Epigenetics and developmental programming of welfare and production traits in farm animals.

The concept that postnatal health and development can be influenced by events that occur in utero originated from epidemiological studies in humans supported by numerous mechanistic (including epigenetic) studies in a variety of model species. Referred to as the 'developmental origins of health and disease' or 'DOHaD' hypothesis, the primary focus of large-animal studies until quite recently had been biomedical. Attention has since turned towards traits of commercial importance in farm animals. Herein we review the evidence that prenatal risk factors, including suboptimal parental nutrition, gestational stress, exposure to environmental chemicals and advanced breeding technologies, can determine traits such as postnatal growth, feed efficiency, milk yield, carcass composition, animal welfare and reproductive potential. We consider the role of epigenetic and cytoplasmic mechanisms of inheritance, and discuss implications for livestock production and future research endeavours. We conclude that although the concept is proven for several traits, issues relating to effect size, and hence commercial importance, remain. Studies have also invariably been conducted under controlled experimental conditions, frequently assessing single risk factors, thereby limiting their translational value for livestock production. We propose concerted international research efforts that consider multiple, concurrent stressors to better represent effects of contemporary animal production systems.

Effects of season on the reproductive organ and plasma testosterone concentrations in guinea cocks (Numida meleagris).

The physiological basis of seasonal breeding in the guinea fowl (Numida meleagris) still remains unknown, despite the socioeconomic importance of these birds, particularly in Ghana. A study involving a total of 50 local guinea cocks was conducted, and documented gross anatomical and histological differences in the reproductive organs of breeding and non-breeding male guinea fowls. The study also compared peripheral testosterone concentrations in breeding and non-breeding cocks. Seasonal differences in variables measured were determined using two-tailed t-test/Mann-Whitney U-test. All comparisons were made at 5% level of significance. Breeding males had significantly (P = 0.000) higher anatomical biometric parameters than their non-breeding counterparts. Also, breeding birds had thicker (P = 0.000) phalli than their non-breeding counterparts. Histologically, regressing testis was characterized by the presence of sloughed off cells and increased debris in the tubular lumen and within the excurrent duct system, collapsed tubules and reduction in tubular lumen. Germ and Sertoli cell populations and nuclear diameters and actual seminiferous tubular diameter and length in regressing testes were significantly (P = 0.000) lower than in active testes. Leydig cell nuclear diameters and populations were also significantly (P = 0.000) reduced. Relative volume of seminiferous tubules in the testis, testicular sperm production/mg testis and per testis and peripheral testosterone concentrations were all higher (P < 0.05) in breeding than non-breeding testis. The ducts in the epididymal region also saw significant (P < 0.05) reductions in luminal diameters in non-breeding birds. Significant regression in anatomical and histological structures of the guinea cock reproductive tract occurred during the non-breeding season, and lower peripheral testosterone concentrations may be responsible for this phenomenon.

Effects of season on the reproductive organs and steroid hormone profiles in guinea hens (Numida meleagris).

The study documented gross anatomical and histological differences in the reproductive organs of 28 breeding and non-breeding female guinea fowls. Peripheral progesterone and 17ß-oestradiol concentrations were also compared in breeding and non-breeding hens. In non-breeding females, all ovarian and oviducal gross anatomical features had significantly regressed. Histologically, some of the changes in a regressing oviduct include systematic changes in height and size of all epithelial cells in all regions of the duct, absence/sparse ciliation of portions of surface epithelium in the magnum, isthmian and uterine regions, general loss of cytoplasmic mass, reduction in size and degeneration of tubular glands. Mucosal folds in all regions of the oviduct except the infundibular lip were higher in breeding females. No difference was found between the two groups in plasma progesterone concentrations. Breeding females, however, had higher peripheral oestradiol concentrations than non-breeding females. About 2 h prior to oviposition, plasma oestradiol concentrations peaked at 2.4-fold (230 pg/ml) compared with baseline concentration and plasma progesterone concentrations by nearly 9-fold (5.29 ng/ml) of baseline. Significant regression and changes in the histological structure of the ovary and oviduct had occurred in non-breeding females, and lower peripheral oestrogen concentrations may be responsible for this phenomenon.

