Juvenile Stage Of Development Research Articles

Comparison of three hatchery culture methods for the giant clam Tridacna noae

This study provides a first evaluation of different hatchery culture methods for the giant clam Tridacna noae. Three culture methods (intensive, semi-intensive, and extensive) using two tank designs (60 L and 277 L) were successful in producing 30-day old T. noae juveniles. There was no statistically significant interaction between tank design and culture method when considering cumulative survival (P = 0.89) or antero-posterior growth (P = 0.20), and the two tank designs did not influence cumulative survival (P = 0.78) or antero-posterior growth (P = 0.81). Differences in cumulative survival among the extensive (0.027 ± 0.015%), semi-intensive (0.005 ± 0.002%), and intensive (0.004 ± 0.001%) culture methods were non-significant (P = 0.11). The mean antero-posterior measurements at 30 days post-fertilisation among intensive (583.57 ± 12.71 μm), semi-intensive (530.92 ± 11.02 μm), and extensive methods (541.67 ± 10.95 μm), were also non-significant (P = 0.05). Further analysis of survival among different developmental stages identified significantly greater survival of T. noae between the pediveliger and juvenile development stages within the extensive culture method. Our results show that extensive culture methods can achieve comparable hatchery production of T. noae to semi-intensive and intensive culture methods, which require additional managerial, infrastructural, and nutritional inputs.

Relative Quantitation of Neuropeptides at Multiple Developmental Stages of the American Lobster Using N,N-Dimethyl Leucine Isobaric Tandem Mass Tags

Neuromodulators and neurotransmitters play important roles in neural network development. The quantitative changes of these signaling molecules often reflect their regulatory roles in physiological processes. Currently, several commercial tags (e.g., iTRAQ and TMT) have been widely used in proteomics. With reduced cost and higher labeling efficiency, we employed a set of custom-developed N, N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass tags as an attractive alternative for the relative quantitation of neuropeptides in brain tissue of American lobster Homarus americanus at multiple developmental stages. A general workflow for isobaric labeling of neuropeptides followed by LC-MS/MS analysis has been developed, including optimized sample handling procedures. Overall, we were able to quantify 18 trace-amount neuropeptides from 6 different families using a single adult brain as a control. The quantitation results indicated that the expressions of different neuropeptide families had significant changes over distinct developmental stages. Additionally, our data revealed intriguing elevated expression of neuropeptides in the early juvenile development stage. The methodology presented here advanced the workflow of DiLeu as an alternative labeling approach and the application of DiLeu-based quantitative peptidomics, which can be extended to areas beyond neuroscience.

High density brood of Australian gall-inducing Acacia thrips aid in fungal control

Kladothrips (Froggatt) is a genus of gall-inducing thrips that develop broods, and in some species, long-lived colonies within galls they form on phyllodes of Acacia in arid and semi-arid Australia. The gall interior is a stable environment for thrips in an otherwise inhospitable environment, but these conditions may also be favorable for fungal parasites. This fungal threat is corroborated by the observation that Kladothrips produce highly effective antifungal compounds. Here we investigated antifungal production in three Acacia thrips species, two gall-inducers: Kladothrips arotrum and Kladothrips tepperi, and one kleptoparasitic thrips, Koptothrips dyskritus. Using a spectrophotometer, the germination of a suspension of Cordyceps bassiana spores (an entomopathogenic fungus) can be detected by observing an abrupt increase in light absorption by the suspension. The addition of thrips exterior washes to these fungal spore suspensions resulted in significant delays in fungal germination for all three species. Foundresses in both Kladothrips species strongly delay fungal germination before their brood has matured. Young and maturing colonies with less than 50 adult individuals (the remainder of the brood are in juvenile stages of development) produced some antifungal effects but within the range produced by the foundress alone. Mature colonies (>100 adults) delayed germination for the duration of our observational window (48 h), suggesting a possible group size threshold for antifungal effectiveness. Koptothrips dyskritus antifungals were observed to be the strongest of the three species when <50 individuals were present. The strong antifungal abilities of the invading Ko. dyskritus would allow this species to invade older or damaged galls, which has been observed in the field. This pattern of antifungal activity in Acacia thrips suggests that effective defense against fungal pathogens is strongly associated with group size and colony maturity.

