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BACKGROUND: Over recent decades, recurring efforts have been devoted to developing testicular cell or tissue cultures for basic and clinical research. However, there remains much confusion, particularly concerning the fate of human germ cells in culture. OBJECTIVE: To reassess the status of human testicular cell types as well as the ability of germ cells to divide and differentiate in organotypic culture. METHODS: Human testicular fragments were maintained for 2 weeks in culture. The viability and functionality of testicular cells were assessed using light and electronic microscopy, apoptotic cell labelling, 5-bromo-2′-deoxyuridine (BrdU) incorporation, immunohistochemistry and quantitative PCR against specific cell markers. RESULTS: A gradual loss of meiotic and post-meiotic germ cells occurred throughout the culture period, irrespective of the presence of gonadotrophins. However, all germ cell types remained traceable for up to 16 days, some still dividing and differentiating at a rate compatible with the in vivo situation. Good maintenance of the general architecture of the explants associated with clearly quantifiable levels of several somatic cell markers was observed. CONCLUSION: Although this culture model is clearly unsuitable for preparing germ cells for therapeutic purposes, it does represent a most valuable tool for testing the effects of biological and chemical agents on testicular tissue. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in metabolic tissues (muscle and liver) and has been identified as a modulator of the female reproductive functions. However, its function in the testis has not yet been clearly defined. We have investigated the potential role of AMPK in male reproduction by using transgenic mice lacking the activity of AMPK catalytic subunit α1 gene [α1AMPK knockout (KO)]. In the testis, the α1AMPK subunit is expressed in germ cells and also in somatic cells (Sertoli and Leydig cells). α1AMPK KO male mice show a decrease in fertility, despite no clear alteration in the testis morphology or sperm production. However, in α1AMPK(-/-) mice, we demonstrate that spermatozoa have structural abnormalities and are less motile than in control mice. These spermatozoa alterations are associated with a 50% decrease in mitochondrial activity, a 60% decrease in basal oxygen consumption, and morphological defects. The α1AMPK KO male mice had high androgen levels associated with a 5- and 3-fold increase in intratesticular cholesterol and testosterone concentrations, respectively. High concentrations of proteins involved in steroid production (3β-hydroxysteroid dehydrogenase, cytochrome steroid 17 alpha-hydroxylase/17,20 lysate, and steroidogenic acute regulatory protein) were also detected in α1AMPK(-/-) testes. In the pituitary, the LH and FSH concentrations tended to be lower in α1AMPK(-/-) male mice, probably due to the negative feedback of the high testosterone levels. These results suggest that total α1AMPK deficiency in male mice affects androgen production and quality of spermatozoa, leading to a decrease in fertility.

Avian influenza virus (AIV) infections of the chicken occur via the respiratory route. Unlike ducks which are considered as a natural AIV reservoir, chickens are highly susceptible to AIV infections and do not possess the RIG-I pattern recognition receptor involved in triggering the antiviral interferon response. To study the chicken innate immune response to AIV in the respiratory tract, we established an epithelial cell line (CLEC213) from lung explants of white leghorn chickens. CLEC213 cells exhibited a polyhedral morphology and formed cohesive clusters bound through tight junctions as assessed by electron microscopy. Expression of E-cadherin but not vimentin could be detected as expected for cells of epithelial origin. In addition, CLEC213 cells showed characteristics similar to those of mammalian type II pneumocytes, including the presence of intracytoplasmic vacuoles filled with a mucopolysaccharide material, alkaline phosphatase activity, transcription of chicken lung collectins genes (cLL and SPA), and some intracytoplasmic lamellar-like bodies. CLEC213 cells showed a constitutive expression level of TLR3 and TLR4 and were responsive to stimulation with the respective agonists, poly (I:C) and LPS: between 4h and 24h after treatment, a strong increase in the expression of IFN-α, IFN-β and IL-8 genes could be detected. Furthermore, CLEC213 cells supported efficient growth of the low pathogenicity avian influenza virus H6N2 (A/duck/France/05057a/2005) in the presence or the absence of trypsin in the culture media. At 4h post-infection, the H6N2 virus induced highly elevated levels of expression of IFN-α and IL-8, moderately elevated levels of LITAF, TGF-β4 and CCL5. However, an increase of IFN-β gene expression could not be detected in response to AIV infection. In conclusion, like mammalian type II pneumocytes, CLEC213 are able to mount a robust cytokine and chemokine immune response to microbial patterns and viral infection. We hypothesize that they could derive from lung atrial granular cells. The involvement of such type of lung epithelial cells in the respiratory tract defence of the chicken can thus be further studied., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a wide range of diseases in humans and animals. Using in vitro invasion assays and transmission electron microscopy, we showed that BEN2908, an ExPEC strain of avian origin (also termed APEC for Avian Pathogenic E. coli), is able to usurp cellular endocytic pathways to invade A549 human type II pneumocytes and LMH avian hepatocytes where it is able to survive over several days. Although type 1 fimbriae are the major adhesin of BEN2908, proportions of adherent fimbriated or afimbriated bacteria that entered cells were comparable. Internalization of BEN2908 into human pneumocytes reinforces previous studies indicating that APEC strains could represent a zoonotic risk., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