Disrupted seasonal biology impacts health, food security and ecosystems.

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Aging causes a slowing in ciliary beat frequency, mediated by PKCε.

The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and is the third most common reason for hospitalization in the elderly. One reason that elderly people may be more susceptible to pneumonia is a breakdown in the lung's first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however, little is known about the mechanism(s) involved. We compared the CBF in BALB/c and C57BL/6 mice aged 2, 12, and 24 mo and found that CBF diminishes with age. Cilia in the mice at age 12 and 24 mo retained their ability to be stimulated by the ß2 agonist procaterol. To help determine the mechanism of ciliary slowing, we measured protein kinase C alpha and epsilon (PKCα and PKCε) activity. There were no activity differences in PKCα between the mice aged 2, 12, or 24 mo. However, we demonstrated a significantly higher PKCε activity in the mice at 12 and 24 mo than the in the mice 2 mo of age. The increase in activity is likely due to a nearly threefold increase in PKCε protein in the lung during aging. To strengthen the connection between activation of PKCε and ciliary slowing, we treated tracheas of mice at 2 mo with the PKCε agonist 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with ß2 agonists. The mechanisms for the slowing of baseline CBF have not been previously determined. In this mouse model of aging we were able to show that decreases in CBF are related to an increase in PKCε activity.

Masculinization of the distal tubular and external genitalia in female sheep with prenatal androgen exposure.

Prenatal exposure to endogenous or exogenous androgens alters the development of the female reproductive tract. Although lesions in ovaries and external genitalia of androgenized female sheep have been reported, lesions of the tubular genitalia have not. Testosterone propionate (TP) or dihydrotestosterone (DHT) was administered by intramuscular injection twice weekly to 32 ewes from 30 to 90 days of pregnancy. The ewes lambed normally. The reproductive tracts from 24 treated and 13 control postpubertal female offspring were examined at 10 months of age. The ovaries, oviducts, and uteri were grossly and histologically normal in both TP- and DHT-exposed sheep. However, in the DHT-treated sheep, the uterus connected to a misshapen, saccular vagina that opened into the urethra; in the TP-treated sheep, it ended in a blind sac. In both TP- and DHT-treated sheep, the urethra was approximately 5 times longer than that of control sheep, and it resembled a male urethra with bilateral male accessory genital glands. The urethra terminated in a fully developed penis in both TP- and DHT-treated sheep, and a scrotal sac was present (without testes). These results show that prenatal exposure of female sheep to exogenous androgens results in masculinization of the tubular and external genitalia.

Plasmacytic differentiation of circulating Epstein-Barr virus-infected B lymphocytes during acute infectious mononucleosis.

During the acute phase (1 wk of symptoms or less) of infectious mononucleosis (IM), 70--80% of circulating Epstein-Barr virus nuclear antigen (EBNA)-positive cells have differentiated toward plasma cells. Thus the characteristics of the infected cells in the majority of IM patients during early disease are indistinguishable from EBNA-positive tumor cells of a previously reported child who developed lymphoma during IM. IgA and IgG were the most frequent and IgM the least frequent immunoglobulin isotypes detected in EBNA-positive cells. In acute disease EBNA was present in 5.5--20% of T cell-depleted blood lymphocytes but in the 2nd or 3rd wk of illness the number of EBNA-positive cells sharply decreased to 0.4--1.4%. At the same time the fraction of antigen-positive cells containing cytoplasmic immunoglobulins also diminished, suggesting either that differentiation of infected cells was altered during the disease or that nondifferentiated antigen-positive cells had a survival advantage. Both the high proportion of plasmacytic EBNA-positive cells seen during acute disease and the apparent loss of differentiation by these cells later in disease may be regulated by host immunologic factors. Immunoglobulin-producing EBNA-positive cells may be the source of heterophile antibodies and other seemingly inappropriate antibodies usually found in serum during IM; however, increased numbers of noninfected plasma cells were present in some patients and may also be a potential source of these unusual antibodies.

