Full-term Embryos Research Articles

Spatial and temporal variation in the reproductive biology of gummy shark Mustelus antarcticus (Chondrichthyes : Triakidae) harvested off southern Australia

Mustelus antarcticus, endemic to southern Australia, exhibits matrotrophic aplacental viviparity. Differences in synchronous ovarian and parturition cycles, mostly annual west and biennial east of longitude 138°E, are explained by environmental differences. Ovulation and parturition peak during November–December and the gestation period is ~12 months. Largest ovarian follicle diameter ranges from 15 to 28 mm at ovulation, and mean wet mass gain is 10-fold from in utero egg (~10 g) to full-term embryo (~100 g) at ~330 mm total length. The sex ratio of embryos in utero is 1:1, and litter size (1 to 57 embryos) rises curvilinearly with maternal length. Length-at-maternity and length-at-maturity increased with rising fishing mortality and subsequently decreased with falling fishing mortality. These patterns are explained by the hypothesis on the ‘phenomenon of apparent change of size-at-maternity’ (and size-at-maturity) caused by gill-net length-selective fishing mortality, which masks any potential density-dependent responses. Male length-at-maturity estimates from seminal vesicle condition, testis development and spermatogenesis stages are similar, but are less than estimates from clasper calcification. Maximum body mass of females is double that of males and, at any length >700 mm, mean body mass of females exceeds that of males.

Thermally induced torpor in fullterm lizard embryos synchronizes hatching with ambient conditions

Eggs inside an underground nest have limited access to information about above-ground conditions that might affect the survival of emerging hatchlings. Our measurements of heart rates of embryos inside the intact eggs of montane lizards (Bassiana duperreyi, Scincidae) show that low temperatures induce torpor in fullterm embryos, but do not do so during earlier embryogenesis or later, post-hatching. Because above-ground conditions affect soil temperatures, this stage-dependent torpor effectively restricts hatching to periods of high ambient temperatures above ground. Torpor thus can function not only to synchronize activity with suitable environmental conditions during post-hatching life (as reported for many species), but also can occur in embryos, to synchronize hatching with above-ground conditions that facilitate successful emergence from the nest.

Microinsemination with first-wave round spermatids from immature male mice.

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.

One hundred years of zygotic embryo culture investigations

Isolation of zygotic embryos from seeds and their culture in a defined medium, initiated by Hanning in 1904, has proved to be a promising method to study the factors that control growth and differentiation of embryos. Using this technique, several investigations have focused on the carbohydrate and nitrogen nutrition during germination of cultured seed embryos and on the effects of plant hormones on their morphogenesis. Culture of immature embryos leads to their germination into weak seeldings, skipping the later stages of embryogenesis, by a process known as precocious germination. Progressively smaller embryos have been cultured by supplementation of the medium with coconut milk or hormonal additives or by osmotic adjustment of the medium by high concentrations of sucrose or mannitol. Although methods have not been developed for large-scale isolation and culture of zygotes, zygotes of maize isolated from embryo sacs and those obtained by in vitro fertilization have been grown in culture into full-term embryos. Embryo culture techniques are widely used to rescue embryos from seed of wide crosses which usually abort and to overcome dormancy of recalcitrant seeds.

Notochord anomalies in the adriamycin rat model: A morphologic and molecular basis for the VACTERL association

Background/Purpose: The Adriamycin rat model (ARM) is a reliable model of the VACTERL association. The notochord is structurally abnormal in the region of the foregut, midgut, and hindgut in the ARM. The authors hypothesised that notochord anomalies allow ectopic expression of molecular signals in the developing embryo and thus lead to VACTERL malformations. This study was designed to investigate this hypothesis. Methods: Adriamycin (1.75 mg/kg) was administered intraperitoneally to pregnant rats on days 7, 8, and 9 of gestation. Control animals were given saline. Embryos were recovered on gestational days 10.5 to 14 at½-day intervals and at full term. The first group of embryos were embedded in resin, and sagittal sections stained with Toluidine blue were studied for morphologic abnormalities. The second group of embryos were examined using in situ hybridization for the expression of Sonic Hedgehog (Shh), a patterning gene implicated in the etiology of the VACTERL association. Results: Twenty-seven of the 28 (96.4%) full-term embryos showed VACTERL anomalies. Forty-five of the 50 (90%) experimental embryos (gestational days 10.5 to 14) showed notochord abnormalities. Abnormal ventral branches from the notochord toward the gut were a commonly observed abnormality. These were seen to impinge on the developing foregut, midgut, dorsal aorta, and kidney. In situ hybridization for Shh showed that these branches from the notochord expressed Shh in 66.6% of experimental embryos. This abnormal Shh expression was not seen in the control embryos. Conclusions: Adriamycin diffusely induces altered notochord morphology in the rat embryo. The abnormal notochord morphology may allow ectopic expression of Sonic Hedgehog, and, thus, contribute to the malformations found in the VACTERL association. J Pediatr Surg 38:469-473. Copyright 2003, Elsevier Science (USA). All rights reserved.

