote a call to action for bystanders who are not or not sufficiently trained in cardiopulmonary resuscitation (CPR) and witness an adult out-of-hospital sudden collapse probably of cardiac origin. These bystanders should provide chest compression without ventilation (so-called compression-only CPR). If bystanders were previously trained and thus confident with CPR, they should decide between conventional CPR (chest compression plus ventilation at a ratio of 30:2) and chest compression alone. However, considering current evidence-based medicine and latest scientific data both the European Resuscitation Council (ERC) and the German Resuscitation Council (GRC) do not at present intend to change or supplement the current resuscitation guidelines "Basic life support for adults". Both organisations do not see any need for change or amendments in central European practice and continue to recommend that only those lay rescuers that are not willing or unable to give mouth-to-mouth ventilation should provide CPR solely by uninterrupted chest compressions until professional help arrives. It is also stressed that the training of young people especially teenagers as lay rescuers should be promoted and the establishment of training programs through emergency medical organizations and in schools should be encouraged.

Evaluation of a commercially available radioimmunoassay and enzyme immunoassay for the analysis of progesterone and estradiol and the comparison of two extraction efficiency methods.

The measurement of progesterone (P4) and estradiol (E2) is essential for monitoring reproductive cycles and can aid in diagnosing the cause of poor reproductive performance in dairy cattle. Readily available, reproducible, accurate, non-radioactive assays are needed for the assessment of P4 and E2 in bovine serum. The gold standard for hormone assessment, radioimmunoassay (RIA), was compared with enzyme-linked immunoassay (EIA). Serum collected from various points in the estrous cycle was extracted with radiolabeled P4 (ie, 3H-P4; HE) and without 3H-P4 (CE) before being used in the assay. For the assessment of P4, there is a great degree of correlation between the RIA and EIA (adjusted R-square = 0.95; Pearson correlation coefficient (PCC) = 0.98, P < 0.001). A difference between the RIA and EIA methods was not detected for E2 concentrations (P = 0.16), but the correlation between techniques was poor (adjusted R-squared = 0.73; PCC = 0.87, P = 0.002). There was no difference in the serum extraction efficiency as measured with 3H-P4 as opposed to without (P = 0.94). The two methods for the measurement of serum extraction efficiency were highly correlated (adjusted R-square = 0.83; PCC = 0.92, P < 0.001). The concordance correlation coefficient (CCC) showed an excellent agreement between RIA and EIA for P4 determination (0.89) and between HE and CE methods (0.90). Although the 95% limits of agreement of the Bland-Altman plots encompassed 89% (8/9) and 92% (12/13) of the differences between methods for P4 quantification and extraction respectively, the CCC indicated an excellent agreement among them. The CCC between RIA and EIA for E2 quantification was 0.68 which corresponds with a fair agreement; however, the 95% limits of agreement of the Bland-Altman plot encompassed 100% (9/9) of differences between methods. The EIA and CE methods are comparable alternatives to the RIA and HE methods, respectively and can be used to quantify P4 and E2 for bovine serum.

Outcome of cardiopulmonary resuscitation in intensive care units in a university hospital.

UNLABELLED: The purpose of this study is to evaluate the demographic characteristics of patients who suffered cardiac arrest in our intensive care units (ICUs) as well as to identify those factors influencing outcome after resuscitation following cardiac arrest. METHODS: We reviewed the records of all patients who underwent cardiopulmonary resuscitation (CPR) in our ICUs at the Georg-August University Hospital, Goettingen, Germany, from January 1, 1999 to December 31, 2003. RESULTS: One hundred and sixty-nine patients underwent CPR. Severity of illness assessed by SAPS II score on admission was 51.8+/-18.5 (predicted mortality 46.6%). The initially monitored rhythm at the time of arrest was asystole in 51 (30.2%) patients. Ventricular tachycardia/fibrillation (VT/VF) was recorded in 65 (38.5%) and pulseless electrical activity in 49 (29.0%) patients. Twenty (23.8%), 28 (33.3%) and 33 (39.3%) patients with initially recorded asystole, VT/VF and pulseless electrical activity (PEA) rhythms, respectively, survived to ICU discharge. Eighty of the 169 patients survived to hospital discharge giving a survival rate of 47.3%. The highest ICU mortality was seen in patients admitted for neurosurgery (80%) followed by major vascular surgery (77.8%), non-surgical patients (67.4%) and patients with severe sepsis (66.7%). The occurrence of cardiac arrest within the first 24h was associated with a significantly lower ICU mortality compared to a later incident. At hospital discharge 66 patients (82.5% of the survivors) achieved good cerebral recovery, 12 patients (15.0%) were severely disabled (CPC 3) while 2 (2.5%) remained unconscious. CONCLUSION: Several factors affect the outcome from CPR. However, quicker triage to ICU, closer monitoring along with prompt intervention might minimise the consequences of cardiac arrest and its complications.