Potential for adaptation to climate change in a coral reef fish

Predicting the impacts of climate change requires knowledge of the potential to adapt to rising temperatures, which is unknown for most species. Adaptive potential may be especially important in tropical species that have narrow thermal ranges and live close to their thermal optimum. We used the animal model to estimate heritability, genotype by environment interactions and nongenetic maternal components of phenotypic variation in fitness-related traits in the coral reef damselfish, Acanthochromis polyacanthus. Offspring of wild-caught breeding pairs were reared for two generations at current-day and two elevated temperature treatments (+1.5 and +3.0°C) consistent with climate change projections. Length, weight, body condition and metabolic traits (resting and maximum metabolic rate and net aerobic scope) were measured at four stages of juvenile development. Additive genetic variation was low for length and weight at 0 and 15days posthatching (dph), but increased significantly at 30 dph. By contrast, nongenetic maternal effects on length, weight and body condition were high at 0 and 15 dph and became weaker at 30 dph. Metabolic traits, including net aerobic scope, exhibited high heritability at 90 dph. Furthermore, significant genotype x environment interactions indicated potential for adaptation of maximum metabolic rate and net aerobic scope at higher temperatures. Net aerobic scope was negatively correlated with weight, indicating that any adaptation of metabolic traits at higher temperatures could be accompanied by a reduction in body size. Finally, estimated breeding values for metabolic traits in F2 offspring were significantly affected by the parental rearing environment. Breeding values at higher temperatures were highest for transgenerationally acclimated fish, suggesting a possible role for epigenetic mechanisms in adaptive responses of metabolic traits. These results indicate a high potential for adaptation of aerobic scope to higher temperatures, which could enable reef fish populations to maintain their performance as ocean temperatures rise.

Prediction of time to harvest and its variability in broccoli (Brassica oleracea var. italica) Part I. Plant developmental variation and forecast of time to head induction

Field experiments showed a strong variability in time of head induction within broccoli plantings. This appears to be the cause of the high variability in head sizes at harvest. To quantify head induction, variation in head induction and the juvenile development cabinet experiments were carried out. One cabinet trial quantified the end of the juvenile development stage by using plants of different ages and size. Every three days a new set of seedlings was raised (26°C) and after youngest seedlings showed 1,5 leaves >1cm on average plants of different ages and with different number of visible leaves were transferred to cool conditions (10°C).Results suggest that the juvenile phase is already passed at planting. A second trial analyzed head induction in relation to temperature. Head induction was determined by microscopical analysis of apical meristems and was considered to have occurred when first floral buds were clearly visible on the apical meristem. With the results of the cabinet experiments we parameterised a dynamic model which considered the measured variation. The vernalisation phase is simulated as an optimum function of temperature by a piecewise linear regression. The variability in development is implemented by measured coefficients of variation combined with a normal distribution function. The model was evaluated using independent data of field crops of broccoli crops, grown in different regions, on different soil types, with different transplant raising systems and over a period of three years throughout the whole growing season. The model predicted the time to head induction well and was able to predict the variability in vernalisation occurring in the field.

Provenance by site interaction and stability analysis of European beech (Fagus sylvatica L.) provenances grown in common garden experiments