Peripubertal GnRH and testosterone co-treatment leads to increased familiarity preferences in male sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) treatment is effective for the medical suppression of the hypothalamic-pituitary-gonadal axis in situations like central precocious puberty and gender dysphoria. However, its administration during the peripubertal period could influence normal brain development and function because GnRH receptors are expressed in brain regions that regulate emotions, cognition, motivation and memory. This study used an ovine model to determine whether chronic peripubertal GnRHa-treatment affected the developmental shift from preference of familiarity to novelty. Experimental groups included Controls and GnRHa-treated rams. To differentiate between effects of altered GnRH signaling and those associated with the loss of sex steroids, a group was also included that received testosterone replacement as well as GnRHa (GnRHa + T). Preference for a novel versus familiar object was assessed during 5-min social isolation at 8, 28 and 46 weeks of age. Approach behavior was measured as interactions with and time spent near the objects, whereas avoidance behavior was measured by time spent in the entrance zone and attempts to escape the arena via the entry point. Emotional reactivity was measured by the number of vocalizations, escape attempts and urinations. As Control and GnRHa-treated rams aged, their approach behaviors showed a shift from preference for familiarity (8 weeks) to novelty (46 weeks). In contrast, relative to the Controls the GnRHa + T rams exhibited more approach behaviors towards both objects, at 28 and 46 weeks of age and preferred familiarity at 46 weeks of age. Vocalisation rate was increased in GnRHa treated rams in late puberty (28 weeks) compared to both Control and GnRHa + T rams but this effect was not seen in young adulthood (46 weeks). These results suggest that the specific suppression of testosterone during a developmental window in late puberty may reduce emotional reactivity and hamper learning a flexible adjustment to environmental change. The results also suggest that disruption of either endogenous testosterone signalling or a synergistic action between GnRH and testosterone signalling, may delay maturation of cognitive processes (e.g. information processing) that affects the motivation of rams to approach and avoid objects.

Circadian rhythms of melatonin and behaviour in juvenile sheep in field conditions: Effects of photoperiod, environment and weaning.

Entrainment of circadian rhythms (CR) to the light dark cycle has been well described under controlled, experimental conditions. However, studies in rodents have reported that rhythms in the laboratory are not always reproduced under field conditions. The aim of this study was to characterise the CR of sheep maintained under conditions of standard UK farm animal husbandry and to investigate the effects of environmental challenges presented by season, weaning and changes in housing on CR. Male sheep (n = 9) were kept at pasture, or group housed in barns, under natural photoperiod for one year. CR in locomotor activity were monitored using accelerometry, and 24 h patterns in plasma cortisol and melatonin were measured every 4 h by ELISA. CR was measured before and after weaning, in summer and winter, and at pasture and by barn housing. Cosinor analysis revealed high amplitude, diurnal rhythms in locomotor activity that were disrupted by weaning and by barn housing. Rhythms in winter showed an interrupted night time activity pattern, but only when the sheep were kept at pasture. Cortisol and melatonin secretion followed typical circadian patterns in winter and summer. The CR of the sheep under the field conditions of this study were strikingly robust under basal conditions, but easily disrupted by environmental challenges. Interrupted patterns of activity during the long nights of wintertime, not previously reported for sheep kept in experimental conditions were recorded. Based on these findings, we propose that animals require exposure to more complex environments than the laboratory in order to exhibit their true circadian phenotype.

Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120.

There is increasing evidence that sera from HIV-1-infected individuals contain antibodies that enhance infection by HIV-1 in vitro. Previous work has demonstrated that complement receptors on T lymphoid cells and Fc receptors for IgG (Fc gamma R) on monocytic cells are required for enhanced infection by antibody-complexed HIV-1. Characterization of such infection-enhancing antibodies is essential because immunogenic epitopes which induce enhancing antibodies should be excluded from HIV-1 vaccines. This study was conducted to identify enhancing antibodies involved in Fc R-mediated enhancement of HIV-1 infection employing IgG human monoclonal antibodies (HMAbs) reactive against gp120 of HIV-1, which were produced by B cell lines derived from an HIV-1-infected individual. A potent neutralizing HMAb N70-1.5e did not enhance infection by HIV-1 (IIIB and MN strains), whereas HMAb N70-2.3a mediated enhancement of HIV-1 infection, but had little neutralizing activity. A competition radio immunoassay demonstrated that the two antibodies bind to distinct epitopes. These results indicated that enhancing and neutralizing antibodies can be induced by different epitopes on gp120, suggesting the potential for development of safe vaccines against HIV-1 by exclusion of immunogenic epitopes for enhancing antibodies. We made attempts to identify the epitope on gp120 that is recognized by the enhancing antibody N70-2.3a by using recombinant HIV-1 proteins and found that the antibody binds to a conformational site of nonvariable sequences in the carboxyl half (aa 272-509) of gp120.