Growth and Reproduction in Spiny Dogfish Squalus Acanthias L. (Elasmobranchii: Squalidae), from the West Coast of Ireland

The age, growth characteristics, maturity, and reproductive cycle of commercially caught spiny dogfish Squalus acanthias were investigated over a 12 month period. The specimens ranged in age from 4 to 30 years, with asymptotic lengths of 79 and 112 cm estimated for males and females, respectively. The lower age range reported here when compared with previous North Atlantic studies is attributed to fishing pressure. Fifty per cent maturity was estimated at 57.5 cm in males and 78.2 cm in females, and fecundity was positively correlated with female size. Although recently formed candles and full-term embryos were observed throughout the year, parturition occurred mainly from August to January. The minimum fecundity recorded was four (determined from developing ova/candled embryos), while the maximum was 16 (determined from embryo counts), and fecundity was found to increase by up to four between pregnancies. The overall sex ratio of embryos was 1:1, although this balance was rarely observed in any one female. The results presented here indicate that neither growth rate, size at maturity, nor fecundity have significantly changed due to fishing pressure.

Changes in Composition During Embryo Development of the Gulper Shark, Centrophorus Granulosus (Elasmobranchii, Centrophoridae): An Assessment of Maternal-embryonic Nutritional Relationships

Centrophorus granulosus is a deep sea shark that reproduces through aplacental viviparity. Its fecundity is one of the lowest described with only one embryo in a pregnancy lasting about two years. Its mature ovarian egg reaches one of the largest cellular sizes (> 350 g) described for any animal species. A previous report suggested a loss of organic matter during development of about 50% (Ranzi 1932), the highest rate reported for any elasmobranch. We measured the amounts of water, organic and inorganic matter in a complete series of embryos, by drying and later incinerating separately the external yolksac, eviscerated body, internal yolksac, liver and digestive tract. Wet weight of uterine ova ranged from 143.2–370.4 g, was positively and significantly correlated with maternal size, and the size of full-term embryos increased with maternal size. A graphical method was developed to allow weight comparison between uterine ova and full-term embryos while taking into account the initial variability in uterine ova size. Total wet weight of the embryo system increased during development by +31/+34%. Changes in percentage composition (from initial values) were: water +99/+101%; organic matter −18/−25%; inorganic matter +114/+170%. The rate of decrease of organic matter was much lower than previously suggested, and was similar to values described for oviparous species. These results suggest that C. granulosus is a strictly lecithotrophic species, with no maternal contribution of organic matter during development, although the female does provide both water and inorganic material. Other factors that might influence the accuracy of this assessment are discussed.

Reproductive biology of the mosquitofish in a permanent natural lagoon in south-west Spain: two tactics for one species

Introduced Gambusia holbrooki in a natural lagoon of southern Spain consisted of two age groups: 1992 cohort, 7-11 months old and 1993 cohort, <4 months old. In the 1992 cohort, females grew even during the gestation period at about 0.30 mm day-1. In the 1993 cohort, females displayed a high growth rate (0.55 mm day-1) and reached reproductive size in a few weeks, but stopped growing when they matured. All the 1992 cohort reproduced from mid-May to mid-June, but only 50% of the 1993 cohort reproduced, from mid-August to mid-September. Reproducing females were significantly larger in the 1992 cohort (39.8 mm) than in the 1993 one (34.8 mm). The largest 1992 females cohort had reproduced previously; the 1993 cohort had not. The mean dry weight of intra-ovarian embryos decreased to a minimum immediately before birth. These metabolic costs represented 29.8 and 31.4% of the initial weights of the 1992 and 1993 embryos, respectively. Mean dry weight of full-term embryo was significantly higher in the 1992 cohort (0.80+/-0.129 mg; 95% CL) than in the 1993 one (0.70+/-0.086 mg; 95% CL). With more females in 1992, cohort fecundity was considerably higher (number of embryos=7151; 63%) than in the 1993 (4193; 37%) cohort. The population completed two generations each year (spring and summer). The spring stock grew slower than the summer one but continued to grow during the gestation period, reaching larger final lengths, with more synchronous reproduction and clearer evidence of a second reproductive event. Each reproductive stock displayed its own life-history characteristics, with significant differences between mean length of reproducing females, growth rate, mean brood size, offspring size, standard fecundity and minimum length at reproduction.