Ion transporters for fluid reabsorption in the rooster (Gallus domesticus) epididymal region.

Testicular fluid is highly condensed during its passage through the epididymal region in the avian species. In the present study, major ion transporters that are responsible for condensation mainly by water resorption in the reproductive tract as identified in the mammalian epididymis were localized within the rooster (Gallus domesticus) epididymis by immunohistochemistry. The results show that the efferent ductule epithelium expressed sodium-potassium ATPase (Na(+),K(+)-ATPase), carbonic anhydrase II (CAII) and sodium hydrogen exchanger isoform 3 (NHE3) and that the connecting ductule and epididymal duct epithelia expressed Na(+),K(+)-ATPase and CAII. These data suggest that a model proposed for reabsorption in mammalian efferent ductules can be applied to avian efferent ductules.

Outcome of cardiopulmonary resuscitation in the intensive care units of a university hospital.

The purpose of this study is to evaluate the demographic characteristics of patients who suffered cardiac arrest in our ICUs and to identify those factors influencing outcome after resuscitation following cardiac arrest. We reviewed the records of all patients who underwent CPR in the two ICUs at the Georg-August University Hospital Goettingen, Germany from 1 January, 1999 to 31 December, 2003. During the study period 169 patients underwent CPR and 80 of the 169 patients survived to hospital discharge, giving a survival to hospital discharge rate of 47.3%. The initial monitored rhythm recorded at the time of arrest was asystole in 99 (58.6%) patients, ventricular tachycardia/fibrillation in 59 (34.9%) and pulseless electrical activity in 7 (4.1%) patients. The respective survival rates were 46 (54.8%), 31 (36.9%) and 5 (6.0%) to hospital discharge. Of the 80 patients that survived to hospital discharge 75 (93.8%) achieved good cerebral recovery (CPC 1 or 2) and were alert and fully oriented on discharge; 4 patients (5.0%) were severely disabled (CPC 3), while 1 (1.2%) remained unconscious and was reported dead five days after discharged to another local hospital. Illness severity as assessed by SAPS II score on admission was 38.8 +/- 16.0. None of our patients with > 40 SAPS II score 24 hours after CPR survived to be discharged from the ICU. Our study showed that nearly half the patients that had cardiac arrest in our hospital ICUs had a favourable outcome despite initial rhythms that are traditionally associated with a poor outcome. This confirms that good results are achievable in these groups of patients.

Localization of progesterone receptors in pre- and postovulatory follicles of the domestic hen.

Progesterone may act locally to modulate follicular maturation and ovulation in the domestic hen. The distribution of progesterone receptors (PR) in the pre- and postovulatory follicles was determined in hens by immunocytochemistry and Western blot analysis. Monoclonal antibodies to chicken PR, PR6, and PR 13, were used. PR were localized in nuclei of theca externa fibroblasts and germinal epithelial cells in stigma and nonstigma regions of the third largest preovulatory follicle (F3). In the largest preovulatory follicle (F1), PR were present in theca externa fibroblasts, germinal epithelial cells, and also in granulosa cells and some of the theca interna fibroblasts in the stigma and nonstigma region. Twenty-four hours after ovulation, PR in the fibroblasts of the theca externa of the postovulatory follicle (POF) were remarkably reduced, but the amount of PR in the granulosa cells was similar to that observed in the F1. A high density of PR was also found in the fibroblasts, arterial wall, and smooth muscle fibers in the loose connective tissue of pre- and postovulatory follicles. Western blot analysis indicated that PR in the granulosa and theca tissue were identical in molecular weight to PR in the shell gland. Western blot analysis also confirmed the changes in the amounts of PR in the pre- and postovulatory follicles as determined by immunocytochemistry. The relative amounts of PR in the granulosa cells as determined by Western blot analysis was F2 less than F1 = POF, and in theca tissue was F2 = F1 greater than POF. The presence of PR in specific ovarian tissues suggests that these tissues are target tissues for progesterone and that progesterone may have a role in regulating follicular maturation and ovulation through receptor-mediated pathways.