The objective of this study was to analyze the validity of the stereomicroscopic evaluation of vitrified-warmed (V-W) porcine blastocysts. Unhatched blastocysts were obtained from Large-white gilts (n=10). Blastocysts (n=156) were vitrified using the Open Pulled Straw technology. After warming, V-W blastocysts were cultured for 24h (V24). Then, their developmental progression was morphologically assessed by stereomicroscopy and classified as: V24 viable re-expanded blastocysts; V24 viable hatched blastocysts or V24 degenerated. Blastocysts which re-expanded or hatched after warming were considered viable. Some fresh blastocysts were not vitrified and were evaluated after 24h in culture (F24). By stereomicroscopic analysis all the fresh blastocysts were considered viable. Some F24, V24 re-expanded viable, V24 hatched viable and V24 degenerated blastocysts were processed for transmission electron microscopy (n=13, 19, 9 and 9, respectively) or assessed by TUNEL for cell-death evaluation (n=16, 21, 11 and 21, respectively). All V24 hatched blastocysts showed similar ultrastructure to fresh blastocysts. However, some V24 re-expanded blastocysts considered viable (6/19) revealed ultrastructural alterations. Degenerated V24 blastocysts showed ultrastructural disintegration. Hatched V24 blastocysts did not differ (p>0.05) from F24 hatched blastocysts with regard to the ratio of dead cells (2.8+/-0.5% versus 1.9+/-0.3%, respectively). However, V24 expanded blastocysts had higher (p<0.01) cell death levels (4.3+/-3.4%) than those observed in the F24 expanded blastocysts (1.1+/-0.3%). The degenerated blastocysts showed the highest cell-death index (19.4+/-6.3%). In summary, V-W blastocyst hatching during in vitro culture appears to coincide with good ultrastructure and low cell-death index, suggesting that the hatching rate assessed by stereomicroscopy is more appropriate than embryo re-expansion for an evaluation of V-W blastocyst quality.

Many studies have shown a correlation between transferrin (Tf) concentration and sperm yield in several mammalian species. We have used transgenic mice expressing human Tf (hTf) to investigate if overexpression of Tf increases the efficiency of mouse spermatogenesis. We demonstrated that a 36% increase of Tf does not ameliorate the efficiency of mouse spermatogenesis but on the contrary resulted in a 36% decrease of testis sperm reserves. Tf overexpression had no effect on testicular determination and development, however testicular function of these transgenic mice was affected in an age-dependent manner. At 16 months of age, testicular and epididymal weights were significantly reduced. While spermatogenesis was qualitatively normal, testicular functions were perturbed. In fact, testosterone rate after human chorionic gonadotropin (hCG) stimulation was lower in Tf overexpressing mice. Intratesticular concentration of estradiol-17beta was increased and fluid accumulation after ligation of rete testis was more abundant in these transgenic mice. Surprisingly, we found that endogenous Tf levels were also increased in Tf overexpressing mice and we demonstrated for the first time that Tf may serve to upregulate its own expression in testis. Collectively, our data show that Tf overexpression has negative effects on testicular function and that Tf levels require strict regulation in the testis.

Background: Mater (Maternal Antigen that Embryos Require), also known as Nalp5 (NACHT, leucine rich repeat and PYD containing 5), is an oocyte-specific maternal effect gene required for early embryonic development beyond the two-cell stage in mouse. We previously characterized the bovine orthologue MATER as an oocyte marker gene in cattle, and this gene was recently assigned to a QTL region for reproductive traits., Results: Here we have analyzed gene expression during folliculogenesis and preimplantation embryo development. In situ hybridization and immunohistochemistry on bovine ovarian section revealed that both the transcript and protein are restricted to the oocyte from primary follicles onwards, and accumulate in the oocyte cytoplasm during follicle growth. In immature oocytes, cytoplasmic, and more precisely cytosolic localization of MATER was confirmed by immunohistochemistry coupled with confocal microscopy and immunogold electron microscopy. By real-time PCR, MATER messenger RNA was observed to decrease strongly during maturation, and progressively during the embryo cleavage stages; it was hardly detected in morulae and blastocysts. The protein persisted after fertilization up until the blastocyst stage, and was mostly degraded after hatching. A similar predominantly cytoplasmic localization was observed in blastomeres from embryos up to 8-cells, with an apparent concentration near the nuclear membrane., Conclusion: Altogether, these expression patterns are consistent with bovine MATER protein being an oocyte specific maternal effect factor as in mouse.

Background: Over recent decades, recurring efforts have been devoted to developing testicular cell or tissue cultures for basic and clinical research. However, there remains much confusion, particularly concerning the fate of human germ cells in culture., Objective: To reassess the status of human testicular cell types as well as the ability of germ cells to divide and differentiate in organotypic culture., Methods: Human testicular fragments were maintained for 2 weeks in culture. The viability and functionality of testicular cells were assessed using light and electronic microscopy, apoptotic cell labelling, 5-bromo-2'-deoxyuridine (BrdU) incorporation, immunohistochemistry and quantitative PCR against specific cell markers., Results: A gradual loss of meiotic and post-meiotic germ cells occurred throughout the culture period, irrespective of the presence of gonadotrophins. However, all germ cell types remained traceable for up to 16 days, some still dividing and differentiating at a rate compatible with the in vivo situation. Good maintenance of the general architecture of the explants associated with clearly quantifiable levels of several somatic cell markers was observed., Conclusion: Although this culture model is clearly unsuitable for preparing germ cells for therapeutic purposes, it does represent a most valuable tool for testing the effects of biological and chemical agents on testicular tissue.