Reproductive cycle of the common guitarfish, Rhinobatos rhinobatos (Linnaeus, 1758), in Alexandria waters, Mediterranean Sea

The common guitarfish, Rhinobatos rhinobatos, is an ovoviviparous fish having a gestation period of about nine months and a mean reproductive rate of 8 to 14 progeny per pregnancy. Data on gonadosomatic index, maximum ovum diameter, size frequency of ova, and ovarian and uterine contents for 438 specimens (236 females and 202 males), analysed on a monthly basis, indicated a distinct seasonal reproductive cycle for R. rhinobatos. Females reached a maximum size of 181 cm and 20 kg, whereas the maximum size of males was 172 cm and 16 kg. Lengths at which 50% of specimens were mature were 87 and 70 cm for females and males, respectively. Ovarian egg size and male gonadosomatic index both peaked in July and August, indicating that mating occurred in summer. Uterine eggs were present from late February through July, but no embryonic development was evident. Females with full-term embryos were observed in late August, and parturition was followed immediately by ovulation. Ovarian fecundity ranged from 8 to 27 (mean 18) eggs per fish, whereas uterine fecundity ranged from 8 to 14 (mean 12) eggs or embryos per fish.

Expression of FGFR-4 mRNA in developing mouse tissues.

We have previously reported the isolation of human FGFR-4 cDNA encoding a receptor for aFGF and shown that the expression patterns of FGFR-1 through FGFR-4 mRNAs are overlapping, but distinct in human fetal tissues (Partanen et al., 1991). In order to define cells and tissues expressing FGFR-4 more accurately, we have cloned mouse FGFR-4 cDNA and used it in Northern and in situ hybridization of mouse embryonic tissues. Our results show that the 3.5 kb FGFR-4 mRNA is expressed in day 10 to full term embryos and newborn mice, with maximal expression levels during days 14-16 p.c. Adult mice express the FGFR-4 mRNA predominantly in the liver, kidney and lung. In in situ hybridization of 10-13 day p.c. mouse embryos, FGFR-4 mRNA is present primarily in tissues of mesodermal origin and in the developing lung and gut. Particularly the mesenchymal tissue in association with the first pharyngeal arch and along the prevertebrae (in developing myotomes) which will differentiate into muscle tissue express high amounts of FGFR-4 mRNA. In addition, the developing metanephros contains FGFR-4 transcripts. These results suggest that FGFR-4 may be particularly important for the differentiation of skeletal muscle and endodermally derived organs.

Porosity of eggshells from wild and captive, pen-reared alligators (Alligator mississippiensis).

Artificially incubated fertile eggs from wild alligators have a significantly better hatch rate than those of captive, pen-reared alligators, possibly due to differences in the morphology of the eggshells. We compared the morphology of eggshells of wild alligators to those of captive alligators living in semi-natural environmental pens. Lengths and widths of eggs were measured and volume was determined, assuming an ellipsoid shape. Eggs were also evaluated for the quality of the eggshell (the presence or absence of rough deposits). Pieces of shell were cut from unincubated eggs and from eggs incubated for 55 days (just before hatching) and examined by scanning electron microscopy. Open pores on the outer surface of the shells were counted and thickness of the pieces was measured from micrographs. Results indicated that the number of pores on eggshells was lowest in eggs of captive alligators with early embryonic death. The number of pores was intermediate in eggs with early embryonic death from wild alligators, and the number of pores was highest in eggs with full-term embryos from wild or captive alligators. It is suggested that decreased porosity of eggshells may be associated with early embryonic death, is more prevalent in captive animals, and may, therefore, be related to poor hatch rate among penreared alligators.