Abstract Fifteen provenances of European beech (Fagus sylvatica L.) were evaluated for stability and adaptability by height growth at four test sites in Bosnia and Herzegovina (1), Croatia (1) and Serbia (2). Provenance trials were established in spring 2007 by planting 2- and 3-yearold seedlings and arranged in a randomized complete block design. The data from seven 6- year-old and eight 5-year-old provenances were obtained in 2009 and analyzed separately. Finlay and Wilkinson’s regression analysis and Additive main effects and multiplicative interaction model (AMMI model) were used to assess provenance by site interaction (P × S) and to identify beech provenances that have high and stable performance in different environments, at the juvenile stage of development. Analysis of variance showed that effects of provenance, site, and P×S interaction are highly significant (p&lt;0.001) in both age groups. Linear regression model evidenced that most of the provenances had regression coefficients not significantly different from unity (b=1.0), except for provenances Sjeverni Dilj Caglinski (HR24) and Vranica-Bistrica (BA59). The partitioning of the total sum of squares (SS) exhibited that the site effect was the predominant source of variation in both age groups of provenances (50.7% and 38.5%, respectively). Additionally, regression analysis explained 15.8% and 33.2% of provenance by site interaction terms, in provenances age six and five years, respectively, while the AMMI analysis accounted for 62.2% and 78.7% in P×S interaction. The results of AMMI showed that the first principal component (PC1) was statistically significant in both age groups. Adaptability and stability of provenances to the test sites were estimated with AMMI1 and AMMI2 biplots. Provenance Sjeverni Dilj Caglinski (HR24) showed constant performance over tested sites, characterizing with aboveaverage height growth at low yielding environments. Provenances HR25 and BA61 showed the opposite type of adaptation, being adapted to high yielding sites. Provenance Valkonya (HU42) was characterized by IPCA1 score close to zero and above average mean height growth, suggesting general adaptation to the tested environments. The implication of P×S was discussed in light of impact of climate change on beech and selection of most suitable provenances for future reforestation programs.

Intranasal delivery of bone marrow mesenchymal stem cells improved neurovascular regeneration and rescued neuropsychiatric deficits after neonatal stroke in rats.

Neonatal stroke is a major cause of mortality and long-term morbidity in infants and children. Currently, very limited therapeutic strategies are available to protect the developing brain against ischemic damage and promote brain repairs for pediatric patients. Moreover, children who experienced neonatal stroke often have developmental social behavior problems. Cellular therapy using bone marrow mesenchymal stem cells (BMSCs) has emerged as a regenerative therapy after stroke. In the present investigation, neonatal stroke of postnatal day 7 (P7) rat pups was treated with noninvasive and brain-specific intranasal delivery of BMSCs at 6 h and 3 days after stroke (1 × 10(6)cells/animal). Prior to transplantation, BMSCs were subjected to hypoxic preconditioning to enhance their tolerance and regenerative properties. The effects on regenerative activities and stroke-induced sensorimotor and social behavioral deficits were specifically examined at P24 of juvenile age. The BMSC treatment significantly reduced infarct size and blood-brain barrier disruption, promoted angiogenesis, neurogenesis, neurovascular repair, and improved local cerebral blood flow in the ischemic cortex. BMSC-treated rats showed better sensorimotor and olfactory functional recovery than saline-treated animals, measured by the adhesive removal test and buried food finding test. In social behavioral tests, we observed functional and social behavioral deficits in P24 rats subjected to stroke at P7, while the BMSC treatment significantly improved the performance of stroke animals. Overall, intranasal BMSC transplantation after neonatal stroke shows neuroprotection and great potential as a regenerative therapy to enhance neurovascular regeneration and improve functional recovery observed at the juvenile stage of development.

Fine‐tuning of a mowing regime, a method for the management of the invasive plant, Ambrosia artemisiifolia, at different population densities

Common ragweed (Ambrosia artemisiifolia) is an invasive annual plant with highly allergenic pollen. Its spread in introduced and native ranges often occurs on roadsides, where it builds stable and rapidly growing populations. The most sustainable way of controlling the population size of this species is to prevent seed production in order to deplete the soil seed bank. Populations on roadsides are submitted to regular mowing management, which can even exacerbate the situation by inducing resprouting after cutting or by accidentally spreading seeds along the road. The population density in the juvenile stages of development could play an important role in the success of cutting regimes, as it might influence the resprouting capacity of this plant. The influence of the juvenile population density and of seven cutting regimes, differing in the timing and frequency of cuts, on easily measurable reproductive traits was investigated in a glasshouse experiment. The cutting regimes had a strong influence on the reproductive success and on the phenology of the development stages of ragweed. The population density in the juvenile stages did not play a role in further phenological development, but did influence the reproductive traits. The reproduction of ragweed can be lowered by locally adapted combinations of the timing and frequency of mowing. As the optimal management option for the reduction of both the male and female flowers, the authors suggest a first cut just before the start of male flowering, followed by subsequent cuts every 3–4 weeks.