Recent data have suggested that P-glycoprotein (Pgp), working as membrane efflux "pumps", plays a major role in the transport of anthelmintic drugs in parasitic nematodes of ruminants. Flow cytometry analyses has shown that active Pgp is probably present in the external layers of Haemonchus contortus eggshells, following staining with the mouse monoclonal anti-human MDR1 antibody UIC2, which binds to Pgp in its active conformation. We evaluated the presence and distribution of this protein in the envelopes (eggshells and cuticles) of H. contortus and compared the various stages (eggs, L1-L2 larvae, L3 larvae, adult male and female worms). Electrophoresis revealed a 170-kDa band, corresponding to the molecular weight of Pgp in all stages. Indirect immunofluorescence staining with UIC2 showed Pgp to be located in the external layer of eggshells or cuticles. Transmission electron microscopy was used to localise Pgp more accurately in the three layers of the eggshells and cuticles. The conformation and biological functions of this protein, which we did not expect to find in such structures, remain to be determined.

Adult transgenic mice overexpressing human insulin-like growth factor-binding protein-1 in the liver present reproductive abnormalities in both sexes. In the present work, we have investigated the mechanisms responsible for limiting breeding capacity in these transgenic male mice. Homozygous adult transgenic male mice (3-6 months old) exhibited irregular copulatory behavior and a reduction of the number of pregnancies per female as well as of litter size per pregnancy. Genital tract weight, more specifically epididymal and seminal vesicle weights, were reduced by 45% in homozygous transgenic vs. nontransgenic mice. Homozygous transgenic mice exhibited a 30% reduction of the length of seminiferous tubules (P = 0.007), a 30% decrease in daily sperm production per testis (P = 0.019), and a 50% decrease in the number of spermatozoa in testis (P = 0.037), associated with morphological abnormalities of the sperm heads leading to an approximately 50% reduction of fertilized two-cell eggs (P = 0.002) and of implanted embryos on d 5.5 after mating (P = 0.004). The round spermatids also appeared altered in their morphology. In addition, Leydig cells in homozygous transgenic mice exhibited an altered appearance, with a 1.8-fold increase in lipid droplets in their cytoplasm (P < 0.001). Moreover, the concentration of 3beta-hydroxysteroid dehydrogenase was 66% lower in testis from transgenics compared with those from normal mice (P = 0.01), leading to a tendency toward lower plasma testosterone levels (P = 0.1). Interestingly, LH concentrations were increased by 40% in transgenic pituitary extracts (P = 0.02), and basal LH secretion by pituitary explants in vitro was increased by 60% in homozygous transgenic vs. normal mice (P = 0.04), suggesting an alteration of LH pulsatile secretion in vivo. In conclusion, these data suggest that the breeding impairment of human insulin-like growth factor-binding protein-1 transgenic males is due at least in part to an alteration of the process of spermatogenesis, leading to a diminution of sperm production and of its quality. Minor impairment of steroidogenesis may also contribute to the reduced reproductive capacity of these animals. Our observations are consistent with the idea that normal spermatogenesis and perhaps also steroidogenesis are dependent on the actions of sufficient concentrations of unbound IGF-I.

In this study we tested the hypothesis that photoperiod can modulate steroid access to the brain in a seasonal breeder. To this goal, we compared the passage of exogenous progesterone to the brain of female sheep maintained under short (SD) or long (LD) daylengths. In the first experiment, we studied two groups of ovariectomized females maintained under SD or LD, for three artificial cycles, consisting of bearing a subcutaneous oestradiol implant (E2-treated) and an intravaginal device releasing progesterone (CIDR). During the third cycle, the concentrations of progesterone and of its metabolites 5alpha-dihydroprogesterone and 3alpha-hydroxy-5alpha-pregnan-20-one were measured in the preoptic area (POA). The levels of progesterone in the POA were higher in ewes under LD than under SD while the amounts of metabolites were unchanged. In the second experiment, we compared ovariectomized female sheep equipped with a cannula in the third ventricle to sample the cerebrospinal fluid (CSF) under LD vs. SD. After progesterone (1 mg and 10 mg) was injected into the carotid artery, it was only detectable in the cerebrospinal fluid in sheep under LD. In the third experiment, we compared progesterone concentration in plasma and CSF in two groups of SD vs. LD ovariectomized E2-treated ewes for 2 h under CIDR treatment. Despite similar progesterone plasma concentrations, concentration in the CSF was 2.5 times higher in SD than in LD. Our results suggest a physiological modulation of the passage of progesterone to the brain according to the photoperiod.