Distribution of galanin receptor-2 immunoreactive neurones in the ovine hypothalamus: no evidence for involvement in the control of gonadotrophin-releasing hormone secretion.

Galanin is a small neuropeptide that mediates its effects via three receptor isoforms: galanin receptor-1, galanin receptor-2 and galanin receptor-3 (Gal-R1, Gal-R2 and Gal-R3). Galanin is thought to be an important intermediate in signalling in the hypothalamic-pituitary-gonadal axis and has been widely detected in the ovine hypothalamus. The expression of galanin and Gal-R1 has been reported to fluctuate during the reproductive cycle. Although the distribution of Gal-R1 has been determined in the ovine hypothalamus, the distribution of Gal-R2 was hitherto unknown. Using immunohistological and immunofluorescence techniques, we have mapped the distribution of Gal-R2 in the ovine hypothalamus, collected during the follicular phase of the oestrous cycle and examined colocalisation of Gal-R2 with oestrogen receptor alpha (ERalpha) and gonadotrophin-releasing hormone (GnRH). Gal-R2 was expressed in several regions of the hypothalamus (supraoptic nucleus, paraventricular nucleus, ventromedial nucleus, arcuate nucleus) but not as widely expressed as Gal-R1. Areas of Gal-R2 expression overlapped with those reported for Gal-R1. We observed that, in certain defined regions of the hypothalamus, up to 50% of neurones that express Gal-R2 also express ERalpha. No neurones coexpressed Gal-R2 and GnRH. Thus, we conclude that, in follicular phase animals, this receptor plays little or no role in direct intermediary signal transmission in GnRH-mediated control of the reproductive cycle.

A reduction in long-term spatial memory persists after discontinuation of peripubertal GnRH agonist treatment in sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) administration is used where suppression of hypothalamic-pituitary-gonadal axis activity is beneficial, such as steroid-dependent cancers, early onset gender dysphoria, central precocious puberty and as a reversible contraceptive in veterinary medicine. GnRH receptors, however, are expressed outside the reproductive axis, e.g. brain areas such as the hippocampus which is crucial for learning and memory processes. Previous work, using an ovine model, has demonstrated that long-term spatial memory is reduced in adult rams (45 weeks of age), following peripubertal blockade of GnRH signaling (GnRHa: goserelin acetate), and this was independent of the associated loss of gonadal steroid signaling. The current study investigated whether this effect is reversed after discontinuation of GnRHa-treatment. The results demonstrate that peripubertal GnRHa-treatment suppressed reproductive function in rams, which was restored after cessation of GnRHa-treatment at 44 weeks of age, as indicated by similar testes size (relative to body weight) in both GnRHa-Recovery and Control rams at 81 weeks of age. Rams in which GnRHa-treatment was discontinued (GnRHa-Recovery) had comparable spatial maze traverse times to Controls, during spatial orientation and learning assessments at 85 and 99 weeks of age. Former GnRHa-treatment altered how quickly the rams progressed beyond a specific point in the spatial maze at 83 and 99 weeks of age, and the direction of this effect depended on gonadal steroid exposure, i.e. GnRHa-Recovery rams progressed quicker during breeding season and slower during non-breeding season, compared to Controls. The long-term spatial memory performance of GnRHa-Recovery rams remained reduced (P<0.05, 1.5-fold slower) after discontinuation of GnRHa, compared to Controls. This result suggests that the time at which puberty normally occurs may represent a critical period of hippocampal plasticity. Perturbing normal hippocampal formation in this peripubertal period may also have long lasting effects on other brain areas and aspects of cognitive function.