Carbon, nitrogen and phosphorus ecological stoichiometry of Lateolabrax macultus and Acanthogobius ommaturus in the Estuary of Yangtze River, China

Most stoichiometric models do not consider the importance of ontogenetic changes in body nutrient composition. Among aquatic animals, body elemental ontogenetic changes may be particularly important in fish due to bone formation at larval–juvenile development stage. We investigated the stoichiometric characteristics and ontogenetic variation of carbon (C), nitrogen (N), phosphorous (P) and calcium (Ca) in the whole fish without gut of Lateolabrax macultus and Acanthogobius ommaturus from the Yangtze Estuary. Some differences were found in the whole-fish nutrient content across species as well as fish length and mass groups. Mean body content of L. macultus was 41.2% for C (34.9–44.9%), 11.8% for N (10.3–12.9%), and 3.3% for P (1.4–5.4%) and the molar ratio of C:N:P was 32:8:1. A. ommaturus had the following mean compositions: carbon, 43.4% (40.0–45.9%); nitrogen, 13.2% (11.7–14.1%); phosphorus, 2.9% (1.6–6.0%) and molar ratio of C:N:P was 38:10:1. The variation of C content in of L. macultus was minimum (0.05), followed by N (0.06), and the variation of P content was the highest (0.22). The variation of C content in A. ommaturus was minimum (0.03), followed by N (0.04), and the variation of P content was also the highest (0.27). Both species varied the least in C, moderate in N and the most in P. Comparing the two species, A. ommaturus was significantly higher for C, N, C:P, N:P and significantly lower for P and C:N than L. macultus. The positive correlation between C and N contents was significant (P<0.01) for both species. In contrast, C and P concentrations showed little correlation (P>0.05). The contents of C, N in L. macultus showed significant negative correlation with total length and wet mass, respectively, while C:N ratio showed significant positive correlation with them. As for A. ommaturus, the content of C and the ratio of C:N showed significant negative correlation with total length and wet mass, respectively. The two estuarine fishes in this study mainly consumed fish, shrimps and crabs, which could be the reason that the stoichiometry of L. macultus and A. ommaturus are higher than other freshwater fishes that have been reported previously. In both species, the content of calcium was highly correlated with the content of body P, which indicated that the change of the content of P is associated with bone formation in ontogeny. Fish shape, scale type, the ratio of head length/standard length and the degree of ossification of the internal skeleton and outer integument might have caused the results that L. macultus had higher contents of P and Ca than A. ommaturus.

Developmental change in the function of movement systems: transition of the pectoral fins between respiratory and locomotor roles in zebrafish.

An animal may experience strikingly different functional demands on its body’s systems through development. One way of meeting those demands is with temporary, stage-specific adaptations. This strategy requires the animal to develop appropriate morphological states or physiological pathways that address transient functional demands as well as processes that transition morphology, physiology, and function to that of the mature form. Recent research on ray-finned (actinopterygian) fishes is a developmental transition in function of the pectoral fin, thereby providing an opportunity to examine how an organism copes with changes in the roles of its morphology between stages of its life history. As larvae, zebrafish alternate their pectoral fins in coordination with the body axis during slow swimming. The movements of their fins do not appear to contribute to the production of thrust or to stability but instead exchange fluid near the body for cutaneous respiration. The morphology of the larval fin includes a simple stage-specific endoskeletal disc overlaid by fan-shaped adductor and abductor muscles. In contrast, the musculoskeletal system of the mature fin consists of a suite of muscles and bones. Fins are extended laterally during slow swimming of the adult, without the distinct, high-amplitude left-right fin alternation of the larval fin. The morphological and functional transition of the pectoral fin occurs through juvenile development. Early in this period, at about 3 weeks post-fertilization, the gills take over respiratory function, presumably freeing the fins for other roles. Kinematic data suggest that the loss of respiratory function does not lead to a rapid switch in patterns of fin movement but rather that both morphology and movement transition gradually through the juvenile stage of development. Studies relating structure to function often focus on stable systems that are arguably well adapted for the roles they play. Examining how animals navigate transitional periods, when the link of structure to function may be less taut, provides insight both into how animals contend with such change and into the developmental pressures that shape mature form and function.