Preovulatory GnRH secretion in ewes, measured in portal blood and cerebrospinal fluid, starts at the time of the LH surge, approximately 4 h after the onset of estrous behavior, and lasts as long as receptivity (36-48 h), which is much longer than the LH surge. This study tested the hypothesis that the extended GnRH secretion is involved in the maintenance of receptive behavior, prolonging the initial triggering effect of E2. Ovariectomized ewes were subjected to artificial estrous cycles and infused intracerebroventricularly either with a water soluble GnRH antagonist (Teverelix, Exp 1 and 2) or GnRH (Exp 3 and 4) after preovulatory E2 challenges of various intensity. The GnRH antagonist infused for 20 h (0.5 mg/ml, flow rate 3 microl/min) following a treatment with 2 x 30-mm E2 implants for 24 h (Exp 1) significantly reduced receptivity 36-48 h post E2 compared with vehicle infusion. By contrast, when the GnRH antagonist was infused with E2 implants still present (Exp 2: E2 for 48 h, GnRH antagonist given 24-44 h after E2 insertion, n = 14) receptivity was not affected. Administration of GnRH (0.5 mg/ml, flow rate 3 microl/min) when receptivity began to decline (Exp 3: 30-48 h after a 6-h 2 x 30-mm E2 implants n = 12) resulted in significantly higher receptivity scores at 48 and 52 h post E2 in GnRH treated animals compared with vehicle treated. GnRH infusion of ewes under subthreshold E2 treatment (Exp 4: GnRH 6-24 h after implantation of 1 x 30-mm E2 for 3 h, n = 12 in a cross-over design) significantly increased their receptivity compared with vehicle administration at 18 and 24 h post E2 insertion, but receptivity remained lower than when induced by high doses of E2. Our results demonstrate for the first time that GnRH is involved in the control of receptivity in a ruminant species and suggest that in the cycling ewe the sustained preovulatory GnRH secretion plays a physiological role in extending the duration of estrous behavior. They also indicate that it is possible to dissociate a direct effect of E2 on estrous behavior from its effect via stimulation of GnRH secretion.

Seasonal anestrus in ewes results from an increase in response to the negative feedback action of estradiol (E(2)). This increase in the inhibitory effects of E(2) is controlled by photoperiod and appears to be mediated, in part, by dopaminergic neurons in the retrochiasmatic area of the hypothalamus (A15 group). This study was designed to test the hypothesis that E(2) increases multiunit electrical activity (MUA) in the A15 during inhibitory long days. MUA was monitored in the retrochiasmatic area of 14 ovariectomized ewes from 4 h before to 24 h after insertion of an E(2)-containing implant subcutaneously. In six of these ewes, MUA activity was also monitored before and after insertion of blank implants. Three of the 14 ewes were excluded from analysis because E(2) failed to inhibit LH. When MUA was recorded within the A15, E(2) produced a gradual increase in MUA that was sustained for 24 h. Blank implants failed to increase MUA in the A15 area, and E(2) did not alter MUA if recording electrodes were outside the A15. These data demonstrate that E(2) increases MUA in the A15 region of ewes and are consistent with the hypothesis that these neurons mediate E(2) negative feedback during long photoperiods.

Estrogen exerts important feedback effects upon the biosynthetic and secretory behavior of gonadotropin-releasing hormone (GnRH) neurons to control reproductive functioning. The mechanism of estrogen action upon these neurons is unclear and seems likely to involve the transsynaptic regulation of GnRH neurons. The objective of the present study was to identify the estrogen-receptive neural populations which project to the general vicinity of the GnRH perikarya in the rostral preoptic area and diagonal band of Broca (rPOA/DBB) of the ewe. Intact breeding-season ewes received an injection of the retrograde tracer fluorogold (FG) into the rPOA/DBB, and their hypothalami and brainstems examined for the presence of FG and estrogen receptor alpha (ERalpha) immunocytochemistry. Retrogradely labeled neurons were identified principally within the lateral septum (LS), lamina terminalis, bed nucleus of the stria terminalis, POA, arcuate nucleus (ARN), ventromedial nucleus (VMN) and median eminence. Smaller numbers of FG-immonoreactive cells were found in the caudal brainstem where they resided mostly in the ventrolateral medulla (VLM). Dual-labeled cells exhibiting both FG and ERalpha staining were prominent in the POA, LS and at all rostrocaudal levels of the VMN and ARN. Small numbers of dual-labeled cells were found in the VLM. These observations indicate that a number of distinct ERalpha-expressing neural populations project to the rPOA/DBB where the majority of the GnRH perikarya are found in the ewe. Although it is not possible to determine the direct connectivity of these projections with GnRH neurons, the findings provide an initial neuroanatomical framework through which the transsynaptic actions of estrogen on ovine GnRH neurons may be tested.

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0-12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12-24 hr, P implant removed; 24-36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3alpha-hydroxy-5alpha-pregnan-20-one, which operates through the type A gamma-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.