Spatial memory is impaired by peripubertal GnRH agonist treatment and testosterone replacement in sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) is used therapeutically to block activity within the reproductive axis through down-regulation of GnRH receptors within the pituitary gland. GnRH receptors are also expressed in non-reproductive tissues, including areas of the brain such as the hippocampus and amygdala. The impact of long-term GnRHa-treatment on hippocampus-dependent cognitive functions, such as spatial orientation, learning and memory, is not well studied, particularly when treatment encompasses a critical window of development such as puberty. The current study used an ovine model to assess spatial maze performance and memory of rams that were untreated (Controls), had both GnRH and testosterone signaling blocked (GnRHa-treated), or specifically had GnRH signaling blocked (GnRHa-treated with testosterone replacement) during the peripubertal period (8, 27 and 41 weeks of age). The results demonstrate that emotional reactivity during spatial tasks was compromised by the blockade of gonadal steroid signaling, as seen by the restorative effects of testosterone replacement, while traverse times remained unchanged during assessment of spatial orientation and learning. The blockade of GnRH signaling alone was associated with impaired retention of long-term spatial memory and this effect was not restored with the replacement of testosterone signaling. These results indicate that GnRH signaling is involved in the retention and recollection of spatial information, potentially via alterations to spatial reference memory, and that therapeutic medical treatments using chronic GnRHa may have effects on this aspect of cognitive function.

ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity.

It has been postulated that synaptic potentiation during waking is offset by a homoeostatic reduction in net synaptic strength during sleep. However, molecular mechanisms to support such a process are lacking. Here we demonstrate that deficiencies in the RNA-editing gene Adar increase sleep due to synaptic dysfunction in glutamatergic neurons in Drosophila. Specifically, the vesicular glutamate transporter is upregulated, leading to over-activation of NMDA receptors, and the reserve pool of glutamatergic synaptic vesicles is selectively expanded in Adar mutants. Collectively these changes lead to sustained neurotransmitter release under conditions that would otherwise result in synaptic depression. We propose that a shift in the balance from synaptic depression towards synaptic potentiation in sleep-promoting neurons underlies the increased sleep pressure of Adar-deficient animals. Our findings provide a plausible molecular mechanism linking sleep and synaptic plasticity.

Progesterone can block the preovulatory gonadotropin-releasing hormone/luteinising hormone surge in the ewe by a direct inhibitory action on oestradiol-responsive cells within the hypothalamus.

Elevated oestradiol concentrations during the follicular phase stimulate a surge in gonadotropin-releasing hormone (GnRH) and luteinising hormone (LH) concentrations, which leads to ovulation. Progesterone can block the oestradiol-induced GnRH/LH surge, but the mechanism that is involved is unclear. We examined the effect of progesterone on oestradiol-induced activation of cells within the ovine hypothalamus/preoptic area (POA) to determine: (i) in which regions progesterone acts to block the GnRH/LH surge and (ii) whether progesterone directly or indirectly prevents activation of oestradiol-responsive cells. Cellular activation was assessed by measuring the number of cells that expressed Fos (an immediate early gene). Exposure to increased oestradiol concentrations in the absence of progesterone (which normally stimulates a LH surge) did not cause any region-specific changes in hypothalamic Fos expression during the activation stage of the LH surge-induction process (Experiment 1). The same treatment significantly increased cellular activation within the POA, lateral septum (LS), and arcuate nucleus at the time of surge onset (Experiment 2). Concurrent exposure to increased oestradiol and progesterone concentrations during the activation stage of the surge-induction process (which normally blocks the LH surge) was associated with significantly reduced cellular activation within the ventromedial hypothalamus and anterior hypothalamic area, relative to the positive controls (oestradiol increment alone) and arcuate nucleus relative to the negative controls (no increment in oestradiol) during the activation stage (Experiment 1). At the time of surge onset (Experiment 2), exposure to progesterone during the activation period prevented the oestradiol-induced increase in cellular activation that occurred in the POA, LS and arcuate nucleus of the positive controls. These results demonstrated that oestradiol and progesterone induced differential region- and time-specific effects on cellular activation within the regions of the ovine brain that generate the preovulatory GnRH/LH surge. Moreover, the lack of cellular activation within the POA, LS and arcuate nucleus at the time of surge onset in animals exposed to progesterone during the activation stage is consistent with the hypothesis that progesterone can block the preovulatory surge by direct inhibition of oestradiol-induced cellular activation in these areas.

