The new dysmorphology: application of insights from basic developmental biology to the understanding of human birth defects.

Information obtained from studies of developmental and cellular processes in lower organisms is beginning to make significant contributions to the understanding of the pathogenesis of human birth defects, and it is now becoming possible to treat birth defects as inborn errors of development. Mutations in genes for transcription factors, receptors, cell adhesion molecules, intercellular junctions, molecules involved in signal transduction, growth factors, structural proteins, enzymes, and transporters have been identified in genetically caused human malformations and dysplasias. The identification of these mutations and the analysis of their developmental effects have been greatly facilitated by the existence of natural or engineered models in the mouse and even of related mutations in Drosophila, and in some instances a remarkable conservation of function in development has been observed, even between widely separated species.

Organismal duplication, inclusive fitness theory, and altruism: understanding the evolution of endosperm and the angiosperm reproductive syndrome.

For almost a century, events relating to the evolutionary origin of endosperm, a unique embryo-nourishing tissue that is essential to the reproductive process in flowering plants, have remained a mystery. Integration of recent advances in phylogenetic reconstruction, comparative reproductive biology, and genetic theory can be used to elucidate the evolutionary events and forces associated with the establishment of endosperm. Endosperm is shown to be derived from one of two embryos formed during a rudimentary process of "double fertilization" that evolved in the ancestors of angiosperms. Acquisition of embryo-nourishing behavior (with accompanying loss of individual fitness) by this supernumerary fertilization product was dependent upon compensatory gains in the inclusive fitness of related embryos. The result of the loss of individual fitness by one of the two original products of double fertilization was the establishment of endosperm, a highly modified embryo/organism that reproduces cryptically through behavior that enhances the fitness of its associated embryo within a seed. Finally, although triploid endosperm remains a synapomorphy of angiosperms, inclusive fitness analysis demonstrates that the embryo-nourishing properties of endosperm initially evolved in a diploid condition.

Radioisotopes in agriculture : animal husbandry, fertilizer uptake, plant physiology, photosynthesis, and entomology : a selected list of references /

This bibliography contains 151 selected references on the uses of radioactive and stable isotopes in animal husbandry and in studies of fertilizer uptake by plants, plant physiology, photosynthesis, and entomology. These references were selected from scientific journals published during the period 1957-1958. An author index is included.

Radioisotopes in agriculture : animal husbandry, fertilizer uptake, plant physiology, photosynthesis, and entomology : a selected bibliography /

This annotated bibliography contains 148 selected references on the uses of radioactive and stable isotopes in animal husbandry and in studies of fertilizer uptake by plants, plant physiology, photosynthesis, and entomology. These references were selected from scientific journals published during the period 1957-1958. Subject and author indexes and a list of the periodicals from which the references were selected are included.

Studies on the cryopreservation of common wombat (Vombatus ursinus) spermatozoa

In an attempt to develop a gamete-recovery protocol for the northern hairy nosed wombat (Lasiorhinus krefftii), spermatozoa were removed from the cauda epididymides of four common wombats (Vombatus ursinus) and cryopreserved following a variety of prefreeze storage conditions. Spermatozoa stored for 72 h at 4 degrees C within the testicle before cryopreservation tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability (% motile P< 0.01; rate of movement P< 0.01; % live P< 0.01) than sperm recovered on the day of post-mortem, stored in a test tube for 72 h at 4 degrees C and then frozen. The effect of post-thaw dilution with Tris citrate fructose (TCF) diluent on the survival of epididymal common wombat spermatozoa was also investigated. Motility (P< 0.05), rate of sperm movement (P< 0.01) and the percentage of live spermatozoa (P< 0.05) were all significantly greater when spermatozoa were thawed and diluted immediately in TCF than when thawed without dilution. The present study also reports, for the first time, a successful pellet method of freezing wombat spermatozoa on dry ice; volumes of 0.25 and 0.5mL resulted in higher post- thaw survival compared with 0.1-mL pellets.

