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To evaluate the effects of repeated intravitreal dexamethasone implant.

Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function.

Severe hyperbilirubinemia is often associated with acute kidney injury (AKT). Considering the potential renal toxicity of bilirubin, it is possible that bilirubin removal per se could represent a way to improve renal function in patients experiencing jaundice-related AKI. Here we present the case of a 47-year-old male patient, who was admitted due to severe jaundice and ascites. The patient presented a history of idiopathic myelofibrosis, associated with liver disease with stable bilirubin values of about 4 mg/dL.At admission, blood tests showed plasma bilirubin of 45 mg/dL, normal liver enzymes and AKI (creatinine 2.1 mg/dL vs a previous value of 0.7 mg/dL). Instrumental examinations and liver biopsy showed a picture of diffuse inflammatory cholangitis. During hospitalization, bilirubin levels further raised (up to 59.7 mg/dL) and, simultaneously, kidney function declined. We excluded functional causes of renal failure and hepatorenal syndrome and thus, suspecting a bilirubin-associated nephrotoxicity, we decided to treat the patient with plasma adsorption perfusion (PAP), a technique based on plasma adsorption by a bilirubin-specific adsorbent. After the treatment plasma bilirubin, as well as creatinine serum levels, progressively decreased. Unfortunately, about 10 days after the beginning of PAP the patient died of septic shock. This case represents an additional proof of the potential nephrotoxicity of bilirubin, suggesting that PAP could be a valuable therapeutic option in patients with jaundice-related AKI.

The human kidney-specific chloride channels ClC-Ka (rodent ClC-K1) and ClC-Kb (rodent ClC-K2) are important determinants of renal function, participating to urine concentration and blood pressure regulation mechanisms. Here we tested the hypothesis that these chloride channels could represent new drug targets for inducing diuretic and antihypertensive effects.

The KEAP1/Nrf2 pathway is a master regulator of several redox-sensitive genes implicated in resistance of tumor cells against chemotherapeutic drugs. Recent data suggest that epigenetic mechanisms may play a pivotal role in the regulation of KEAP1expression. We performed a comprehensive genetic and epigenetic analysis of the KEAP1 gene in 47 non-small cell lung cancer tissues and normal specimens. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real time. Methylation at the KEAP1promoter region was detected in 22 out of the 47 NSCLCs (47%) and in none of the normal tissues analyzed. Somatic mutations were detected in 7 out of the 47 tumors (15%) and loss of heterozygosity (LOH) in 10 out of the 47 (21%) of the cases. Overall, we found at least one molecular alteration in 57% of the cases. Approximately one third of the tumors had two alterations and this feature was associated with higher risk of disease progression in univariate COX regression analysis (HR = 3.62; 95% CI 1.24–10.65, p = 0.02). This result was confirmed by Kaplan-Meier analysis, which demonstrated an association between worst outcome and KEAP1double alterations (p = 0.01, Log rank test). Our results further suggest that deregulation of the NRF2/KEAP1 system could play a pivotal role in the cancerogenesis of NSCLC. In addition identifying patients with KEAP1genetic and epigenetic abnormalities may contribute to disease progression prediction and response to therapy in lung cancer patients.

Achalasia is a primary esophageal motor disorder characterized by degenerative changes of the myenteric plexus. The pathophysiologic abnormalities may be the final result of several intermeshing mechanisms, and more than one single factor may cause the motor abnormalities.