First results of larval rearing and development of the bluestriped angelfishChaetodontoplus septentrionalis(Temminck &amp; Schlegel) from hatching through juvenile stage with notes on its potential for aquaculture

The aim of this study was to report the first successful results on larval rearing of Chaetodontoplus septentrionalis, a description of larval and juvenile development stages, and appropriate first-feed items for pilot-scale larviculture systems. Fertilized eggs were obtained from natural spawnings from communally reared captive wild broodstock. Newly hatched larvae were 2.11 ± 0.06 mm (mean ± SD) in total length (LT) with 27–28 myomeres. Larvae completed yolk absorption within 3 days post hatching (dph) at 2.92 ± 0.11 mm LT. Transformation of larvae to the juvenile stage was completed at 16.51 mm LT, all fins had the adult complement of rays and spines. A statistical model: Y = (X × 0.0112) + (X2 × 0.0086) − (X3 × 0.00004) + 2.8065, where Y is mean LT (mm) and X represents dph, explained 99.8% of variation in growth (P < 0.001, R2 = 0.998). The effects of live feed on survival was significantly (P < 0.05) higher in larvae fed a combination of diet (ciliates, rotifers and copepods) than the others. These results indicate that C. septentrionalis is a potential species for captive breeding programs and the use of a combination of diet may be a suitable first food for the larviculture.

Clever strategists: Australian Magpies vary mobbing strategies, not intensity, relative to different species of predator

Anti-predator behaviour of magpies was investigated, using five species of model predators, at times of raising offspring. We predicted differences in mobbing strategies for each predator presented and also that raising juveniles would affect intensity of the mobbing event. Fourteen permanent resident family groups were tested using 5 different types of predator (avian and reptilian) known to be of varying degrees of risk to magpies and common in their habitat. In all, 210 trials were conducted (across three different stages of juvenile development). We found that the stage of juvenile development did not alter mobbing behaviour significantly, but predator type did. Aerial strategies (such as swooping) were elicited by taxidermic models of raptors, whereas a taxidermic model of a monitor lizard was approached on the ground and a model snake was rarely approached. Swooping patterns also changed according to which of the three raptors was presented. Our results show that, in contrast to findings in other species, magpies vary mobbing strategy depending on the predator rather than varying mobbing intensity.

Prenatal exposure to bisphenol A impacts midbrain dopamine neurons and hippocampal spine synapses in non-human primates

Prevalent use of bisphenol-A (BPA) in the manufacture of resins, plastics and paper products has led to frequent exposure of most people to this endocrine disruptor. Some rodent studies have suggested that BPA can exert detrimental effects on brain development. However as rodent models cannot be relied on to predict consequences of human exposure to BPA during development, it is important to investigate the effects of BPA on non-human primate brain development. Previous research suggests that BPA preferentially targets dopamine neurons in ventral mesencephalon and glutamatergic neurons in hippocampus, so the present work examined the susceptibility of these systems to low dose BPA exposure at the fetal and juvenile stages of development in non-human primates. Exposure of pregnant rhesus monkeys to relatively low levels of BPA during the final 2 months of gestation, induced abnormalities in fetal ventral mesencephalon and hippocampus. Specifically, light microscopy revealed a decrease in tyrosine hydroxylase-expressing (dopamine) neurons in the midbrain of BPA-exposed fetuses and electron microscopy identified a reduction in spine synapses in the CA1 region of hippocampus. In contrast, administration of BPA to juvenile vervet monkeys (14–18 months of age) was without effect on these indices, or on dopamine and serotonin concentrations in striatum and prefrontal cortex, or on performance of a cognitive task that tests working memory capacity. These data indicate that BPA exerts an age-dependent detrimental impact on primate brain development, at blood levels within the range measured in humans having only environmental contact with BPA.