Enhancement of in vivo humoral immunity by estrogen: permissive effect of a thymic factor.

Physiological levels of estrogen enhance humoral immune responses. Several in vitro studies indicate the hormone to have a direct effect on immune cells, and other studies show that estrogen may affect humoral immunity indirectly through the thymus. Therefore, we have conducted experiments to investigate the requirement of the thymus in the enhancement of humoral immune responsiveness by estrogen. In Exp I, adult ovariectomized Lewis rats were thymectomized or sham thymectomized and given estradiol (E2; 0.25 microgram E2 in sesame oil, sc, once every 4 days) or the oil vehicle in a 2 x 2 factorial design, and their antifluorescein responses were followed by enzyme-linked immunosorbant assay across 21 days. Only animals that were thymus intact and given estrogen replacement showed significantly (P less than 0.05) greater serum antifluorescein titers than all other treatments. In Exp II, ovariectomized thymectomized rats were submitted to a 2 x 3 factorial design of oil vehicle or E2 replacement and saline, gelatin, or thymus replacement (thymosin fraction 5; 1 mg/kg in saline, sc). As described above, only the animals receiving both thymosin fraction 5 and E2 replacement displayed antifluorescein titers that were significantly (P less than 0.03) increased over titers of all other treatment groups. These results indicate that the enhancement of in vivo humoral immunity by estrogen requires the thymus, and that a constitutive thymic factor, found in thymosin fraction 5, exerts a permissive influence on the action of E2 outside the thymus to increase a specific humoral immune response.

Modulation of the primary and secondary antifluoresceyl antibody response in rats by 17 beta-estradiol.

Estradiol (E2) enhances humoral immunity, but the quantitative and temporal relationship between serum E2 and antibody titers is unclear. The present study was therefore designed to determine whether the sex of the animal, ovariectomy (OVX), and replacement with various tonic levels of serum E2 influenced the primary and secondary antifluorescein antibody titers of rats. Two experiments were performed. In Exp I, intact male and intact female Lewis inbred rats were given primary and booster (secondary) immunizations with fluorescein-keyhole limpet hemocyanin. Primary and secondary bleedings for antifluorescein antisera were obtained. In Exp II female Lewis inbred rats were randomly distributed among the following treatment groups: sham OVX, sham implanted; OVX, sham implanted; and three groups of OVX and implanted with E2-filled Silastic capsules of 0.5, 1.5, or 5.0 cm lengths. Immunization with fluorescein-keyhole limpet hemocyanin and bleedings for antifluorescein antisera followed the same schedule as in Exp I. In Exp I, females had significantly higher antifluorescein antibody titers than males during the primary and secondary response. In Exp II, OVX significantly depressed the primary and secondary antifluorescein antibody titers relative to sham OVX controls. During the primary antifluorescein antibody response, a significant quadratic relationship was seen between antibody titers and serum E2 when measurements included the pharmacological range of the serum E2 concentrations. A significant linear relationship existed between serum E2 and antifluorescein antibody titers when the same data were examined over a range of 15-130 pg E2/ml. This linear relationship is more significant during the primary response than the secondary response. It is concluded that females generate higher antifluorescein antibody titers than males; OVX significantly depresses the antifluorescein antibody response; E2 is immunoenhancing in the physiological range and immunosuppressive in the pharmacological range; and the presence of E2 during the primary response may be more critical to immunoenhancement.

Preovulatory depletion of ovarian catecholamines in the rat.