The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2mRNA expression and hence prostaglandin E2synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the national dairy herd. The aims of this study were to determine the effect of copper and/or copper chelating thiomolybdates on LH-induced differentiation by looking at the effects on androgen production, steroidogenic enzymes (cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage) and lysyl oxidase mRNA expression in cultured theca cells maintained under serum-free conditions.The effect of thiomolybdates and copper on LH differentiation was investigated by supplementing (ammonium) tetrathiomolybdate to optimum theca cell culture media at 0–100 μg/ml, copper (chloride) at equimolar concentrations (0–51.6 μg/ml) or equimolar combinations of both media. Lysyl oxidase mRNA expression was investigated with semi-quantitative RT-PCR, whilst expression of cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage mRNA was examined using real time PCR. Both PCRs used bovine specific primers and cell lysates obtained from bovine theca cells cultured for 6 days and in the presence or absence of the 100 μg/ml dose of thiomolybdate and equimolar copper thiomolybdate.Thiomolybdate depressed androstenedione production in a dose-dependent manner at doses greater than 1 μg/ml at 96 h (P<0.05); doses above 20 μg/ml were all greatly reduced at all time points and at 192 h when related to numbers of cells (P<0.001). Copper alone had no effect at physiological doses, but the use of the equimolar copper thiomolybdate reversed the effect of tetrathiomolybdates on androstenedione production at the 20 μg/ml dose. Thiomolybdate supplementation, with and without copper, had no significant effect on the level of lysyl oxidase or cytochrome P450 side-chain cleavage expression. However, cytochrome P450 17α-hydroxylase expression was significantly increased (P<0.05) by tetrathiomolybdate, possibly due to a local regulatory system.In conclusion, these results demonstrate that thiomolybdates can prevent LH-induced differentiation of bovine theca cells in vitroand that these effects can be ameliorated by copper supplementation. Our results also indicate that it is unlikely that the effects of thiomolybdate are mediated at the transcriptional level and further work is required to determine if thiomolybdate exerts its effects through post-translation processing or some other unrelated mechanism. Overall, these data support the hypothesis that copper responsive subfertility results from perturbation of the normal pattern of ovarian steroidogenesis.

We conducted an interview in the summer and autumn of 1991 with Dr Enrico Chaim Pajes, a Jewish Polish doctor who trained in Italy before World War II. Pajes was arrested when Italy entered the war in 1940. For the next three years he was rotated through various prisons. He witnessed the bombing of Campagna in 1943 and risked his life to treat the wounded. After the war he started a new life as the head of a medical clinic for Jewish displaced persons in Grottaferrata, in the suburbs of Rome. How did a Jew of Polish origin end up practising medicine in Italy for over 40 years? This oral history is the story of Enrico Pajes's life.

The purpose of this research is micropropagation of Ginkgo bilobaL. Apical and nodal meristems removed from plantlets or apical buds from a tree were used as explants. Meristems produced an extensive callus and single or rare multiple shoots on Murashige and Skoog medium with different growth regulators and endosperm extract (En) obtained from mature seeds of the same species. For successful root production it was necessary to transfer the shoots to a rooting medium with En.

Improved nutrition increases ovulation rate in sheep and there is evidence that intra-ovarian pathways mediate responses to nutrition. An experiment was conducted to examine the effect of dietary energy on folliculogenesis. Anoestrous Merino ewes were fed a diet of wheat straw alone (control, n = 5), or wheat straw supplemented with lupins (500 g day(-1), n = 5). Other ewes were fed wheat straw and infused with glucose (50 mmol h(-1), n = 5) or with glucosamine (3.5 mmol h(-1), n = 5). Intravaginal progestagen sponges were inserted for 12 days, and nutritional treatments were started 5 days before sponge removal. At sponge removal, the ewes were injected with a regimen of GnRH pulses (500 ng every 4 h from 0 to 12 h; 250 ng every 2 h from 14 to 24 h; and 200 ng every 1 h from 25 to 36 h) to simulate normal follicular development. Thirty-six hours after sponge removal, the animals were killed and the ovaries were collected and stored at -80 degrees C. The ovaries were sectioned serially every 10 microm. Every 20th section was stained (to estimate number and diameter of follicles) and every 17-19th section was probed by in situ hybridization for P(450) aromatase. Data were analysed using ANOVA and chi-squared tests. There was an effect of treatment (P < 0.05) on the number of follicles 2-3, 3-4 and 6-7 mm in diameter. Aromatase-positive follicles (1.6-7.9 mm) were detected in 31 follicles from 15 ewes across all four groups. In ten animals, the largest follicle was aromatase-positive. The diameters of aromatase-positive follicles were larger (P = 0.004) in lupin fed compared with glucose-infused ewes (4.9 +/- 0.5, 3.6 +/- 0.7, 5.3 +/- 0.5 and 4.2 +/- 0.5 mm for control, glucose-infused, lupin-fed and glucosamine-infused groups, respectively). Treatment did not affect the plasma concentration of FSH when compared with controls, indicating that the energy supplements were modifying recruited (2-3 mm and 3-4 mm) and selected follicles (> 6 mm) directly. In conclusion, dietary energy can directly stimulate folliculogenesis in recruited and selected follicles, and this effect may be mediated by changes in systemic leptin concentrations and the hexosamine energy-sensing pathway in the follicle.

