orchard: Paternity program for autotetraploid species.

Advances in molecular marker technology have provided new opportunities to study the population genetics of polyploid taxa. Paternity analysis using microsatellite markers can be used in detection of gene flow between individuals and populations, in mating system analysis, to identify factors that influence fecundity and fertility, to identify behaviour of parent-offspring relationships and in the analysis of the reproductive success of different ecological groups. As there is no specific program for carrying out paternity analysis in tetraploid species, specialized software was designed for the assignment of paternity for autotetraploid species. orchard is a novel implementation of exclusion and likelihood statistics for carrying out paternity analysis of autotetraploids. First, the program performs an exclusion method, and then, a likelihood statistic is used with nonexcluded candidate fathers. Optional features include estimation of allele dosage of known mother trees and the estimation of pollen flow distances. orchard was tested using a data set of microsatellite data of Dipteryx odorata, a tetraploid Amazonian tree species.

Remnant Pachira quinata pasture trees have greater opportunities to self and suffer reduced reproductive success due to inbreeding depression.

Habitat fragmentation is extensive throughout the world, converting natural ecosystems into fragments of varying size, density and connectivity. The potential value of remnant trees in agricultural landscapes as seed sources and in connecting fragments has formed a fertile area of debate. This study contrasted the mating patterns of bat-pollinated Pachira quinata trees in a continuous forest to those in pasture through microsatellite-based paternity analysis of progeny. The breeding system was determined by analysis of pollen tube growth and seed production from controlled pollinations. Fitness of selfed and outcrossed seed was compared by germination and seedling growth. There was more inbreeding within pasture trees (outcrossing=0.828±0.015) compared with forest trees (0.926±0.005). Pasture trees had fewer sires contributing to mating events, but pollen dispersal distances were greater than those in the forest. Paternity analysis showed variation in outcrossing rates among pasture trees with high proportions of external and self pollen sources detected. A leaky self-incompatibility system was found, with self pollen having reduced germination on stigmas and slower growth rate through the style. Controlled pollinations also showed a varied ability to self among trees, which was reflected in the selfing rates among pasture trees shown by the paternity analysis (0-80% selfing). Self pollination resulted in lower seed set, germination and seedling growth compared with outcrossing. While remnant trees in agricultural landscapes are involved in broader mating patterns, they show increased but varied levels of inbreeding, which result in reduced fitness.

Long-term impacts of selective logging on two Amazonian tree species with contrasting ecological and reproductive characteristics: inferences from Eco-gene model simulations.

The impact of logging and subsequent recovery after logging is predicted to vary depending on specific life history traits of the logged species. The Eco-gene simulation model was used to evaluate the long-term impacts of selective logging over 300 years on two contrasting Brazilian Amazon tree species, Dipteryx odorata and Jacaranda copaia. D. odorata (Leguminosae), a slow growing climax tree, occurs at very low densities, whereas J. copaia (Bignoniaceae) is a fast growing pioneer tree that occurs at high densities. Microsatellite multilocus genotypes of the pre-logging populations were used as data inputs for the Eco-gene model and post-logging genetic data was used to verify the output from the simulations. Overall, under current Brazilian forest management regulations, there were neither short nor long-term impacts on J. copaia. By contrast, D. odorata cannot be sustainably logged under current regulations, a sustainable scenario was achieved by increasing the minimum cutting diameter at breast height from 50 to 100 cm over 30-year logging cycles. Genetic parameters were only slightly affected by selective logging, with reductions in the numbers of alleles and single genotypes. In the short term, the loss of alleles seen in J. copaia simulations was the same as in real data, whereas fewer alleles were lost in D. odorata simulations than in the field. The different impacts and periods of recovery for each species support the idea that ecological and genetic information are essential at species, ecological guild or reproductive group levels to help derive sustainable management scenarios for tropical forests.

Impacts of selective logging on inbreeding and gene flow in two Amazonian timber species with contrasting ecological and reproductive characteristics.

Selective logging in Brazil allows for the removal of up to 90% of trees above 50 cm diameter of a given timber species, independent of a species' life history characteristics or how quickly it will recover. The genetic and demographic effects of selective logging on two Amazonian timber species (Dipteryx odorata Leguminosae, Jacaranda copaia Bignoniaceae) with contrasting ecological and reproductive characteristics were assessed in the same forest. Genetic diversity and gene flow were characterized by genotyping adults and seed sampled before and after logging, using hypervariable microsatellite markers. Overall, there were no short-term genetic impacts on the J. copaia population, with commercial application of current Brazilian forest management regulations. In contrast, for D. Odorata, selective logging showed a range of genetic impacts, with a 10% loss of alleles, and reductions in siring by pollen from trees within the 546-ha study area (23-11%) and in the number of pollen donors per progeny array (2.8-1.6), illustrating the importance of the surrounding landscape. Asynchrony in flowering between D. odorata trees led to trees with no breeding partners, which could limit the species reproduction and regeneration under current regulations. The results are summarized with other published studies from the same site and the implications for forest management discussed. The different types and levels of impacts associated with each species support the idea that ecological and genetic information by species, ecological guild or reproductive group is essential in helping to derive sustainable logging guidelines for tropical forests.