Establishment of metanephros transplantation in mice highlights contributions by both nephrectomy and pregnancy to developmental progression

Background: It has been demonstrated that embryonic kidneys (metanephroi) xenotransplanted into the omentum of adult recipients continue to develop and display immune protection due to their more nave immune presentation. To date, this has been achieved using rat, pig and human metanephroi, with unilateral nephrectomy (UNX) of recipient rats a requisite of renal development. The aim of this study was to adapt this approach for use in mice and examine the parameters affecting successful onward development in this species. Methods: Metanephroi at embryonic age (E) 13.5 were transplanted either onto the body wall, abdominal fat pads or omentum of recipient isogenic C57/Bl6 mice using either sutures or polyglycolic acid mesh. Having established greatest success with polyglycolic acid mesh on the body wall, E12.5 and 15.5 days metanephroi from C57/Bl6 mice were then transplanted onto the body wall of control (non-pregnant non-UNX), UNX or 12.5 days post-coitum pregnant isogenic recipients. After 7 days, implanted tissue was harvested and examined using histology and immunohistochemistry for markers of renal maturation. The mean number of S-shaped bodies and glomeruli per section were recorded and statistically analysed for significant differences between all recipient groups and untransplanted metanephroi. The degree of development was scored qualitatively. Results: Transplanted E12.5 metanephroi developed S-shaped bodies and glomeruli in all recipient groups, although there were statistically higher numbers of S-shaped bodies in UNX (n = 2) and pregnant recipients (n = 9) than in control recipients (n = 4). Continued development, as indicated by mature vascularized glomeruli, was only observed in those E15.5 metanephroi transplanted into pregnant recipients (n = 11) with a 15.5-fold increase in S-shaped bodies and 4-fold increase in glomeruli compared with control transplants (n = 12). Conclusions: We have successfully established metanephros transplantation in mice and demonstrated enhancement of onward development of E12.5 metanephroi in response to both pregnancy and UNX. Using E15.5 metanephroi, continued development only occurred in pregnant recipients, implying pregnancy provides an environment conducive to continued organogenesis. This murine assay, when coupled with transgenically-tagged strains of mice, will allow the investigation of the relative contribution of donor and recipient cells to this process. Copyright (C) 2005 S. Karger AG, Basel.

To self, or not to self ... A review of outcrossing and pollen-mediated gene flow in neotropical trees

Despite the typically low population densities and animal-mediated pollination of tropical forest trees, outcrossing and long-distance pollen dispersal are the norm. We reviewed the genetic literature on mating systems and pollen dispersal for neotropical trees to identify the ecological and phylogenetic correlates. The 36 studies surveyed found >90% outcrossed mating for 45 hermaphroditic or monoecious species. Self-fertilization rates varied inversely with population density and showed phylogenetic and geographic trends. The few direct measures of pollen flow (N = 11 studies) suggest that pollen dispersal is widespread among low-density tropical trees, ranging from a mean of 200 m to over 19 km for species pollinated by small insects or bats. Future research needs to examine (1) the effect of inbreeding depression on observed outcrossing rates, (2) pollen dispersal in a wide range of pollination syndromes and ecological classes, (3) and the range of variation of mating system expression at different hierarchical levels, including individual, seasonal, population, ecological, landscape and range wide.

Collection, seminal characteristics and chilled storage of spermatozoa from three species of free-range flying fox (Pteropus spp.)

This study reports observations on the collection and characteristics of semen from free-range populations of flying fox in Brisbane, Australia. Semen was successfully recovered by electroejaculation from 107 of 115 wild flying foxes (Pteropus alecto, Pteropus poliocephalus and Pteropus scapulatus). A proportion of ejaculates collected from all three species contained seminal vesicle secretions, the incidence of which appeared related to breeding season. Ejaculate volume was small (5-160 mu L), requiring a specialised collection vessel and immediate extension to avoid desiccation. Sperm morphological abnormalities and characteristics are described for the first time. In two species (P. scapulatus and P. alecto), sperm quality varied with breeding season. Dilution in Tris-citratefructose buffer and subsequent incubation (37 degrees C) of Pteropus semen for 2-3 h appeared to have a negative impact on sperm motility and the percentage of sperm with intact plasma membranes and acrosomes and represents a concern for the potential development and use of assisted breeding technology in these species. Preliminary attempts to develop a short-term chilled preservation protocol for flying fox semen revealed that spenn viability (percentage motility and percentage live sperm with intact acrosomes) was significantly reduced after 102 h chilled storage at 5 degrees C; nevertheless, approximately 40% of the spermatozoa were still motile and contained intact acrosomes. Glycerol was neither protective nor detrimental to sperm survival during chilled storage. Microbial flora of the prepuce, urethra and semen of all species were isolated and their antibiotic susceptibility tested. Tetracycline, penicillin, ciprofloxacin, and ceftazidime were the most effective antibiotics in preventing growth of all identified bacteria; however, their effects on sperm survival were not investigated. (c) 2005 Elsevier Inc. All rights reserved.