Kaposi's sarcoma (KS) develops upon reactivation of human herpesvirus 8 (HHV8) infection and virus dissemination to blood and tissue cells, including endothelial and KS spindle cells where the virus is mostly present in a latent form. However, this may likely require the presence of compromised host immune responses and/or the evasion of infected cells from the host immune response.In this regard, mechanisms of evasion of productively infected cells from both CTL and NK cell responses, and resistance of latently infected cells from specific CTL, have already been shown. Here we show that cells which are latently infected by HHV8 are indeed efficiently lysed by NK cells from individuals with a normal immune response. Notably, NK cell-mediated immunity was found to be significantly reduced in AIDS patients with progressing KS as compared to both HIV-negative patients with indolent classic KS or normal blood donors. However, it was restored after treatment with the highly active antiretroviral therapy (HAART) in AIDS-KS patients, that showed regression and clearance of HHV8 from PBMC. By contrast, AIDS-KS patients with a more aggressive disease and no clinicalresponse had persistent HHV8 viremia associated with reduced NK cell cytotoxicity. These results suggest a key role for NK cells in the control of HHV8 latent infection, KS development, and in disease remission upon HAART.

The stimulatory effect of nutritional supplementation on ovarian activity in sheep has been linked to an increase in glucose availability that, with insulin, directly decreases follicular steroidogenesis. Glucose uptake occurs by glucose transporters, but it is not known which glucose transporters are present in the sheep ovary or whether they are affected by nutritional stimulation. The aim of this study was to determine whether widely distributed glucose transporter 1 (GLUT1) or insulin-responsive GLUT4 are present in the granulosa or theca cells of sheep ovarian follicles, and whether their concentrations are affected by nutritional stimulation. Merino ewes (n = 49-51 per group) were stimulated nutritionally for 5 days before luteolysis with lupin grain or with one of two regimens of a glucogenic mixture, administered orally, which increases blood glucose concentrations towards the upper end of the normal range. Water was used as a control. Ovaries (n = 3 per group) were dissected and the granulosa cells and thecal shell from individual follicles were examined for glucose transporters using western blotting. GLUT1 concentration was 7-18 times higher in the granulosa than in the theca cells. GLUT4 was detected at a similar concentration in both types of cell. Nutritional treatment had no effect on the concentration of GLUT1 or GLUT4 in either tissue, and did not increase ovulation rate, despite increased concentrations of glucose and insulin. Concentrations of glucose transporters were not correlated with follicular concentrations of oestradiol or androstenedione. The presence of GLUT1 and GLUT4 in the granulosa and theca of sheep follicles indicates that the transporters have a role within the ovary in the modulation of follicular function.

A previous study demonstrated for the first time that a drug such as caffeine, administered prior to ovulation and genomic activation, causes a quantitative difference in growth-promoting energy utilization in a proportion of 5-day-old blastocysts. The objective of the present study was to investigate whether developmental changes induced by caffeine administered throughout the estrus cycle prior to fertilization are sustained throughout pregnancy and after birth. Caffeine was administered to rats throughout the estrus cycle prior to fertilization, with control and experimental groups subdivided into preimplantation and postimplantation categories. Preimplantation fertilization rate was assessed on day 4 of pregnancy by a pregnancy-induced elevation in maternal plasma progesterone concentration, or by flushing each uterine horn on day 5 of pregnancy to determine the presence or absence of a litter. Postimplantation fetuses were collected on gestational day 12 or allowed to go to term. Preconceptual caffeine exposure significantly reduced maternal fertility by the failure of a proportion of the litters to implant, rather than curtailing preimplantation development or postimplantation losses. Postnatal mortality between weeks 0 and 1 was elevated and the weekly incremental growth rate of the pups from week 3 through week 7 was significantly reduced in the preconceptually caffeine-treated offspring. Experimental females reached puberty at the same age as the controls but at a significantly lower body weight. Gestation length, birthweight, litter size, sex ratio, and anogenital distance (a measure of prenatal androgenization) were not affected by preconceptual caffeine treatment. It was concluded that the reduced fertility rate in preconceptually caffeine-exposed rats was due to the failure of litters to implant rather than to a reduced fertilization rate, which was normal. It was further concluded that the growth rate over the neonatal and prepubertal periods of surviving pups in the caffeine-treated group was subnormal. J. Matern.-Fetal Med. 1999;8:220–224. © 1999 Wiley-Liss, Inc.