Impacts of aggregate dredging on sediment composition and associated benthic fauna at an offshore dredge site in the southern North Sea.

Dredging and associated screening at a dredge site in the southern North Sea (Area 408) is associated with areas of well-sorted fine sand that extend for up to 3 km to the south-east of the dredged area and overlay sediments with a more variable particle size composition. This well-sorted fine sand may reflect deposition and transport of material mobilised by the dredging and screening processes at the dredge site. Multivariate analysis of the benthic community structure suggests that marine aggregate dredging, at the level of intensity employed in the study area prior to sample collection, has had a limited impact on benthic community composition compared with that reported from studies elsewhere. This is ascribed to the likely rapid rates of recolonisation by the mobile opportunistic polychaetes and crustaceans that dominate the macrofauna of the sandy gravel deposits at this particular dredge site. Analysis of variance showed, however, that significant differences existed between the sample treatments in terms of species evenness (Pielou's J). Dredged samples were found to have the lowest mean species evenness (0.71) when compared to controls (0.77). The present study highlights the inherent difficulties in the application of general impact/recovery predictions to dredged sites with varying environmental characteristics.

Antibody-dependent cell-mediated cytotoxicity directed by a human monoclonal antibody reactive with gp120 of HIV-1.

We used a human monoclonal antibody (MAb; 15e) to identify an antibody-dependent cell-mediated cytotoxicity (ADCC) epitope on HIV-1 gp120. 15e has been shown to recognize a conformation-dependent epitope on gp120 which is important in both CD4 binding and neutralizing of HIV-1 infection. 15e binds to gp120 of HIV-1IIIB but not HIV-1RF. Using a standard ADCC assay, 15e was found to mediate ADCC against cells infected with HIV-1IIIB but not HIV-1RF. 15e did not mediate ADCC against cells with recombinant gp120 bound to surface CD4, indicating that 15e does not mediate innocent bystander ADCC against uninfected CD4 cells. To better define the 15e epitope, we performed ADCC against target cells infected with a vaccinia vector which expresses processed HIV-1IIIB gp160 from which the third variable region was deleted (amino acids, 312-328). MAb 15e efficiently mediated ADCC against cells expressing this altered form of gp120, indicating that this region is not contributing to the conformational epitope defined by 15e. 15e defines an important epitope in the human immune response to HIV-1 infection. Antibodies with 15e-like activity may be useful in immunoprophylaxis or immunotherapy of HIV-1 infection.

In utero exposure of female lambs to testosterone reduces the sensitivity of the gonadotropin-releasing hormone neuronal network to inhibition by progesterone.

Exposure of the female ovine fetus to exogenous androgens during early gestation permanently masculinizes the reproductive anatomy, physiology, and behavior of the adult ewe. In utero testosterone exposure has been shown to act centrally on the GnRH neuronal network to alter the response to both the stimulatory and inhibitory actions of estrogen. It is currently unknown whether fetal androgens alter other mechanisms that are critical for the regulation of GnRH release and, specifically, other important regulatory steroid feedback loops. Three studies were performed on gonadectomized postpubertal sheep to determine whether the inhibitory actions of progesterone on episodic LH release are also sex-specific and engendered by early in utero exposure to testosterone. In each study, the pulsatile pattern of LH release was determined both before and after the sc implantation of a progesterone releasing CIDR device. The studies involved 7 female, 7 male, and 12 androgenized female sheep (T60 (n = 7) and T30 (n = 5) groups; 200 mg testosterone propionate/week im to the mother for 60 or 30 days, respectively, from day 30-90 or 60-90 of pregnancy). The first two studies were performed in the anestrous season in the presence (Exp 1) or absence (Exp 2) of a low circulating concentration of estradiol. Exp 3 was carried out in the breeding season in the absence of exogenous estrogen. In all three studies progesterone inhibited LH pulse frequency only in the females. Progesterone had no action on mean LH concentrations or the frequency or amplitude of LH pulses in the males or either group of androgenized ewes. We conclude that the inhibition of episodic LH release by progesterone is sexually differentiated in the sheep, males being less responsive than females to steroid negative feedback. Further, these sex differences are a consequence of in utero exposure to androgens for a period as short as 30 days between days 60 and 90 of a 147-day pregnancy.