Leaf morphometric characteristics variability of different beech provenances in juvenile development stage

The taxonomic status of beech from the Balkan Peninsula is not yet clearly defined. There is no agreement among different authors about the morphological characteristics discriminating between the Balkan and European and/or Eastern beech. For most characteristics, the mean values are different but the ranges of variation overlap considerably. Provenance trial of beech established in Serbia, at the locality Debeli Lug, has provided an opportunity for research of interprovenance variability at the level of leaf morphometric characteristics in juvenile development stage. Research included 10 provenances originating from the Western Balkans (Serbian provenance 36 and 38; Croatian provenance 24 and 25; Bosnian provenance 30 and 32) and from Central Europe (German provenance 47 and 49; Austrian provenance 56 and Hungarian provenance 42), where following morphometric characteristics were analyzed: leaf length (Ll), leaf width (Lw), petiole lenght (Pl), leaf base width on 1 cm (Blw), number of veins - left (Vl), number of veins - right (Vr), distance between 3rd and 4th vein - left (Dv 3-4). The results of this research show existence of clear differentiation among provenances from the Western Balkan and from Central Europe, from the point of leaf dimensions, number of veins and leaf base width.

Spawning and Development of Larvae and Juveniles of the Indian Ocean Oriental Sweetlips, Plectorhinchus vittatus (Linnaeus, 1758), in the Aquarium

The aim of this study was to report the successful results on voluntary spawning of Plectorhinchus vittatus, a description of larval and juvenile development stages, and appropriate first‐feed items for pilot‐scale larviculture systems. Spontaneous spawning of P. vittatus in captivity from 11 April to 22 June, 2009, was described for the first time. Over a 73‐d period, the female produced 49 spawns, 75.1% of which resulted in fertile eggs. The average fecundity per spawn was 4170 eggs (range 2090–7080 eggs). Fertilized eggs were spherical, buoyant, and had a diameter of 0.80 ± 0.02 mm (mean ± SD). Newly hatched larvae were 1.90 ± 0.06 mm in total length (TL) with 28 myomeres. Transformation of larvae to the juvenile stage was completed at 9.58 ± 0.69 mm TL. A statistical model: Y = (X× 0.5034) − (X2× 0.0004) − 0.5883, where Y is mean TL (mm) and X represents days post hatch, explained 99.57% of variation in growth (P &lt; 0.001, R2 = 0.9957). The effects of live feed on survival was significantly (P &lt; 0.05) higher in larvae fed a combination of diet (rotifers, copepods, and Artemia) than the others. These results indicate that P. vittatus is a potential species for captive breeding programs and the use of a combination of diet may be a suitable first food for the larviculture.

Age‐dependent trade‐offs between immunity and male, but not female, reproduction

Immune function is costly and must be traded off against other life-history traits, such as gamete production. Studies of immune trade-offs typically focus on adult individuals, yet the juvenile stage can be a highly protracted period when reproductive resources are acquired and immune challenges are ubiquitous. Trade-offs during development are likely to be important, yet no studies have considered changes in adult responses to immune challenges imposed at different stages of juvenile development. By manipulating the timing of a bacterial immune challenge to the larvae of the cotton bollworm moth, we examined potential trade-offs between investment into immunity at different stages of juvenile development (early or late) and subsequent adult reproductive investment into sperm or egg production. Our data reveal an age-dependent trade-off between juvenile immune function and adult male reproductive investment. Activation of the immune response during late development resulted in a reduced allocation of resources to eupyrene (fertilizing) sperm production. Immune activation from the injection procedure itself (irrespective of whether individuals were injected with an immune elicitor or a control solution) also caused reproductive trade-offs; males injected early in development produced fewer apyrene (nonfertilizing) sperm. Contrary to many other studies, our study demonstrates these immune trade-offs under ad libitum nutritional conditions. No trade-offs were observed between female immune activation and adult reproductive investment. We suggest the differences in trade-offs observed between male sperm types and the absence of reproductive trade-offs in females may be the result of ontogenetic differences in gamete production in this species. Our data reveal developmental windows when trade-offs between immune function and gametic investment are made, and highlight the importance of considering multiple developmental periods when making inferences regarding the fundamental trade-offs expected between immune function and reproduction.