Improving ewe nutrition even for short periods will increase ovulation rate. The increased nutrients must in some way affect the number of follicles that develop to the pre-ovulatory stage. One possible mechanism is that a nutrient or a metabolic hormone that responds to nutrition might act directly on the ovary to influence follicle development and/or follicle selection. In the study described here, insulin and glucose, alone or together, were infused directly into the ovarian artery of ewes with an autotransplanted ovary, for 13.5 h on day 11 of the oestrous cycle. The pattern of androstenedione and oestradiol secretion in response to a GnRH-stimulated LH pulse was measured 2.5 h before and 12.5 h and 24.5 h after the start of the infusion. Glucose or insulin infused alone had no effect on the secretion of androstenedione and oestradiol. However, when infused together, they decreased significantly the secretion of androstenedione and, to a lesser extent, oestradiol. We suggest that the sudden availability of additional glucose and insulin increases insulin-stimulated glucose uptake by the follicle. This leads to an inhibition of LH-stimulated steroidogenesis by the ovarian follicle which occurs in the absence of any detectable changes in circulating plasma concentrations of FSH. These results show that insulin and glucose act together to influence ovarian function directly and suggest that the effects of short-term nutrition on ovulation rate may be mediated by a direct ovarian action of insulin and glucose.

The GnRH-antagonist suppression–ovarian autotransplant model (n= 18) was used to examine the relative roles of temporal changes in FSH and LH stimulation on follicle development and selection. Follicle development was stimulated by infusion with oFSH for 3 days and treatments applied for 60 h after progestagen sponge withdrawal and before delivery of an ovulatory stimulus. In Expt 1, there was continuous infusion of FSH with or without small amplitude high frequency LH pulses, or withdrawal of FSH with or without pulsatile LH. In Expt 2, there was acute or gradual withdrawal of FSH at sponge withdrawal with pulsatile LH. The patterns of follicle development and basal and pulsatile ovarian hormone secretion were determined. The maintenance of FSH throughout the artificial follicular phase resulted in multiple follicle development and ovulation (3.3 ± 0.3). Pulsatile LH stimulated steroid secretion (P< 0.001) but had little effect on ovulation rates (3.8 ± 0.8) when FSH was maintained. However, withdrawal of FSH in the absence of LH resulted in atresia of the ovulatory follicles and anovulation whereas, when FSH was withdrawn in the presence of LH, preovulatory follicle development was maintained in some animals (3/6 and 5/9 in Expts 1 and 2, respectively) and these ewes had lower (P< 0.05) ovulation rates (1–2 ovulations per ewe). When FSH was withdrawn gradually in the presence of pulsatile LH, 9/9 animals ovulated with ovulation rates in the normal range. These results indicate that ovulatory follicles can transfer their gonadotrophic dependence from FSH to LH. It is hypothesized that the ability of a follicle to respond to this switch in gonadotrophic support is central to the mechanism of follicle selection.