Noradrenergic neurons are implicated in the estrogen-dependent neural regulation of luteinizing hormone secretion in a variety of mammalian species. The current study has used immunocytochemical methods to determine whether estrogen receptors (ER) are expressed within the brainstem of the ewe and to establish their relationship to noradrenergic neurons. Using a monoclonal mouse antiserum directed against the N-terminal of ERa, four distinct populations of ER alpha-immunoreactive cells were identified in ovine medulla and pons. The largest population was found in the superficial laminae of the spinal nucleus of the trigeminal nerve, followed by the nucleus tractus solitarius, lateral area postrema, and ventrolateral medulla. Double-labelling immunocytochemistry using antisera directed against the ER alpha and dopamine-beta-hydroxylase revealed that noradrenergic neurons expressing ER immunoreactivity were only found in ventrolateral medulla (A1 cell group) and nucleus tractus solitarius (A2 cell group). No double-labelled cells were identified in the A5, A6, or A7 noradrenergic cell groups. ERs were expressed with a clear rostrocaudal topography within the A1 and A2 populations, with 80-90% of noradrenergic neurons expressing ERA alpha in the caudalmost medulla as compared with less than 5% rostral to the obex. Our findings demonstrate that, as in the rat, the ovine A1 and A2 neurons express ERs in a defined topographical manner, while, dissimilar to the rat, ER alpha is not synthesized by noradrenergic neurons in the other cell groups. These observations indicate that A1 and A2 noradrenergic neurons in the ovine brainstem are likely to be influenced by circulating estrogens and lay the neuroanatomical foundations for investigating the functional role of these cell populations within the gonadotropin-releasing hormone neuron network of the sheep.

Although a neural site of action for estradiol in inducing a LH surge via a surge of GnRH is now well established in sheep, the precise target(s) for estrogen within the brain is unknown. To address this issue, two experiments were conducted during the breeding season using an artificial model of the follicular phase. In the first experiment, bilateral 17beta-estradiol microimplants were positioned in either the medial preoptic area (MPOA) or the mediobasal hypothalamus (MBH), and LH secretion was monitored. An initial negative feedback inhibition of LH secretion was observed in ewes that had estradiol microimplants located in the MPOA (6 of 6 ewes) or caudal MBH in the vicinity of the arcuate nucleus (4 of 4). In contrast, a normal LH surge was only found in animals bearing estradiol microimplants in the MBH (5 of 10). Detailed analysis of estradiol microimplant location with respect to the estrogen receptor-alpha-immunoreactive cells of the hypothalamus revealed that 4 of the 5 ewes exhibiting a LH surge had microimplants located bilaterally within or adjacent to the area of estrogen receptor-expressing cells of the ventromedial nucleus. Two of these ewes exhibited a LH surge without showing any form of estrogen negative feedback. In the second experiment, we used the technique of hypophyseal portal blood collection to monitor GnRH secretion directly at the time of the LH surge induced by estradiol delivered either centrally or peripherally. Central estradiol implants induced the GnRH surge. The duration and mean plasma concentration of GnRH during the surge were not different between animals given peripheral or central MBH estradiol implants. Cholesterol-filled MBH microimplants did not evoke a GnRH surge. We conclude that the ventromedial nucleus is the primary site of action for estradiol in stimulating the preovulatory GnRH surge of the ewe, whereas the MPOA and possibly the caudal MBH are sites at which estrogen can act to inhibit LH secretion. These data provide evidence for the sites within the ovine hypothalamus responsible for mediating the bimodal influence of estradiol on GnRH secretion and suggest that different, and possibly independent, neuronal cell populations are responsible for the negative and positive feedback actions of estradiol.

The equilibrium of the brain-pituitary-testicular axis is controlled by negative feedback exerted primarily through changes in the circulating concentrations of gonadal steroids. This is usually studied in gonadectomised animals treated with single large doses or constant low levels of exogenous steroid. However, the feedback system probably also contains dynamic components, perhaps expressed as delays to changes in GnRH secretion following a change in steroid concentration. These delays must be measured without interference from surgical procedures, including anaesthesia, bias associated with changes in pituitary responsiveness (which affect the efficiency of pulse detection), and chronic side-effects of gonadectomy. We used a GnRH antagonist ['Antarelix': Ac-D-Nal, D-Cpa, D-Pal, Ser, Tyr, D-Hci, Leu, Lys-(iPr), Pro, D-Ala-NH2] to transiently block LH and steroid secretion (in effect, inducing and reversing castration) in mature male sheep, and measured GnRH secretion into cerebrospinal fluid (CSF) in the third cerebral ventricle. The CSF was withdrawn with a peristaltic pump at a rate of 2 ml/h and pooled every 20 min. Jugular plasma was sampled every 20 min and analysed for testosterone and LH pulses. The antagonist (500 microg i.v.) was injected after 6 h of baseline sampling and the study continued for a further 24 h. The pulses of LH and testosterone disappeared shortly after antagonist injection, with delays of 20 +/- 12 min for LH and 80 +/- 29 min for testosterone. This led to an increase in GnRH pulse frequency, starting 300 +/- 54 min after antagonist injection. Secretion of LH and testosterone pulses resumed at 553 +/- 38 and 530 +/- 30 min (after antagonist injection), and GnRH pulse frequency returned to baseline values after 170 +/- 42 min (relative to LH) and 117 +/- 35 min (relative to testosterone). The consistent nature of these responses across the group of animals suggests that this can be used to test the effects of exteroceptive factors on the dynamics of negative feedback.