Genetic resource impacts of habitat loss and degradation; reconciling empirical evidence and predicted theory for neotropical trees.

The theoretical impacts of anthropogenic habitat degradation on genetic resources have been well articulated. Here we use a simulation approach to assess the magnitude of expected genetic change, and review 31 studies of 23 neotropical tree species to assess whether empirical case studies conform to theory. Major differences in the sensitivity of measures to detect the genetic health of degraded populations were obvious. Most studies employing genetic diversity (nine out of 13) found no significant consequences, yet most that assessed progeny inbreeding (six out of eight), reproductive output (seven out of 10) and fitness (all six) highlighted significant impacts. These observations are in line with theory, where inbreeding is observed immediately following impact, but genetic diversity is lost slowly over subsequent generations, which for trees may take decades. Studies also highlight the ecological, not just genetic, consequences of habitat degradation that can cause reduced seed set and progeny fitness. Unexpectedly, two studies examining pollen flow using paternity analysis highlight an extensive network of gene flow at smaller spatial scales (less than 10 km). Gene flow can thus mitigate against loss of genetic diversity and assist in long-term population viability, even in degraded landscapes. Unfortunately, the surveyed studies were too few and heterogeneous to examine concepts of population size thresholds and genetic resilience in relation to life history. Future suggested research priorities include undertaking integrated studies on a range of species in the same landscapes; better documentation of the extent and duration of impact; and most importantly, combining neutral marker, pollination dynamics, ecological consequences, and progeny fitness assessment within single studies.

Increased pollen flow counteracts fragmentation in a tropical dry forest: an example from Swietenia humilis Zuccarini.

Habitat destruction and the resultant fragmentation of the remaining forest are a common phenomenon in the tropics. Most investigations emphasize the potential dangers of fragmentation in isolating patches of forest and exposing populations to loss of species diversity through founder effects, genetic drift, inbreeding, and restricted gene flow. However, a limited number of studies have shown that gene flow may be extensive in tropical trees, suggesting that it may occur between forest fragments and also "isolated" remnant trees. There is an urgent need to quantify pollen flow within and between forest fragments to test the veracity of such views and determine the genetic value of such fragments for in situ conservation. Microsatellite markers are used to genotype individuals of Swietenia humilis from a highly fragmented forest mosaic to directly quantify pollen-mediated gene flow. Distances of pollen flow more than 10 times greater than previously reported were detected. Our results show that some tropical angiosperm tree species may be much more adaptable and resilient to habitat destruction and fragmentation than previously considered. The description of many remnant trees as isolated or "living dead" may be more a conditioning of human perception than a true reflection of their potential conservation value.

Immunohistochemical localization of 3 beta-hydroxysteroid/delta 5-delta 4-isomerase, tyrosine hydroxylase and phenylethanolamine N-methyl transferase in adrenal glands of sheep fetuses throughout gestation and in neonates.

Immunoreactive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) was localized in adrenal glands of sheep fetuses in cortical-type cells, but not in medullary-type cells, from day 43 of gestation to term and in 2-4-day-old neonates. From day 54 of gestation, the formation of distinct zones within the adrenal cortex was apparent and immunoreactive 3 beta-HSD was found in cortical cells in the zona fasciculata and in groups and cords of cortical cells within the developing medulla, with weak positive staining in the zona glomerulosa. At this stage, most medullary cells were positive for immunoreactive tyrosine hydroxylase, and some of these cells with a juxtacortical distribution also stained positively for immunoreactive phenylethanolamine N-methyl transferase (PNMT). Between days 65 and 130, the adrenal medulla increased in size with little change in the width of the cortex. Organization and zonation of immunoreactive 3 beta-HSD staining cells were evident in the zona fasciculata and in groups of cells in the medulla. Between day 130 and term, uniform immunoreactive 3 beta-HSD staining was found throughout the zona fasciculata, and there was also staining in single cells and small clusters of cells throughout the medulla. At this stage, immunoreactive tyrosine hydroxylase was distributed in most cells throughout the medulla, but in two distinct patterns: cells staining intensely for immunoreactive tyrosine hydroxylase in the central region of the medulla, and cells exhibiting weaker staining for immunoreactive tyrosine hydroxylase localized in a juxta-cortical position. These juxta-cortical cells were also positive for immunoreactive PNMT.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of hypothalamic lesions on the length of gestation in fetal sheep.