Two experiments were conducted during the anoestrous period in Border Leicester × Merino ewes with ovarian autotransplants to study the effects of a single injection of 20 mg progesterone on follicular steroid secretion. The aim of these experiments was to determine whether pretreatment with a 20 mg intramuscular injection of progesterone could reduce GnRH-induced ovarian steroid secretion in anoestrous ewes. In both experiments, an injection of 150 ng GnRH induced an LH pulse in all ewes with a maximum concentration 10 min (the first post-injection sample) after injection. Oestradiol and androstenedione secretion increased progressively after the GnRH-induced LH pulse and reached maximum rates of secretion between 60 and 90 min before decreasing slowly to pre-injection rates at 150 min. There were no differences in the pattern of secretion of oestradiol (measured in both experiments) or androstenedione (measured only in Expt 2). In Expt 1, the injection of progesterone 72 h before the challenge with GnRH had no effect on the maximum rate of oestradiol secretion from the autotransplanted ovary. However, in Expt 2, when progesterone was given either 36 or 60 h before GnRH, there was a significant suppression in the maximum rate of secretion of both oestradiol and androstenedione between 60 and 90 min after GnRH injection. These data show that pretreatment of anoestrous sheep with progesterone can suppress LH-stimulated steroid secretion from the ovary and indicate that progesterone may have a direct effect on oestrogenic follicles in sheep.

Plasma luteinising hormone (LH) concentrations were measured during the ovulatory cycle in lines of Australorps and White Leghorns selected for reduced oviposition interval and maintained under continuous light and noise.2. Selection significantly increased plasma LH concentrations in mid-sequence ovulatory cycles of Australorps but not in the White Leg-horns.3. Selection in the Australorps apparently increased the rate of ovarian follicular maturation, resulting in more frequent LH peaks.4. The effect of selection on plasma LH concentrations is a function of the lighting condition to which the hens are exposed.

1. The effects of selection under continuous light for reduced oviposition interval within a sequence of eggs on plasma luteinising hormone (LH) concentrations during the ovulatory cycle were investigated in lines of Australorps and White Leghorns maintained on a 24-h light:dark cycle.2. Selection did not significantly alter LH concentrations during mid-sequence cycles in either breed.3. In the control lines, concentrations were higher in the Australorps than in the White Leghorns before and at peak in mid-sequence cycles.4. The increase in egg production resulting from selection was associated with an advance in the mean time of lay of mid-sequence eggs in both breeds and, in the Australorps, with more pre-ovulatory LH peaks being initiated at the beginning of the open period of the ovulatory cycle.5. Selection reduced the time an egg spends in the oviduct and, in the Australorps, significantly reduced the interval between a pre-ovulatory LH peak and the oviposition of the egg currently in the shell gland.6. We suggest that selection under continuous light for reduced oviposition interval increased the rate of ovarian follicular maturation by a mechanism which did not involve an increase in pre-ovulatory concentration of plasma LH in a 24-h light-dark cycle.

Based upon very large differences in the steroid content of blood obtained by concurrent cannulations of both ovarian veins, Shaikh and Harper (1972) have implied that one member of a pair may act predominantly as the steroid-secreting ovary. To test this hypothesis, estradiol, testosterone, and progesterone were measured by radioimmunoassay in the ovarian venous plasma of six rabbits in which right and left ovarian veins were cannulated concurrently and in eight others in which right and left ovarian veins were cannulated successively. The duration of blood collections ranged from 5 to 20 min/ovary and the resultant calculated amounts of whole blood obtained/ovary/h ranged between 54 and 252 ml. In most cases, paired ovaries secreted comparable amounts of steroid. Relative to steroid concentration, the highest ratios obtained between right and left ovarian veins were 1.0–3.0 for estradiol, 1.0–1.6 for testosterone, and 1.0–1.8 for progesterone. The highest ratios of secretion rate/ovary/h ranged from 1.0 to 3.0 for estradiol, 1.0 to 1.4 for testosterone, and 1.0 to 1.5 for progesterone. These results are in sharp contrast to those reported by Shaikh and Harper and fail to support their hypothesis of marked differential secretion rates by the two ovaries.