Corticotrophin-releasing hormone (CRH) has been proposed as a mediator of the antireproductive effects of stress through an action within the hypothalamus to inhibit GnRH secretion. This hypothesis was tested in sheep by studying the responses to central administration of CRH in both sexes and in both seasons. Sexually mature, Ile-de-France ewes and Romanov rams that had been gonadectomized and implanted with a permanent guide cannula into the third cerebral ventricle were used. Ewes were studied in the presence and absence of exogenous oestradiol plus progesterone, in both the breeding and anoestrous seasons. All rams were treated with testosterone and were studied only during the breeding season. Each observation involved serial samples (every 10 min) of jugular blood for 5 h before (control) and 5 h after an intracerebroventricular (i.c.v.) injection of either saline (vehicle) or 5 nmoles CRH in 20 microliters vehicle. The saline injections did not affect any of the endocrine variables measured; however, CRH always increased cortisol concentrations in jugular plasma. In the absence of treatment with replacement sex steroids, icv injection of CRH had no effect on pulsatile LH secretion in females either during the breeding season or during anoestrus. However, LH pulse frequency and mean LH concentrations increased significantly on every occasion on which animals were treated with sex steroids. Treatment with CRH also increased LH secretion in the testosterone-treated rams. It is concluded that, contrary to the hypothesized role of CRH as an inhibitor of reproductive activity, this neuropeptide stimulates pulsatile LH (and thus GnRH) secretion, at least in this species. The fact that gonadal steroids seem to be obligatory for the expression of this effect suggests that the protocols used in past studies need to be reassessed.

In the present study we have identified a site of action of estradiol in the inhibition of LH secretion during anestrus in the ewe. In the first experiment, we studied six sites: the medial preoptic area, the lateral preoptic area, the ventromedial hypothalamus, the ventrolateral hypothalamus, the retrochiasmatic area (RCh), and the periventricular posterior hypothalamus. We compared the changes in parameters of pulsatile LH secretion (interpulse interval, mean nadir, mean amplitude, and mean area under curve) during three 6-h sampling periods: before and 30-36 h and 9 days after intracerebral implantation of crystalline estradiol. Animals that received estradiol in the RCh (n = 5) showed a significantly greater increase in both the intervals between pulses of LH (up 116%, p < 0.03) and the area under the curve (up 180%, p < 0.01) than any of the other groups of 7 animals. In the second experiment, implantation of estradiol in the RCh (n = 6) induced an increase in the intervals between pulses of LH (p < 0.03), whereas receiving an empty implant (n = 6) had no effect, showing that estradiol specifically induced increases in the intervals between pulses. Thus, estradiol appears to act in the RCh where the dopaminergic A15 nucleus, known to inhibit pulsatile LH release, is located.

In the ewe, the inhibition of pulsatile LH secretion by oestradiol during long days depends on dopaminergic activity and could involve amino acid transmitters. In the first experiment of the present study we observed the changes in LH secretion in ovariectomised ewes under long days immediately after subcutaneous implantation of oestradiol (peripheral treatment). In the second experiment, in order to identify the site of action of oestradiol, we observed the LH changes following intracerebral infusion of oestradiol through a microdialysis membrane (central treatment) within the preoptic area, the mediobasal hypothalamus (MBH) or the retrochiasmatic area (RCh) and measured amino acids and catecholaminergic transmitters and metabolites within the dialysates. With peripheral treatment, the amplitude, the nadir and the area under the LH pulse curve decreased within 4 to 8 h of the insertion of a subcutaneous oestradiol implant. After 18 h, the amplitude and the area under the pulses increased, as well as the intervals between pulses (from 49.9 + 1.4 min to 75.6 +/- 5.9 min). With central oestradiol treatment. LH changes were similar whatever the site of oestradiol infusion, suggesting either multiple sites of action or diffusion between structures. Twenty hours after the beginning of intracerebral oestradiol treatment, the amplitude and the area under the pulses increased, as did the interval between LH pulses (from 49.5 +/- 4.1 min to 73.2 +/- 14.2 min). Comparison of peripheral with central oestradiol treatment suggested that the long-lasting decrease in the nadir, as well as the transitory decrease in the amplitude and area, before 18 h in experiment 1 are reflections of hypophysial effects. In contrast, the increases in amplitude and area under the LH pulse curve seen 18-20 h after oestradiol in the two experiments could be due to the higher amplitude of LHRH pulses, as a result of an early stimulatory effect of oestradiol. After central oestradiol infusion, there was a decline in the concentration in the dialysate of two metabolites of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the RCh, suggesting an early inhibition of monoamine oxidase by the steroid. During the inhibition of LH pulsatility the concentration of gamma-aminobutyric acid in the dialysate from the RCh and the MBH increased, suggesting the participation of this transmitter in the changes induced by oestradiol under long days.