Aspect of the reproductive biology of the Gummy Shark, Mustelus antarcticus Gunther, from waters off the South coast of Western Australia

The gummy shark (Mustelus antarcticus) is a major target species in two substantial shark fisheries that operate in temperate Australian waters. Data on the reproductive biology of M. antarcticus in the waters off south-western Australia were obtained from samples collected by commercial vessels operating from Albany to Esperance, Western Australia. The samples contained a ratio of four females to one male. M. antarcticus is a viviparous, aplacental species. Males mature at a smaller size than females. Since the overwhelming majority of sharks sampled were mature, it was not possible to estimate precisely the mean size at which sexual maturity was attained. Examination of seasonal changes in the development of ova and testes, in uterine contents, and in embryo growth established that the period of parturition, mating and ovulation occurred over the 3 months between early November and early February. The gestation period was 11-12 months. Full-term embryos ranged in size from 30 to 36 cm total length and occurred in a sex ratio of one male to one female. The ovarian and gestational cycles proceed concurrently, with reproduction occurring annually. Only one of the 224 females analysed for uterine content was considered to be in a true post-partum condition. The number of embryos (N) per mother increased with the length of the mother (L) according to the regression N = exp(-4.13398 + 0.049171L). The reproductive biology of females collected off Albany and Esperance differed in some respects from that of females collected off south-eastern Australia.

Size at maturity, reproductive cycle, fecundity, and depth segregation of the deep sea squaloid sharks Centroscymnus owstoni and C. coelolepis in Suruga Bay, Japan.

Specimens of the deep sea sharks, Centroscymnus owstoni and C. coelolepis, were collected from Suruga Bay, Japan to examine their reproductive habits. Size at maturity for both species was about 700mm TL in males and 1000mm TL in females. Neither species has a well defined breeding season. The reproductive cycle appears to extend over one calendar year. The ratio of males to females was approximately 1:1 for C. owstoni and 1: 2 for C. coelolepis. Litter size ranged from 16 to 28 for C. owstoni and from 15 to 29 for C. coelolepis. Total number of mature ova, fertilized ova and embryos tended to increase with size of the female. Females of both species displayed a well defined pattern of depth segregation by stage of maturity. The catch depths of pregnant females for both species were shallower than those of non-pregnant ones. However, pregnant females with full-term embryos were not caught in shallow waters of Suruga Bay.

Metabolic Response of Mallee Fowl Leipoa ocellata Embryos to Cooling and Heating

Full-term mallee fowl embryos (3 days before hatch) respond to prolonged chilling by increasing oxygen consumption but are unable to prevent egg temperature from dropping. The increase in oxygen consumption is interpreted as the initiation of autonomic thermoregulatory behavior. The transition from purely ectothermic to endothermic behavior takes 3-5 days, a time similar to other precocial birds. However, in mallee fowl, this process occurs before hatching, while, in the other species, it occurs after hatching.

Reproductive Cycle and Embryonic Development of Nerodia taxispilota (Serpentes: Colubridae) at the Northeastern Edge of Its Range

The seasonal reproductive cycle of Nerodia taxispilota in southeastern Virginia resembles that of other temperate zone colubrids. Testes are small during AprilJune, largest in Aug. and then decrease in size during Sept.-Nov. Spermatozoa produced in late summer are stored in the vas deferens through winter and used the following spring. The female cycle is annual. Vitellogenesis occurs during April-early June, ovulation in late June and parturition early to mid-Sept. Fecundity increases with increasing female body size. Mean total clutch size is 33.9 and mean clutch size of full-term embryos is 28.0. Sequential stages of embryonic development are described. Males average significantly smaller than females. Percentage of tail breaks are about equal in both sexes, but adults have higher proportions of tail breakage than juveniles. Sex ratios do not differ significantly from 1:1; however, males predominate in autumn.

A fetal-maternal shift of blood oxygen affinity in an Australian viviparous lizard,Sphenomorphus quoyii (Reptilia, scincidae)

Compared to adults, the oxygen affinity of blood from fetalSphenomorphus quoyii is very much higher:P50 is approximately 70 Torr in adults and 30 Torr in nearly full term embryos\((P_{CO_2 } = 17 Torr, T = 34 ^\circ C)\). Following birth, oxygen affinity decreases gradually and adult values are approached after about 15 weeks, with the onset of winter retreat. Electrophoresis revealed a multiple hemoglobin system in both adults and embryos, but there were no apparent differences between them.

Some aspects of reproduction in the pointed-snout dogfish Squalus japonicus taken off Nagasaki and Choshi.