Merino ewes were immunized against bovine serum albumin (BSA), or oestrone or androstenedione, conjugated to BSA. Immunization against oestrone and androstenedione elevated the mean number of ovulations per ewe ovulating (ovulation rate) from 1.2 to 1.5 and 2.3, respectively. The higher ovulation rate was not reflected in more lambs born per ewe lambing (1.12-1 .I 6 for all groups). The frequency of ovulation and oestrus was depressed in ewes immune to oestrone, and there was a high frequency of abnormalities in the reproductive tract. These effects of immunization were not as frequent in ewes immune to androstenedione. There was no relation between titre and any measure of reproductive performance. The use of immunization against ovarian steroids to improve the reproductive efficiency of commercial flocks is not justified without further investigation.

This study examined the effect of LH pulses, of similar amplitude and frequency to those found in the luteal phase, on the pattern of hormone secretion and follicle development in GnRH antagonist-suppressed ewes stimulated with exogenous FSH. This experiment was conducted on ewes with ovarian autotransplants in a continuous study. Follicle development was suppressed in 18 ewes by 3 weeks of GnRH antagonist treatment (50 micrograms/kg per 4 days s.c.), and was then stimulated by infusion of ovine (o)FSH (5 micrograms NIADDK-oFSH-16/h i.v.) for 3 days. In addition to FSH, 10 animals received pulses of LH (2.5 micrograms NIADDK-oLH-26 i.v.) every 4 h for the entire period of the FSH infusion. The follicle population was determined by daily ultrasound. Samples of ovarian and jugular venous blood were collected at 4-h intervals over the period of the FSH infusion and there were three periods of intensive blood sampling (15-min intervals for 2.5 h at 24, 48 and 72 h after the start of the FSH infusion) when the steroidogenic capacity of the follicles in all 18 ewes was tested around an LH challenge (2.5 micrograms i.v.). GnRH antagonist treatment resulted in a 57% decrease in FSH concentrations and prevented ovarian follicle development beyond 3 mm in diameter. Infusion of FSH resulted in a 60% increase in FSH concentrations and stimulated the development of large antral follicles and a coincident increase in ovarian androstenedione, inhibin and oestradiol secretion in both experimental groups. In the absence of 4-hourly LH pulses basal steroid secretion was negligible (< 1 ng/min; P < 0.001). Daily LH challenges, however, revealed no difference in the steroidogenic capacity of the follicle population in either experimental group. Similarly, LH pulses had no effect on the growth rate and number of antral follicles stimulated by FSH infusion, or the pattern of ovarian inhibin secretion. In conclusion, these results show that while FSH alone can stimulate the development of ovulatory sized follicles in ewes made hypogonadal with GnRH antagonist, physiological patterns of LH stimulation have no deleterious effects on FSH-stimulated follicle development and are essential for normal steroidogenesis.

An experiment was conducted to define the seasonal changes in LH secretion in ovariectomized does with oestrogen implants and the effect of immunization against melatonin. Fifteen mature Australian cashmere goats were ovariectomized and given either no further treatment or one or two implants containing oestradiol; another similar group of 15 does were immunized against melatonin before ovariectomy and oestrogen treatment. LH concentrations and livemasses were recorded every week for 2 years. Livemasses of both groups showed a distinct seasonal pattern with a summer maximum and a winter minimum irrespective of treatment. LH concentrations also showed distinct seasonal patterns with a significant interaction between the number of implants and the time of the year. In the nonimmunized does, the presence of a constant low dose of oestrogen (one implant) resulted in low concentrations of LH except from May to August, the normal period of spontaneous ovulatory activity in intact does. In contrast, nonimmunized does receiving a high dose of oestrogen (two implants) showed a rise in LH concentrations in February, and concentrations remained high until August. Immunization against melatonin abolished this differential LH secretory pattern, and both doses of oestrogen were associated with a short period of high LH concentration between May and September. These results indicate that a negative feedback effect of oestrogen results in low LH secretion for most of the year and that hypothalamic sensitivity to LH decreases for only a short period between May and August. It is suggested that the early increase in LH secretion in does with two oestrogen implants is due to a positive oestrogen feedback mechanism that may depend on a photoperiodic signal for activation.