Identification of neurokinin B-expressing neurons as an highly estrogen-receptive, sexually dimorphic cell group in the ovine arcuate nucleus.

Studies were undertaken to examine the hypothesis that neurons expressing neurokinin B (NKB) may represent an estrogen-receptive input to GnRH neurons in the sheep. Cells immunoreactive for NKB were located almost exclusively within the arcuate nucleus of the ovine hypothalamus. Dual labeling experiments revealed that essentially all NKB neurons (97%) were immunoreactive for estrogen receptor alpha and that NKB-immunoreactive fibers were found in close proximity to approximately 40% of GnRH neurons located in the rostral preoptic area as well as intermingled with GnRH fibers in the median eminence. The analysis of male and female brains revealed a marked female-dominant sex difference in the numbers of NKB neurons, and sections obtained from in utero androgen-treated females indicated that this sex difference resulted from an organizational influence of testosterone during neural development. In adult ovariectomized ewes, in situ hybridization studies failed to detect any significant effect of 8- to 26-h exposure of estrogen on cellular NKB messenger RNA levels. Together, these studies identify the first sexually differentiated neuronal cell population in the ovine hypothalamus and, remarkably, show that essentially all of these female-dominant NKB neurons express estrogen receptors. Although these neurons may be involved in any number of steroid-dependent, sexually differentiated functions in the sheep, the neuroanatomical evidence for potential NKB inputs to GnRH neurons suggests a role for this novel population in the regulation of reproductive function.

Gonadotropin-releasing hormone messenger ribonucleic acid expression changes before the onset of the estradiol-induced luteinizing hormone surge in the ewe.

The preovulatory LH surge in the ewe is stimulated by the massive and sustained release of GnRH into the pituitary portal vessels. This study has examined the temporal relationship between changes in LH secretion and GnRH messenger RNA (mRNA) expression at the time of the estradiol-induced LH surge. Ovariectomized Clun Forest ewes were treated with exogenous progesterone and estradiol (E) to mimic estrous cycle concentrations of these gonadal steroids and to induce the LH surge. Ewes were killed at five time points relative to the time of onset of the LH surge: pre-E, before E insertion (n = 6); presurge, after E insertion and 8-10 h before surge onset (n = 5); ascending limb, 2-6 h after surge onset (n = 5); midpeak, 9-12 h after surge onset (n = 5); and postsurge, 21-27 h after surge onset (n = 5). Control animals (n = 5/group), which received no E, were killed at identical time intervals alongside the E-treated ewes. Coronal sections containing the diagonal band of Broca through to the anterior hypothalamus were processed for cellular in situ hybridization using an 35S-labeled oligonucleotide probe complementary to ovine GnRH. No changes were found in the number of GnRH mRNA-expressing cells detected in the rostral preoptic area or the medial septum in either gonadal steroid-treated or control ewes. In contrast, cellular GnRH mRNA expression (as assessed by silver grain density) decreased significantly (P < 0.05) between presurge and ascending limb groups within both the rostral preoptic area (0.64 +/- 0.06 vs. 0.43 +/- 0.05 silver grain density/microm2) and medial septum cells (1.08 +/- 0.09 vs. 0.77 +/- 0.07). No significant changes were detected in control ewes. These results show that the estradiol-induced LH surge in the ewe is associated with a decrease in GnRH mRNA expression that occurs in advance of the onset of the GnRH surge. This suggests that neural mechanisms controlling GnRH biosynthesis may be distinct from those regulating GnRH secretion.