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly (P < 0.05) over a 24 h period although there was a tendency (P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 mu g IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05-1.10 ng/ml), injection of hCG resulted in an increase (P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69-7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans. (c) 2005 Elsevier B.V. All rights reserved.

Changes to porcine blastocyst vitrification methods and improved litter size after transfer

The objective was to improve the protocol that was used to obtain the first reported piglets from transferred vitrified and warmed zona-intact blastocysts. Blastocysts were collected from superovulated sows and gilts, centrifuged to polarize lipid, vitrified, warmed and cultured for 24 h or transferred immediately. Removing the zona pellucida after warming increased the number of cells in the surviving blastocysts (zona-free 60.8 +/- 4.3, zona-intact 39.1 +/- 2.8; P < 0.05). Thinning the zona pellucida produced similar results to zona removal. Changing the basal medium of the vitrification and warming solutions from modified PBS to phosphate buffered NCSU-23 increased the number of cells (44.7 +/- 2.2 versus 56.0 +/- 3.9, respectively; P < 0.05). Reducing the plunge temperature of the liquid nitrogen from - 196 degrees C to less than -204 degrees C improved the embryo survival rate (61.9% versus 82.9%, respectively; P < 0.05). These modifications were incorporated into the vitrification protocol that was used to vitrify and warm 105 blastocysts (that were subsequently transferred into four recipients). Three recipients became pregnant, farrowing three litters (average litter size, 5.3; 18.8% embryo survival in farrowing sows). Changing the warming protocol to using sucrose rather than ethylene glycol resulted in a trend towards improved embryo survival (73.5% versus 91.2%) but this was not statistically significant. Incorporating this modification, 203 blastocysts were vitrified, warmed and transferred into seven recipients. Five became pregnant and 36 fetuses were recovered (average litter size 7.2; 24.8% embryo survival in pregnant sows) at Day 40 of pregnancy. In conclusion, changes made to the vitrification protocol improved pregnancy rate and in vivo embryo survival compared to an earlier study using the original protocol. (c) 2005 Elsevier Inc. All rights reserved.

Relationship of body condition score and blood urea and ammonia to pregnancy in Italian Mediterranean buffaloes

The relationship of body condition score ( BCS) and blood urea and ammonia to pregnancy outcome was examined in Italian Mediterranean Buffalo cows mated by AI. The study was conducted on 150 buffaloes at 145 +/- 83 days in milk that were fed a diet comprising 14.8% crude protein, 0.9 milk forage units . kg(-1) dry matter and a non- structural carbohydrate/ crude protein ratio of 2.14. The stage of the oestrous cycle was synchronised by the Ovsynch- TAI programme and blood urea and ammonia levels were assessed on the day of AI. Energy corrected milk ( ECM) production and BCS were recorded bi- weekly. The pregnancy risk was 46.7% and was slightly lower in buffaloes with BCS < 6.0 and BCS > 7.5. There were no significant differences in ECM, urea and ammonia between pregnant and non- pregnant buffaloes. However, pregnancy outcome was higher ( P = 0.02) in buffaloes with blood urea < 6.83 mmol . L-1. The likelihood of pregnancy for buffaloes with low urea blood level was 2.6 greater than for high urea level and exposure to a high urea level lowered the probability of pregnancy by about 0.25. The findings indicate that buffaloes are similar to cattle and increased blood levels of urea are associated with reduced fertility when animals are mated by AI.

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta 2 and gamma 1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha 1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha 1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha 1, laminins beta 2 and gamma 1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.