An experiment in which a lupin grain supplement was fed to ewes (n= 11) over days 2–13 of the oestrous cycle was carried out. A group of 12 ewes was used as a control and not fed the supplement. Plasma concentrations of LH and GH (20 min intervals) and FSH, insulin and prolactin (hourly intervals) were determined in plasma samples collected every 20 min over 24 h on day 11 of the oestrous cycle. The changes were related to increases in ovulation rate. Ovulation rate was increased (2.5 ± 0.2 versus 1.9 ± 0.2 for lupin-supplemented and control groups, respectively; P= 0.073) in the group that received the lupin supplement, but this increase was not associated with significant changes in either LH or FSH concentrations on day 11 of the oestrous cycle. Lupin supplementation had significant effects on the plasma concentrations of prolactin, GH and insulin. There was a transient increase (P< 0.05) in the concentration of prolactin 4–8 h after feeding, whereas insulin concentrations were increased immediately after feeding (P< 0.02) and were still high 24 h later (P< 0.02). Growth hormone concentrations were reduced in ewes fed with lupin grain (P< 0.001). These metabolic responses initiated by feeding a high energy and protein supplement such as lupin grain may be related to changes in ovulation rate. In particular, the sustained increases in insulin concentrations suggest that an increased supply of glucose to the follicle mediates nutritionally stimulated increases in ovulation rate.

The response of mouse ovaries maintained in organ culture to prostaglandin E2(PGE2) and prostaglandin F2α(F2α) was assessed using quantitative histological and radioimmunoassay procedures.Prostaglandin E2 induced histological changes in the cultured follicles comparable to those induced by human chorionic gonadotrophin (HCG) and the increase in the number of oocytes undergoing preovulatory maturation over the control value was the same irrespective of the treatment (PGE2 alone, HCG alone, or PGE2 + HCG). The amount of progesterone/ ml of culture medium was also significantly higher with these preparations than in control cultures (about 125 ng/ml compared with 57 ng/ml).By contrast, 5 μg PGF2α/ml medium increased neither the number of oocytes undergoing maturation nor the concentration of progesterone in the culture medium. The latter increased when the dose of PGF2α was increased to 30 μg/ml, although the proportion of oocytes beyond the dictyate stage remained at the control level. There was no augmentation in the response (above the level for HCG alone) when HCG and PGF2α were added to the explant medium simultaneously. These results are discussed in terms of the possible mechanism of action of the various preparations.

Our knowledge of ovulation rate is still very rudimentary, but that is not to say that little information is available. The literature contains a vast body of data on the subject (Cumming, 1977) but a general model, such as those that are now widely accepted for the control of the oestrous cycle (Baird & McNeilly, 1981) or of seasonal reproduction (Goodman & Karsch, 1981), is not available.The ovulation rate of eutherian mammals is defined in two ways: (1) as the average number of eggs released in animals that ovulate, but this definition cannot produce an ovulation rate of less than 1·0 as it makes no allowance for anovulatory animals which should then be reported as a separate datum; and (2) as the average number of eggs released in a group of animals—this definition includes anovulatory animals and therefore can give values of less than 1·0. The physiological requirement for

Summary.The rosette inhibition test has been used to monitor the decrease of an 'early pregnancy factor' in 2 groups of pregnant sheep (19–21 days) in which embryos were removed surgically or by induction of luteolysis with cloprostenol. The rosette inhibition titres of sera taken from sheep of each group declined from high (16–18) to low (8–10) levels within 48 h of treatment. Surgical removal of embryos caused little change in serum progesterone concentration whereas cloprostenol prompted a rapid decrease over the same period. Death of the embryo can therefore be detected by the rosette inhibition test within 48 h of occurrence, but not necessarily by the measurement of progesterone in blood within this period.

Summary.The rosette inhibition test, an established test for determining the immunosuppressive potential of antilymphocyte serum, has been applied to the serum of sheep after mating. The rosette inhibition titre was much higher (12–26) in 7 sheep which were fertilized and remained pregnant for up to 21 days than in 5 sterile ewes mated with intact rams (8–10). The difference was apparent by 24 h after mating. One ewe had high titres for 6 days after mating but these then dropped and she returned to oestrus; early embryonic loss was suspected. Another ewe which returned to oestrus had consistently low titres. The results indicate that the rosette inhibition test can be used to detect fertilization, early embryonic death and continued pregnancy in sheep.