Twenty-nine sheep fetuses were subject to stereotaxic surgery at 106 to 110 days of gestation. Electrolytic lesions were placed bilaterally in the anterior hypothalamus. Sham-operated controls (n = 4) were delivered at 146.3 +/- 4.3 days. Of the fetuses with lesions, two were excluded because histologic confirmation of the lesion was not possible. Those fetuses with bilateral lesions of the suprachiasmatic nuclei (n = 4) were delivered at 148 +/- 10 days. In 10 animals with bilateral lesions of the paraventricular nuclei, delivery was significantly (p less than 0.0001) prolonged to at least 165.6 +/- 5.1 days. In nine animals with lesions not involving the endocrine hypothalamus, delivery was at 148.1 +/- 4.3 days. All animals that were delivered after 157 days (n = 9) had lesions including the paraventricular nuclei bilaterally (p less than 0.01). The adrenal glands of fetuses with prolonged gestation were normal in weight and light microscopic appearance. These observations demonstrate that fetal neural pathways involving the paraventricular nuclei are essential for parturition in the sheep. However, fetal adrenal growth can continue without such influences.

Morphometric analyses of adrenal gland growth in fetal and neonatal sheep. III. Volumes of the major organelles within zona fasciculata steroidogenic cells.

We have undertaken a morphometric analysis of the distribution patterns of the major subcellular organelles of the zona fasciculata in fetal (53, 100, 130, 144 days) and neonatal (2 days) sheep as they may be expressed as volume densities, volumes per cell, volumes per 100 microns 3 of cytoplasm per cell, and volumes per mm3 of steroidogenic cells per gland. Data obtained indicate that certain of the volume densities and the 3 other volume indicators of the 6 organelles considered change with gestational age. While the volume of mitochondria per cell increased in late gestation because of cellular hypertrophy, the real time volumes of mitochondria per unit volume of cell or gland did not vary significantly during the period of development studied. The volume of the SER, however, changed markedly, being minimal at 100 days fetal development (0.68 gestation) and increasing, probably initially during the 123-130 day (0.84-0.88 gestation) period, until the end of the fetal period (term = 147 days). The SER exhibited the most marked variability in the three volume indicators, the volumes correlating closely with cell size and steroidogenic capability. The RER volumes altered little, decreasing per 100 microns 3 of cytoplasm from mid-gestation, probably as a reflection of the cell maturity state attained. Changes in the distribution of the Golgi apparatus paralleled those of the SER; but the lipid storage droplets were sparse and their volumes constant. In the fetal sheep, the Golgi apparatus appears to be a significant component of the steroidogenic organelle complex, while the lipid droplet compartment does not. Consideration of growth during the 2nd major growth phase of the adrenal cortex (from before 0.85 gestation), as expressed in mitotic events, showed that at 130 days (0.88 gestation) cellular replication was randomly spread through the zona fasciculata, whereas at 144 days (0.98 gestation) mitoses were twice as prevalent in the outer half of that zone as they were in the inner half. At 130 and 144 days, the cells of the outer half of the zona fasciculata contained significantly more smooth endoplasmic reticulum than did those of the inner half. The maturation of the zona fasciculata, which allows the maximal production of cortisol in late pregnancy, thus occurs in a centripetal direction. This may result from the fact that the outer cells of the zona fasciculata are first in contact with the rising levels of ACTH and cortisol, two primary determinants of its growth and differentiation during the last 3 weeks of gestation.

Immunohistochemical localization of prostaglandin H synthase in the sheep placenta from early pregnancy to term.