Morphological evidence suggests that LHRH may be secreted into cerebrospinal fluid (CSF), but only in the rhesus monkey has CSF LHRH been found to reflect changes occurring in the LHRH neuroendocrine system. This study investigated whether LHRH is detectable in ovine CSF and, if so, whether its release profile correlates with peripheral LH profiles during pulse and surge conditions. A polyethylene catheter was threaded through a stainless steel guide cannula previously implanted into the third ventricle of an ovariectomized ewe, which enabled continuous CSF withdrawal on repeated occasions. The first experiment (n = 3) showed that peripheral LH concentrations were unaffected by CSF removal at rates of 12, 30, 50, and 100 microliters/min, and the second (n = 4) established that CSF LHRH secretion was pulsatile, with considerable variation in pulse amplitude (6.3 +/- 1.8 pg/ml; range, 1.3-18.7 pg/ml). In the third experiment (n = 6), an endogenous LH surge was induced after progesterone withdrawal and 17 beta-estradiol administration. Although CSF LHRH (15.3 +/- 1.3 h) and peripheral LH (14.8 +/- 1.0 h) surges occurred simultaneously, CSF LHRH concentrations were greater than half-maximal levels for longer (11.0 +/- 0.6 h; P < 0.005) than LH concentrations (6.0 +/- 0.4 h). This is the first study in sheep to reveal the presence of LHRH in CSF and show that it expresses dynamic and longer term changes coincident with peripheral LH fluctuations. CSF LHRH analysis also permits repeated sampling from individuals and, therefore, long term within-individual comparisons.

Of 656 patients with ALL (all types) diagnosed in Switzerland during 4 consecutive 4-year periods (1976-1979, 1980-1983, 1984-1987, 1988-1991), 507 were officially registered on protocols ("study" patients) while 149 were not ("nonstudy" patients). The mean incidence of 3.8/100,000 children < 15 years/year is higher than reported for other Western countries. Evidence is presented suggesting that the 656 patients represent only approximately 90% of all children with ALL residing in Switzerland, indicating that the true incidence of ALL might even be higher. The fraction of "nonstudy" patients fell from 40% (1976-1979) to 15% (1984-1987). The rate of survival at 4 years of all patients with ALL ("study" and "nonstudy") increased by 17% during the three consecutive periods 1976-1979, 1980-1983, and 1984-1987. As expected, a higher increase (20%) was observed in "study" patients and a statistically nonsignificant lower one (10%) in "nonstudy" patients.

Previous studies have demonstrated a neural action of estradiol in inducing a surge of GnRH in the ewe. However, although the GnRH and LH surges began concurrently, the GnRH surge consistently continued well beyond the surge of LH. Three experiments were conducted to test the hypothesis that the termination of the LH surge results from the secretion of a relatively inactive variant of GnRH during the later phases of the GnRH surge. In the first experiment, hypophyseal portal blood collected during an estrogen-induced LH surge was analyzed for GnRH immunoreactivity using two antibodies having specificity for the N- or C-terminal portion of the GnRH molecule. The duration, amplitude, and time course of the GnRH surge were found to be similar irrespective of the antisera used. In a second experiment, a competitive GnRH antagonist was administered at the beginning of the estrogen-induced GnRH/LH surge at a dose capable of blocking pituitary responsiveness for approximately half the duration of the GnRH surge. Antagonist treatment did not result in any change in the time of onset of the GnRH surge, but there was no increase in LH that naturally occurs coincident with onset of the GnRH surge. Rather, a persistent increase in LH secretion was observed during the latter stages of the GnRH surge, indicating that the GnRH molecules secreted at this time were biologically active. Finally, a sensitive and specific ovine pituitary cell bioassay was used to test bioactivity of GnRH in hypophyseal portal blood during different phases of the GnRH surge. GnRH bioactivity in samples collected early in the GnRH surge was greater than that before the onset of the GnRH surge but no greater than that collected during the descending limb of the surge. The results of all three experiments fail to support the hypothesis that the LH surge ends because of a change in the nature of the GnRH secreted. Rather they show that GnRH secreted throughout the surge is biologically active. Thus, the termination of the LH surge before that of the GnRH surge occurs for reasons other than lack of a bioactive GnRH signal.

The preovulatory surge of LH, or positive feedback response to oestradiol, is related to the steroid's direct stimulating action on the pituitary gland, but we have demonstrated that, in sheep, the central nervous system plays an essential role in its action: a preovulatory GnRH surge. Since GnRH neurons appear to contain few, if any receptors for oestradiol the question is raised: Where in the central nervous system does oestradiol act to stimulate GnRH secretion? A series of experiments were conducted with a combination of techniques for sampling pituitary portal blood and stereotaxic brain micro-implantations of oestradiol. Castrated Ile-de-France breed ewes were treated during the pseudo follicular phase of successive artificial cycles, receiving either peripheral oestradiol implants or brain implants containing oestradiol or cholesterol. Administration of oestradiol implants into the ventromedial region of the hypothalamus induced a preovulatory GnRH surge in 5 out of 10 animals. The interval between estradiol and the GnRH surge was comparable with that observed in animals treated with a peripheral implant although the amplitude of the surge was smaller. No GnRH surges were observed in animals treated with cholesterol. Finally, there was an inverse correlation between the response intensity (amplitude of the GnRH/LH surges) and the distance separating implants from cells in the region carrying oestradiol receptors. These results show that the mediobasal hypothalamus is a site of action for the oestradiol-induced preovulatory GnRH surge in the ewe. Immunohistochemical tests should identify the nature of the cells forming the relay for the steroid action.