The purpose of the present investigation was to correlate changes in ovarian catecholamines and serum pituitary hormones and sex steroids. As the experimental model we used the prepubertal rat injected with 7.5 IU PMS on day 28 at 0900 h. Within 24 h after the injection, ovarian weight increased, and on day 30 at 2400 h, it was twice that of controls. On day 31, all of the PMS-treated rats but none of the controls had ovulated, and the average number of ova was 9. Serum estradiol in these rats was elevated significantly on the afternoon of day 30 and was followed by a preovulatory surge of LH and FSH. Serum progesterone was also increased on the evening of day 30, and an elevation in serum PRL on the morning of day 30 in the PMS-treated rats was evident, whereas serum GH was unchanged. Ovarian norepinephrine (NE) dropped from 65.5 +/- 6.9 pg/mg ovary (mean +/- SE) before the PMS injection of 14.4 +/- 1.5 pg/mg ovary on the evening of day 30. This depletion proceeded in two apparent phases: an initial drop within 12 h after PMS injection, and a further reduction coincident with the gonadotropin surge. Throughout this period, ovarian NE in the controls increased 50--60%. There was a 2-fold reduction in ovarian dopamine in the PMS-primed rats, but the levels were 10-fold lower than those of NE. The data document that the hormonal profiles and the number of ovulating follicles of the prepubertal rat primed with a low dose of PMS resemble those of the normal cycling rat. A significant depletion of ovarian catecholamines was observed during the preovulatory period, which is temporally related to increases in serum gonadotropins.

Inhibition of the activities of P450 cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase and the amount of P450 cholesterol side-chain cleavage by testosterone and estradiol-17 beta in hen granulosa cells.

We have found that androgens and estradiol-17 beta (E2) produced by theca cells suppress progesterone (P4) secretion by granulosa cells of the domestic hen in a dose-dependent manner. Furthermore, testosterone (T) and E2 inhibited the conversion of cholesterol to pregnenolone (P5) and of P5 to P4, respectively. The aim of this study was to determine if T and E2 suppress P4 biosynthesis by changing activities of the cytochrome P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (Exp I) and the amount of P450scc (Exp II). Granulosa layers of the largest follicle of two to four hens were obtained 22 h before ovulation, pooled, and isolated granulosa cells were prepared. In Exp I, the specific activities of the P450scc and 3 beta-HSD were measured in mitochondrial and microsomal proteins of granulosa cells, respectively, in the presence of T or E2 (0-10 microM). Addition of T to mitochondrial proteins increased the Michaelis-Menten constant (Km) with no change in the maximum velocity (Vmax) of the P450scc, which suggests competitive inhibition (Ki = 30.9 microM), whereas E2 had no effect on Km and Vmax of the P450scc. Likewise, addition of E2 to microsomal proteins increased the Km with no change in the Vmax of the 3 beta-HSD, which suggests competitive inhibition (Ki = 15.1 microM), whereas T had no effect on Km and Vmax of the 3 beta-HSD. In Exp II, granulosa cells (3 x 10(5)/3 ml.tube) were incubated for 0-12 h in triplicate for each combined treatments of 25-OH-cholesterol (8 microM) and cyanoketone (10 microM), T, or E2 (0-10 microM) in the presence or absence of LH (25 ng). Protein content and P5 secretion were measured and the amount of P450scc was determined by Western blot analysis. Incubation of granulosa cells with T decreased the amount of the P450scc in granulosa cells cultured for 12 h and P5 secretion in granulosa cells cultured for 3 h or longer (P less than 0.05), without a change in protein content and cell viability. Our results suggest that P4 production by granulosa cells is suppressed by T and E2 acting as competitive inhibitors of the P450scc and 3 beta-HSD, respectively, and by T decreasing the amount of the P450scc. We conclude that steroidogenesis in the follicle of the chicken is regulated through the interaction of theca and granulosa layers.

Inhibitory sites of androgens and estradiol in progesterone biosynthesis in granulosa cells of the domestic hen.