Prostaglandin H synthase (PGHS) activity within intrauterine tissues is considered to catalyze a critical step in prostaglandin (PG) biosynthesis at parturition. In sheep, the placenta is a major site of PG production throughout pregnancy, but little information is available concerning the cells that are responsible. Therefore we determined the distribution of immunoreactive (IR-) PGHS in ovine placental tissue obtained at different times of pregnancy using immunohistochemical techniques. In placentomes from early pregnancy (Days 30-54), IR-PGHS was present in maternal epithelial syncytium, but was not detectable in trophoblast cells. Between Day 54 and Day 100, the number of cells that stained positive for PGHS declined in the maternal epithelial layer in the body of the placenta, but IR-PGHS was present in maternal epithelial cells overlying the vascular cones of the placental hemophagous zone. It was also present in the chorionic fibroblasts, but remained undetectable from all classes of trophoblast cells. IR-PGHS was first detectable in the trophoblastic epithelium by Day 114. Between Day 119 and term the trophoblast mononuclear epithelial cells were intensely immunopositive for PGHS, although immunonegative binucleate cells were present. The maternal epithelium was immunonegative except during the last 7-10 days of pregnancy when PGHS immunostaining appeared in both basal and apical regions of the placenta. Thus, the cellular localization of IR-PGHS changes during ovine pregnancy, from predominantly maternal during the first half of gestation to undetectable and then to predominantly trophoblastic between Day 114 and term, suggesting a gestation-dependent change in sites of PG production during ovine pregnancy. Appearance of IR-PGHS in the trophoblast precedes activation of the fetal hypothalamic-pituitary-adrenal axis, generally considered to provide the trigger to the onset of parturition in sheep, and would therefore appear to be regulated through alternative pathways or mechanisms.

Investigations on the morphology and function of adrenocortical tissue regenerated from gland capsular fragments autotransplanted in the musculus gracilis of the rat.

This paper describes the function and morphology of regenerated adrenocortical nodules obtained by implanting, in the musculus gracilis of rats, several (n = 6-7) fragments of the capsular tissue of their excised adrenal glands. Four months after the operation, each bilaterally adrenalectomized rat developed six or seven well encapsulated adrenocortical nodules about 2-3 mm in diameter and always lacking chromaffin cells, and displayed almost complete normalization of basal and stimulated blood levels of corticosterone, but not of aldosterone. In vitro study showed that regenerated nodules were well functioning as far as glucocorticoid production was concerned. Accordingly, electron microscopy and stereology indicated that the majority of the parenchymal cells (independently of their location in the outer subcapsular, middle, or inner portions) closely resembled those of the zonae fasciculata/reticularis of the adrenal gland of age-matched sham-operated rats. By contrast, regenerated nodules evidenced a relative impairment in aldosterone secretion, and this was coupled with the presence of only a few zona glomerulosa-like cells. Such cells were grouped in small islets located near the few connective trabeculae detaching from the capsule, and autoradiography showed that they were the only parenchymal elements of the nodule able to bind [125I]angiotensin-II. The possibility is suggested that the paucity of zona glomerulosa-like cells in regenerated nodules could be ascribed to the absence of zona medullaris, which is currently thought to exert a paracrine control on the growth and secretion of zona glomerulosa in the rat adrenal glands.

Cellular responses of the rat adrenal zona fasciculata to acute ACTH stimulation: a morphometric study.

Long-term ACTH-stimulation of steroidogenesis in the rat adrenal cortex results in time-dependent increases in the surface area per cell of smooth endoplasmic reticulum and mitochondrial cristae. As the morphological responses to short-term ACTH stimulation have not been described, we undertook morphometric analyses of the effects of acute (10 min) ACTH stimulation of rat adrenocortical cells in vivo as they may be expressed in the mitochondria and the smooth endoplasmic reticulum. Six young male Wistar rats were allocated to each of four groups: 1. normal controls; 2. ACTH-treated normal rats; 3. Dexamethasone-inhibited; 4. ACTH-treated Dexamethasone-inhibited. As judged by the radio-immunoassay of trunk blood, levels of ACTH, 11-deoxycorticosterone and corticosterone were appropriate to the treatment state. ACTH activation resulted in no changes in the smooth endoplasmic reticulum; but the mitochondrial inter-membrane space was significantly increased over that of the contrasted pair. The inter-membrane space in the dexamethasone-inhibited rats was significantly less than that of all other groups. No responses to ACTH-activation were shown by the intra-cristal or matrix volumes of the mitochondria. The increased inter-membrane space appears to be caused by a decrease in the surface area of the inner mitochondrial membrane. The significance of these intra-mitochondrial changes to the rate-limiting step of steroidogenesis is discussed.

Morphometric analyses of adrenal gland growth in fetal and neonatal sheep. I. The adrenal cortex.