In 265 patients operated for breast carcinoma the monoclonal antibody serum test CA 15.3 was predictive of metastatic diffusion of the disease. Its level increased in cases of distant metastasis with no significant difference between multiple and single sites (p = 0.014). The concentration of the marker was higher in 21 (23.8%) patients without nodal involvement and in 19 (27.5%) with nodal involvement (p = 0.193). Our study suggests that CA 15.3 may be an aid in the follow-up of patients with metastatic diffusion of breast cancer.

TheStaphylococcus epidermidisplasmid pSK639 is the prototype of a newly described family of small plasmids identified in clinical staphylococcal isolates. pSK639 is 8 kb in length and possesses a composite structure consisting of an IS257-flanked segment mediating trimethoprim resistance (Tpr), and regions responsible for replication and mobilization of the plasmid. Comparative sequence analysis suggests that a pSK639-like plasmid may represent a progenitor of previously identified staphylococcal Tprdeterminants related to the transposon-like structure, Tn4003.In contrast to the small staphylococcal plasmids characterized to date that all utilize a rolling circle mode of replication, the replication region of pSK639 was found to contain features typical of an iteron-controlled theta-mode replicon. pSK639 is the first small plasmid of this type to be identified in the staphylococci.

The effects of oestradiol-17β, androstenedione, progesterone and time of the year on the pulsatile secretion of LH were tested in ovariectomized Merino ewes (n= 32). The steroids were administered by small subcutaneous implants, and the LH pulses were observed in samples taken at intervals of 15 min for 12 h in spring 1979, autumn 1980 and spring 1980, seasons corresponding to successive periods of anoestrus, breeding season and anoestrus.During spring, oestradiol alone was able to reduce the frequency of the LH pulses, while progesterone, either alone or in combination with oestradiol, had little effect. During autumn, on the other hand, neither oestradiol nor progesterone could significantly reduce the frequency of the pulses when administered independently, whereas the combined treatment was very effective. Androstenedione had no significant effect on pulse frequency at either time of the year, either alone or in any combination with oestradiol and progesterone.The basal levels of LH, over which the pulses are superimposed, were reduced by oestradiol alone in both seasons. Progesterone alone had no consistent effects, but interacted significantly with oestradiol and this combined treatment maintained low basal levels most effectively at all times. Androstenedione had no significant effect.The amplitude of the pulses increased throughout the course of the experiment. Within seasons, the amplitudes were significantly higher in the presence of oestradiol and progesterone, but were not significantly affected by androstenedione.It was concluded that certain of the ovarian steroids exert negative feedback on the tonic secretion of LH primarily by reducing the frequency of the pulses, and that the changes in LH secretion attributable to season and phases of the oestrous cycle can be accounted for entirely by the responses of the hypothalamus to oestradiol and progesterone. The role of the androstenedione secreted by the ovary in the ewe remains obscure.

The site of secretion of androstenedione in women was investigated by measuring the concentration of androstenedione in peripheral and ovarian venous plasma by radioimmunoassay at different stages of the menstrual cycle. The concentration in peripheral plasma rose from 1·45 ± 0·11 (s.e.m.) ng/ml on day 1 to a peak on the 7 days around mid-cycle (2·29 ± 0·08) before declining gradually during the luteal phase. The concentration of androstenedione in plasma draining ovaries containing the pre-ovulatory follicle or a corpus luteum (33·2 ± 9·0) was significantly higher than in plasma draining the contralateral ovary (10·1 ± 1·6, P< 0·001). In women in whom ovulation was induced with gonadotrophins the concentration of androstenedione in peripheral plasma rose from 1·42 ± 0·21 before treatment to reach maximum levels (2·5–4·5 ng/ml) in the luteal phase. There was a significant (P< 0·001) correlation between the concentration of androstenedione and oestradiol-17β in peripheral (r= 0·50) and ovarian (r= 0·79) venous plasma. These findings indicate that all three cellular compartments in the ovary secrete significant amounts of androstenedione in women during their reproductive life.