In a previous in vitro study we found that androgens and estradiol (E2) suppress progesterone (P4) production by the granulosa cells isolated from the largest follicle of the domestic hen in a dose-dependent manner. The presence of an aromatase inhibitor did not block the inhibitory action of androgens. The addition of androgen plus E2 to the granulosa cells had an additive effect on suppressing P4 secretion. The aim of this study was to determine the loci in the steroid biosynthetic pathway where androgens and E2 inhibit P4 production by the granulosa cells. Granulosa layers of the largest follicles removed from two or three hens 22 h before ovulation were pooled. Dispersed granulosa cells were incubated for 3 h in triplicate for each treatment, and pregnenolone (P5) and P4 secretion were measured in medium and cells. Experiments were replicated three or four times. Treatment of granulosa cells with cyanoketone (0-100 microM), an inhibitor of 3 beta-hydroxysteroid dehydrogenase, increased P5 production and decreased P4 production in a dose-dependent manner, with maximal production of P5 and suppression of P4 production at 10 microM cyanoketone. The addition of 25-hydroxycholesterol (25OHCh) or P5 (0-16 microM) caused a dose-related increase in basal and LH-stimulated steroid production. The maximal production of P5 or P4 was found at 8 microM 25OHCh or P5. Also, the effect of LH (0-100 ng) on granulosa cell steroidogenesis was examined with or without 8 microM 25OHCh or P5. The half-maximal and maximal doses for P5 or P4 production were 5 and 25 ng LH, respectively. Next, suppression of P5 production by androstenedione, testosterone, dihydrotestosterone, and E2 (each at 0-10 microM) was tested in the presence of 25OHCh plus cyanoketone with or without LH. We found a dose-dependent suppression of P5 production by androgens (1-10 microM), but not by E2. However, when we added the above steroids to granulosa cells in the presence of P5 with or without LH, only E2 (1 and 10 microM) caused a significant suppression of P4 production. Our results suggest that 1) androgens primarily act at the conversion site of cholesterol to P5 to suppress P4 production; and 2) E2 acts at the conversion site of P5 to P4 to suppress P4 production. We conclude that production of androgens and E2 by thecal cells may regulate P4 biosynthesis by granulosa cells in the domestic hen.

Elevated catecholamines in porcine follicular fluid before ovulation.

The purpose of this study was to correlate changes in catecholamine concentrations in porcine follicular fluid and cyclic events in the ovary. Follicular fluid was aspirated from follicles of ovaries obtained from pigs throughout the 21-day estrous cycle and analyzed for norepinephrine (NE), epinephrine (EPI), and estradiol (E2). Serum was obtained from cycling pigs on days 6-10 and 16-20 of the cycle and assayed for NE, EPI, E2, and progesterone. The concentrations of NE in the follicular fluid were relatively constant during days 1-15 of the luteal phase (1.7 +/- 0.2 ng/ml), but were elevated significantly to 2.9 +/- 0.4 ng/ml during the follicular phase (days 16-20). EPI had a similar profile, but a 6- to 10-fold lower concentration. The follicular fluid E2 concentration increased from 15.6 to 76.5 ng/ml during the luteal phase to 630 ng/ml during the follicular phase. Serum NE and EPI concentrations were similar during midluteal and follicular phases, whereas progesterone and E2 were significantly elevated during the luteal and follicular phases, respectively. These results indicate that catecholamines in follicular fluid are elevated significantly during the follicular phase of the estrous cycle and may have a physiological role in preovulatory events as well as during the luteinization process.

Multiple steroidogenic cell populations in the thecal layer of preovulatory follicles of the chicken ovary.

The thecal layer of the preovulatory follicle of the chicken ovary produces primarily androgens and estrogens. However, the precise cellular sites of androgen and estrogen synthesis in the thecal layer have not been identified. Therefore, our aims were 1) to identify steroidogenic cells in the thecal layer of the preovulatory follicles by localizing specific steroidogenic enzymes in these cells by immunocytochemistry, and 2) to confirm that these cells which contained the specific steroidogenic enzymes secreted the expected steroid in a short term cell incubation. Follicles were collected 2 h after oviposition and prepared for immunocytochemistry and short term cell incubation. Immunocytochemistry for cholesterol side-chain cleavage cytochrome P450 (P450SCC), 17 alpha-hydroxylase cytochrome P450 (P450C17), and aromatase cytochrome P450 (P450AROM) was performed to localize pregnenolone-, androgen-, and estrogen-producing cells, respectively, on frozen sections and paraffin sections of the five largest preovulatory follicles. Interstitial cells showed immunoreactivity for both P450SCC and P450C17, whereas a specific cell population of the theca externa, hereafter termed aromatase cells, showed immunoreactivity for P450AROM. Furthermore, fibroblasts in the theca externa indicated immunoreactivity for P450C17. The immunoreactivity of P450C17 and P450AROM enzymes in specific cells in the theca externa appeared to decrease with follicular maturation. The third largest, fourth largest, and fifth largest follicles were selected for short term cell incubations because the immunoreactivity for P450 enzymes in the thecal layer of these follicles was high. Isolated theca interna cells, theca externa cells, and a combination of interna and externa cells were incubated with/without ovine LH (oLH) for 3 h. The medium was assayed for progesterone, testosterone, and 17 beta-estradiol by RIAs. Incubation of theca interna cells with oLH increased progesterone and testosterone production in a dose-dependent manner. However, we did not observe any production of progesterone and testosterone by theca externa cells. Theca externa cells produced 17 beta-estradiol, and its production was increased significantly when theca interna and externa cells were coincubated in the present of oLH. Based on these data, we propose a multiple cell theory for steroidogenesis in the thecal layer of preovulatory follicles of the chicken ovary which states that interstitial cells in the theca interna produce progestins and androgens, fibroblasts in the theca externa may function as an additional site for the conversion of progestins to androgens, and aromatase cells in the theca externa require androgens as substrate to produce estrogens.