This, the first linear morphometric analysis of the epigenesis of the fetal mammalian adrenal cortex, has shown that in the fetal sheep during the latter two thirds of gestation and in the newborn lamb, there are two periods of rapid growth separated by a period of much reduced growth. The fetal ages studies were 53 days (0.36 gestation), a period when the fetal adrenal cortex is actively steroidogenic; 100 days (0.68 gestation), a period of adrenocortical quiescence; 130 days (0.88 gestation), the period of increasing responsiveness to ACTH and cortisol production; 144 days (0.98 gestation), the period of maximal adrenocortical steroidogenesis; and 2 days postpartum, when cortisol production is normally maintained. The first adrenocortical growth period extends to mid-gestation, then growth slows to 0.85 gestation when the second growth period begins. The changes between the first growth period (0.36 gestation) and the period of quiescence (0.68 gestation) are characterised by the attainment of normal adrenocortical zonation and the separation of the medulla. The rate of adrenocortical cell division slows and the zona fasciculata cells become smaller in size. The volume density of the adrenocortical blood sinusoids decreases significantly. The onset of the second growth phase is associated with the previously reported increased levels of fetal plasma ACTH at 0.85 gestation and is expressed initially as a hypertrophic response. Cellular hypertrophy increases from 0.88 gestation to 0.98 gestation and then declines over the birth period. The rate of adrenocortical cell division increases from 0.88 gestation and maintains a maximal rate from 0.98 gestation to 2 days postpartum. These interactions of cellular hypertrophy and hyperplasia, which result in adrenocortical growth, may be explained as a response to fetal ACTH, which has the ability to stimulate the production of peptide growth and differentiation factors, e.g. IGF-II, and cortisol, which then control adrenocortical development in an autocrine and paracrine fashion.

Morphometric analyses of adrenal gland growth in fetal and neonatal sheep. II. The adrenal medulla, with some observations on its ultrastructure.

This account of fetal and neonatal sheep adrenomedullary development is the first such study in mammals using both morphometric and microscopic techniques. At 53 days gestation some cells in the migratory whorls and columns contained noradrenaline (NA) granules whereas by 100 days the medulla, now enclosed by the cortex, was composed of elongated juxtacortical cells and rounded central medullary cells, both populations of cells containing NA granules. In the 130 days glands, many of the juxtacortical cells contained adrenaline granules and had synaptic connection with axons of the preganglionic sympathetic nerve fibres. Later development was essentially growth-related. While the juxtacortical cells decreased from 33% of the medulla at 100 days to 22% at 144 days, the central medullary cells increased from 19% to 30% over the same period. Both cell populations exhibited hypertrophic growth over the study period; but the central cells multiplied at a faster rate. We conclude that the development of the cortical and medullary compartments of the adrenal gland are closely linked, for both showed rapid mid-gestational growth which slowed with the attainment of definitive tissue organisation. Then a second phase of growth, associated with increased and controlled catecholamine secretion in the medulla and cortisol secretion in the cortex, occurred in late gestation.

Morphometric analyses of the effects of thyrotrophin releasing hormone and cortisol on the lungs of fetal sheep.

Morphometric analyses of ovine fetal lung parenchyma were undertaken in order to elucidate the roles of pituitary, thyroid and adrenocortical hormones in promoting the structural changes underlying the increased distensibility and stability present in mature fetal lungs. Twenty-six Romney fetuses were treated with either cortisol for 84 h from 125 days (4), pulsatile TRH for 6.5 days from 122 days (4), cortisol and TRH (12), or 0.9% NaCl solution (6). The left lungs were used for physiological studies (distensibility, V40) and the right lungs were prepared for electron microscopy. Using 32 regions of lung parenchyma per fetus, volume density, surface density and arithmetic mean thickness of the alveolar walls were calculated using point and intersection counts. Of the three regimens, treatment with TRH + cortisol (exposure to raised concentrations of cortisol, T3 and prolactin) induced significantly greater lung distensibility, the largest potential alveolar air space (62% of the parenchyma), the greatest alveolar surface area (113.7 mm2/mm3 x 10(-3)) and the thinnest alveolar walls (6.7 microns). We conclude that cortisol, T3 and prolactin act synergistically to promote maturational changes in the alveolar wall. While cortisol plays the major role, T3 and prolactin enhance the ability of the immature lung to respond to the cortisol.

Assessment of sheep blastocyst effects on neutral lipids in the uterine caruncular epithelium.

Neutral lipids in the maternal uterine caruncular epithelium were studied by histochemical localization with Oil Red O. Results using a scoring system of 1 (negligible lipid) to 5 (maximal lipid) showed that intraepithelial lipid stores were minimal until Days 7-8 of the oestrous cycle and then increased to have a mean score of 4.4 on Day 14-15. In early pregnancy, although relatively high with a mean score of 3.2 at Day 15-16, such neutral lipids were significantly lower than those present at a comparable stage in the oestrous cycle. Thereafter, levels declined to a mean score of 1 on Days 21-23 of pregnancy. Such neutral lipid loss appears to be one of the first signs of the maternal response to the implanting embryo and precedes morphological evidence of transformation of either maternal or fetal tissues.

A comparison of standard lipid staining techniques used in electron microscopic studies of mammalian tissues.

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylene-diamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.