Specimens of the pointed-snout dogfish Squalus japonicus taken off Nagasaki and Choshi, were investigated to study its reproduction. Specimens from Choshi were larger in the size at maturity as well as in the maximum size than those from Nagasaki. The size at first maturity for females was 560-157mm in Nagasaki and 740-759mm in Choshi. Maximum lengths were greater by 14cm for males and 19cm for females in Choshi than in Nagasaki. Testis weights and clasper lengths showed a rapid increase with onset of maturity. The gestation period was estimated to be roughly one year sine both full-term embryos and fertilized eggs occurred in the same period. Litter size ranged from 2 to 8 for each region but average litter size was 3.9in Nagasaki and 5.3in Choshi. Litter size increased with size of the parent. A difference was also noted in the average number of ovarian eggs per adult female between the two regions (4.4in Nagasaki and 6.6in Choshi). Ova usually outnumbered embryos and showed a similar growth pattern to embryos. The ratio of males to females in embryos was approximately 1:1 as a whole in both regions.

Volumetric Reduction in Nest Contents of Loggerhead Sea Turtles (Caretta caretta) (Reptilia, Testudines, Cheloniidae) on the Georgia Coast

Volumetric reduction of nest contents prior to hatchling emergence was deter- mined for natural hatchery nests of loggerhead sea turtles by implantation of a disc apparatus. The hypothesis that significant reduction occurs at hatching has been falsified. Reduction of nest contents occurred prior to hatching due to a loss in egg turgor. Initial reduction occurred in most nests within 7 to 8 days of emergence. The maximum depth of air space created above the eggs was 4.0 + .26 cm (x + SE, n = 52). Amount of egg-mass reduction was significantly correlated to the number of hatchlings and full-term embryos per nest and not correlated to egg number. The rate of volumetric reduction varied seasonally and can be explained by temperature. * * * Incubation length of eggs in natural turtle nests is usually defined as the elapsed time between oviposition and hatchling emergence. This time period includes embryonic development, hatching, possible delayed emergence, and digging to reach the nest surface. Determination of the time required for these events has been made by frequent nest openings. This practice, however, may decrease developmental time if the eggs are moved (Bustard, 1972). Hatchlings of Pseudemys scripta troostii (=elegans) in Louisiana, require up to 5 days for yolk retraction and a concomitant increase in size and firmness of the shell while remaining in the nest (Cagle, 1950). Similar results were found for artifically incubated eggs of Terrapene ornata orata (Legler, 1960). In natural nests of Malaclemys terrapin terrapin on Little Beach Island, New Jersey, hatching requires up to 4 days while emergence occurs 1 to 9 days later (Burger, 1976). The sequence of post-hatching events was observed for one green turtle, Chelonia mydas, clutch from Tortuguero, Costa Rica, trans- planted to a sand-filled wooden box with a glass side (Carr and Ogren, 1960). The eggs began to lose turgor 4 days prior to hatching. Air space above nest contents was created during hatching. Vertical movement of turtles began 2 to 3 days after hatching and emergence occurred 7 days after most of the eggs hatched. The production of an air space between the egg-mass and ceiling of the egg cavity during hatching is well documented for sea turtle nests (Carr and Hirth, 1961; Hendrickson, 1958; Schulz, 1975). The length of time for hatching has not been determined for natural nests. Early hatching

Production of interspecific hybrids inLotusaided by endosperm transplants

Abstract Endosperm transplants were used to aid the in vitro culture of young Lotus embryos. Three tetraploid hybrids of 4x L. pedunculatus × 4x L. tenuis were obtained. Three tetraploid hybrids of 4x L. tenuis × L. corniculatus were grown from full-term embryos on a simple medium. Hybridity for all tetraploids was confirmed by morphological studies and, where appropriate, by the presence of hydrocyanic acid, flavanol polymers (“tannins”), and patterns of phosphatase and peroxidase isozymes.

Influence of exogenous tRNA on growth of transplantable 32P-induced osteosarcomata

The weights of osteosarcomata that arose 22 days after the s.c. injection of cell suspensions (of whold tumours) that had been exposed in vitro to tRNA were significantly different from the weights of those arising from untreated cells. The tRNA was isolated by phenol extraction and DEAE-cellulose chromatography from eviscerated full-term rat embryos, from rat mesenchymal (granulation) tissue and from rat anaplastic osteosarcomata. Tumours developing from osteosarcoma cells treated with either embryonic or normal mesenchymal tRNA were reduced in weight by 76% and 60% respectively. These effects could not be explained as a toxic consequence, because tumour weight was increased by 128% after exposure of cells to osteosarcoma tRNA. In this osteosarcoma model it appears that tumour weights can be influenced in different ways by tRNA from different sources.