Of 54 children with acute lymphoblastic leukemia (ALL) and first hematological recurrence observed between 1985 and 1989, 31 relapsed while still on treatment and 23 after cessation of therapy. Of the former, only one survived. Of the latter, 11 children survived after a minimum follow-up of 25 months. During the same period, a first isolated testicular relapse was observed in nine boys, of whom six survived, and an isolated CNS relapse in eight patients, of whom three survived. As a rule, survivors of a bone marrow or testicular relapse were doing well while those surviving a CNS relapse had considerable neuropsychological sequelae. These results, compared with those of two preceding studies, suggest that with intensification of front-line treatments, it becomes more difficult to rescue children who relapse, particularly those with a bone marrow relapse while on therapy.

The administration of GnRH agonists and antagonists suppresses pituitary LH secretion. However little is known about their effects on endogenous GnRH secretion. To determine if GnRH analogs act on GnRH secretion through a short or ultrashort loop feedback mechanism, experiments were performed to analyze GnRH secretion in hypophyseal portal blood of conscious short-term castrated rams under both agonist or antagonist treatment. In Study 1, six rams were castrated and surgically prepared for portal blood collection on day -7. Portal and peripheral blood were collected simultaneously every 10 min for 14-15 h on day 0. Five hours after the beginning of the portal blood collection, animals were injected im with 5 mg potent GnRH antagonist (Nal-Glu). In Study 2, six rams were treated daily from day -11 to day 0 with the GnRH agonist D-Trp6 GnRH (0.5 mg im). Castration and surgical preparation for portal blood collection were performed on day -7. On day 0 portal and peripheral blood were collected simultaneously every 10 min for 10-11 h. In both studies, to determine whether an increase in GnRH concentration in hypophyseal portal blood can overcome the inhibitory effect of the GnRH analogs, between 5 and 5.5 h after the injection of the analogs, endogenous GnRH secretion was stimulated by Naloxone administration (3 x 100 mg, iv, at 30-min intervals) followed by a bolus of exogenous GnRH (2 x 10 micrograms, iv at 30-min intervals). In Study 1, Nal-Glu administration led to a rapid cessation of pulsatile LH secretion for the duration of blood collection while GnRH pulse frequency and amplitude were not affected. GnRH and LH pulse frequency before and after Nal-Glu administration were, 6.2 +/- 0.6 vs. 5.7 +/- 0.8 (NS) and 5.3 +/- 0.3 vs. 0.3 +/- 0.2 pulses/6 h (P less than 0.001) respectively. In Study 2, peripheral LH secretion was completely suppressed while GnRH secretion (portal blood) remained pulsatile. GnRH pulses frequency and pulse amplitude were 4.3 +/- 0.3 pulses/6 h and 43.0 +/- 4.7 pg/ml, respectively. In both experiments, neither stimulation of endogenous GnRH secretion by naloxone nor administration of exogenous GnRH allowed reinitiation of LH secretion. However, additional studies in two animals of each treatment group (study-III) showed that this was clearly a dose related effect in antagonist treated but not in agonist-treated animals since higher doses of exogenous GnRH (i.e. 100 micrograms or 1000 micrograms) can increase significantly LH levels.(ABSTRACT TRUNCATED AT 400 WORDS)

13 cases of childhood acute lymphoblastic leukaemia (ALL) were studied combining cell surface marker analysis with immunogenotyping by Southern blot hybridisation with a panel of antigen receptor gene probes. The immunophenotypes were unequivocal: 7 patients had B-phenotype and 6 patients T-phenotype ALL. In several patients immunogenotypes were not fully consistent with the respective phenotypes. For example, 2 B-cell precursor ALL had rearranged TCR beta chain genes and 2 T-ALL rearrangement of Ig heavy-chain genes. All cases showed clonal rearrangement or deletions within the TCR delta gene locus. TCR delta gene rearrangements might, therefore, serve as markers of clonality but not of B- or T-lineage in immature lymphoid neoplasms. We conclude that in current diagnostic practice immunogenotyping is a supplement rather than an alternative to immunophenotyping by surface marker analysis.

Reinfusion of undetected tumour cells is a possible cause of relapse after autologous bone-marrow transplantation. In this paper, a system for in-vitro purging of bone marrow is presented which involves the B-cell neoplasia-associated monoclonal antibody anti-Y 29/55 and complement. Five patients with Burkitt's lymphoma were transplanted with purged marrow, demonstrating the clinical feasibility of the method. The pretransplant regimen included vincristine 2 mg/m2, adriamycin 60 mg/m2, four doses of cyclophosphamide 45 mg/kg and total body irradiation with 6 Gy. Tumour control appears to be better in patients with purged bone marrow as compared to an earlier patient group with unpurged marrow.

The incidence of isolated CNS-relapse in the SPOG ALL studies 1976-1986 was analyzed and the prophylaxis of meningosis leucaemica of the different studies was compared. In the SPOG ALL high-risk study 1979-1983, the incidence of isolated CNS-relapse was significantly higher (17/71, 24%) than in the other studies. In this period, radiotherapy was omitted and the prophylactic treatment consisted only of moderately high doses of intravenous methotrexate and intrathecal methotrexate. In other studies, it was shown that the prophylactic combination of CNS-radiotherapy and intrathecal methotrexate, or the periodic administration of combined intrathecal chemotherapy alone, during the whole therapy of 2 1/2 years, produced comparably good results. The prophylaxis with the combined intrathecal chemotherapy was less neurotoxic and allowed the use of a curative radiotherapy in case of a CNS-relapse.