Juvenile Immunotoxicology

Developmental immunotoxicity (DIT) testing is centered around the concern that exposure to immunotoxicants early in development may result in enhanced susceptibility of, or unique or more persistent effects on, the immune system, in comparison to adult exposure. Developmental immunotoxicity has been the focus of numerous workshops and reviews for at least fifteen years. Most of these earlier activities have focused on both environmental chemicals and pharmaceuticals and have concluded that the best approach to DIT is to address the possible impacts of exposure during all of the critical windows of development. This article will emphasize the critical role played by exposure during the juvenile stage of development. This article will also highlight several key issues that distinguish DIT testing of pharmaceuticals. Representatives from the pharmaceutical, biotechnology, academic, and regulatory sectors (both FDA and EMA) were brought together during a two-day workshop in May 2010 to consider the current state of the science of DIT as it pertains to the testing of pharmaceuticals. It is important to emphasize at the onset that there are currently no regulatory guidelines for either drugs or nondrug chemicals specifically focused on assessment of DIT, although some general guidelines are included in both developmental and reproductive toxicity and general immunotoxicology guidance documents.

Sertoli cell differentiation in rhesus monkey (Macaca mulatta) is an early event in puberty and precedes attainment of the adult complement of undifferentiated spermatogonia

In primates, the time course of Sertoli cell proliferation and differentiation during puberty and its relationship with the expansion of undifferentiated type A spermatogonia that occurs at this critical stage of development are poorly defined. Mid and late juvenile and early and late pubertal male rhesus monkeys were studied. Testes were immersion fixed, embedded in paraffin, and sectioned at 5 μm. Sertoli cell number per testis, S-phase labeling (BrdU), and growth fraction (Ki67 labeling) were determined and correlated with corresponding parameters for undifferentiated type A spermatogonia (A dark and A pale). Dual fluorescence labeling was used in addition to histochemistry to monitor spermatogonial differentiation during the peripubertal period using GFRα-1 and cKIT as markers. While the adult complement of Sertoli cells/testis was attained in early pubertal monkeys after only a few weeks of exposure to the elevated gonadotropin secretion characteristic of this developmental stage, the number of undifferentiated type A spermatogonia several months later in mid pubertal monkeys was only 50% of that in adult testes. Both A dark and A pale spermatogonia exhibited high S-phase BrdU labeling at all stages of juvenile and pubertal development. Spermatogonial differentiation, as reflected histochemically and by relative changes in GFRα-1 and cKIT expression, was not observed until after the initiation of puberty. In the rhesus monkey and maybe in other higher primates including human, the pubertal proliferation of undifferentiated spermatogonia is insidious and proceeds in the wake of a surge in Sertoli cell proliferation following termination of the juvenile stage of development.

Hypothalamic Actions and Interactions of Alcohol and IGF-1 on the Expression of Glial Receptor Protein Tyrosine Phosphatase-β During Female Pubertal Development