Inhibition of progesterone secretion from granulosa cells by estradiol and androgens in the domestic hen.

We previously reported no difference in progesterone (P4) secretion from the granulosa layer of the largest follicle (F1) of the domestic hen regardless of the maturity of the F1 follicle. However, coincubation of the granulosa and thecal layers resulted in inhibition of P4 secretion from the less mature F1, but not from the more mature F1. The goal of this study was to determine if estradiol (E2) and androgens secreted by the thecal layer suppress P4 production by the granulosa cells. We removed the granulosa layer from less mature F1 follicles and dispersed granulosa cells (1 x 10(5)) were incubated (3 h) in triplicate with one of these treatments: control, E2, testosterone (T), androstenedione (A), and dihydrotestosterone (DHT; at concentrations of 1 x 10(-7), 1 x 10(-6), and 1 x 10(-5) M), LH (100 ng) as well as LH plus E2, T, A, and DHT at the same concentrations. P4 secretion was measured in the medium and cells, and the experiment was replicated seven times. We found a dose-related suppression of basal and LH-stimulated P4 production by all steroids. In a second experiment (n = 3-5), we tested the specificity of the androgens in suppressing P4 production by granulosa cells by using the aromatase inhibitor 7-(4'-amino)phenylthio-4-androstene-3,17-dione. This compound did not reduce the effectiveness of T in suppressing P4 production. Finally in Exp 3 (n = 4-7), E2 and T were tested individually and in combination at concentrations of 1 X 10(-8)-1 X 10(-5) M. We found a possible synergistic effect, in that the combination of E2 plus T suppressed P4 to a greater degree than either steroid alone. Our results indicate that 1) E2 and androgens suppress basal and LH-stimulated P4 production by granulosa cells in a dose-related manner; 2) androgen suppression of P4 production is not mediated by aromatization to estrogen; and 3) the suppressive effects of E2 and androgens may be synergistic. We conclude that E2 and androgens secreted by the thecal layer may regulate P4 production by the granulosa layer.

Effect of LH injection on plasma and follicular steroids in the chicken.

During the 26-h laying cycle of the hen the three sex steroids, progesterone (P4), estrogen (E), and testosterone (T), show peaks and valleys in both the follicle wall and the peripheral plasma. Furthermore, for E and T (but not for P4) at comparable times, there is an inverse relationship between the size of the follicle and the concentration of these hormones. Shortly before ovulation all three steroids peak in synchrony, at other times only E and T rise and fall together while P4 shows no change. The question arose whether similar changes in steroid synthesis can be produced by exogenous LH. A single injection of 25 mug of ovine LH was given to laying hens at a time of the laying cycle when no natural peaks of the steroids are seen. The hens were killed at 5, 30, or 90 min after injection and the walls of the three largest follicles as well as the plasma of these hens were assayed by RIA for P4, T, and E. Both the follicle walls and the plasma showed highly significant (P smaller than 0.005) increases in T and P4 but not in E, even though E does show significant changes during the normal cycle. The reason for this difference in response of the steroids is being investigated. The 3 largest follicles respond to exogenous LH in the same size order as do untreated hens during the cycle. The conclusion is drawn that peaks of T and P4 normally seen are due to endogenous releases of LH while E seems to be controlled differently.