Hypothalamic glial-neuronal communications are important for the activation of luteinizing hormone releasing hormone (LHRH) secretion at the time of puberty. As we have shown that alcohol (ALC) diminishes prepubertal LHRH secretion and delays puberty, we first assessed the effects of short-term ALC administration on the basal expression of a specific gene family involved in glial-neuronal communications. Second, as insulin-like growth factor-1 (IGF-1) is a critical regulator of LHRH secretion and the pubertal process, we then assessed whether IGF-1 could induce the expression of these signaling genes and determine whether ALC can block this affect. Immature female rats were fed a liquid diet containing ALC for 6 days beginning when 27 days old. Control animals received either the companion isocaloric liquid diet or rat chow and water. Animals were decapitated on day 33, in the late juvenile stage of development. Medial basal hypothalamic (MBH) tissues were obtained for gene and protein analyses of glial receptor protein tyrosine phosphatase-β (RPTPβ) and the 2 neuronal components, contactin and contactin-associated protein 1 (Caspr1). In the second experiment, IGF-1 was administered into the third ventricle (3V) and the MBH removed 6 hours after peptide delivery, and the above-mentioned 3 genes were analyzed by real-time PCR. To determine whether this action was affected by ALC, immature female rats were administered either ALC (3 g/kg) or water via gastric gavage at 0900 hours. At 1030 hours, the ALC and control groups were subdivided such that half of the animals were injected into the 3V with IGF-1 and the other half with an equal volume of saline. Rats were killed 6 hours after the IGF-1 injection and MBHs collected. Real-time PCR showed that when compared with control animals, ALC caused a marked decrease (p < 0.001) in the basal expression of the RPTPβ gene, but did not affect the expression of either contactin or Caspr1. Likewise, analysis by Western blotting demonstrated that ALC caused suppressed (p < 0.001) levels of the RPTPβ protein, with the expressions of both contactin and Caspr1 proteins being unaltered. In the second experiment, results showed that only the RPTPβ gene was stimulated (p < 0.05) by IGF-1 in the MBH 6 hours after peptide delivery. Assessments revealed that the IGF-1 induced increase (p < 0.01) in the expression of the RPTPβ gene was blocked by the presence of ALC. Prepubertal ALC exposure is capable of interfering with hypothalamic glial-neuronal communications by suppressing the synthesis of the glial product, RPTPβ, which is required for binding to the contactin-Caspr1 complex on LHRH neuronal terminals, thus suggesting that this action of ALC contributes to its detrimental effects on the pubertal process.

Prepubertal ethanol exposure alters hypothalamic transforming growth factor-α and erbB1 receptor signaling in the female rat

Glial-derived transforming growth factor alpha (TGFα) activates the erbB1/erbB2 receptor complex on adjacent glial cells in the medial basal hypothalamus (MBH). This receptor activation stimulates the synthesis and release of prostaglandin-E 2 (PGE 2) from the glial cells, which then induces the release of prepubertal luteinizing hormone-releasing hormone (LHRH) secretion from nearby nerve terminals; thus, showing the importance of glial–neuronal communications at the time of puberty. Ethanol (EtOH) is known to cause depressed prepubertal LHRH secretion and delayed pubertal development. In this study, we assessed whether short-term EtOH exposure could alter the hypothalamic glial to glial signaling components involved in prepubertal PGE 2 secretion. Immature female rats began receiving control or EtOH diets beginning when 27 days old. The animals were killed by decapitation after 4 and 6 days of treatment and confirmed to be in the late juvenile stage of development. Blood and brain tissues were collected for gene, protein, and hormonal assessments. Real-time polymerase chain reaction (PCR) analysis demonstrated that EtOH did not affect basal levels of erbB1 gene expression in the MBH. Expression of total erbB1 protein was also unaffected; however, the EtOH caused suppressed phosphorylation of erbB1 protein in the MBH at both 4 and 6 days ( P < .01) as revealed by Western blotting. Phosphorylation and total protein levels of erbB2 receptor were not affected by EtOH exposure. Because this receptor is critical for PGE 2 synthesis/release, which mediates the secretion of LHRH, we assessed whether in vivo EtOH exposure could affect the release of PGE 2. EtOH exposure for 6 days suppressed ( P < .01) basal levels of PGE 2 released into the medium. The effects of 4- and 6-day EtOH exposure on gene and protein expressions of TGFα, an upstream component in the activation of erbB1/erbB2, were also studied. The levels of TGFα mRNA were increased markedly at 4 days ( P < .001), but declined to near basal levels by 6 days in the EtOH-treated animals. The EtOH caused increases in TGFα protein expression at both 4 ( P < .001) and 6 ( P < .01) days; hence, suggesting that the EtOH inhibited release of the peptide. We confirmed this inhibition by showing decreased ( P < .01) TGFα released from MBHs incubated in vitro following 6 days of EtOH exposure in vivo. Thus, these results demonstrate that EtOH is capable of interfering with hypothalamic glial to glial signaling processes involved in prepubertal PGE 2 secretion.