Distribution of immunoreactive thyrotropin-releasing hormone in the forebrain and hypophysis of the bullfrog,Rana catesbeiana

The distribution of immunoreactive thyrotropin-releasing hormone (TRH) in the forebrain and hypophysis of Rana catesbeiana was studied by means of specific radioimmunoassay and immunohistochemistry based on peroxidase-antiperoxidase (PAP) techniques. A relatively high concentration of immunoassayable TRH is present in the hypothalamus. Immunoreactive TRH cell bodies are found in the anterior part of the preoptic nucleus, the dorsal infundibular nucleus, the nucleus of diagonal band of Broca, and the medial part of the amygdala. Immunoreactive nerve terminals are observed in the neurohypophysis and the external layer of the median eminence, where the terminals are in close contact with the capillary loops of the hypophyseal portal vessels. The possible role of TRH in the frog brain is discussed.     

Development of melanin-concentrating hormone-immunoreactive elements in the brain of gilthead seabream (Sparus auratus)

The development of the hypothalamic melanin-concentrating hormone (MCH) system of the teleost Sparus auratus has been studied by immunocytochemistry using an anti-salmon MCH serum. Immunoreactive perikarya and fibers are found in embryos, larvae, and juvenile specimens. In juveniles, most labeled neurons are present in the nucleus lateralis tuberis; some are dispersed in the nucleus recessus lateralis and nucleus periventricularis posterior. From the nucleus lateralis tuberis, MCH neurons project a conspicuous tract of fibers to the ventral hypothalamus; this penetrates the pituitary stalk and reaches the neurohypophysis. Most fibers end close to the cells of the pars intermedia, and some reach the adenohypophysial rostral pars distalis. Immunoreactive fibers can also be seen in extrahypophysial localizations, such as the preoptic region and the nucleus sacci vasculosi. In embryos, MCH-immunoreactive neurons first appear at 36 h post-fertilization in the ventrolateral margin of the developing hypothalamus. In larvae, at 4 days post-hatching, perikarya can be observed in the ventrolateral border of the hypothalamus and in the mid-hypothalamus, near the ventricle. At 26 days post-hatching, MCH perikarya are restricted to the nucleus lateralis tuberis. The neurohypophysis possesses MCH-immunoreactive fibers from the second day post-hatching. The results indicate that MCH plays a role in larval development with respect to skin melanophores and cells that secrete melanocyte-stimulating hormone. Received: 4 April 1995 / Accepted: 17 July 1995     

Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.     

Development of the caudal neurosecretory system of the chum salmon,Oncorhynchus keta,as revealed by immunohistochemistry for urotensins I and II

In order to make an immunohistochemical analysis of the development of the caudal neurosecretory system of the chum salmon, Oncorhynchus keta, we employed the peroxidase-anti-peroxidase technique using antisera specific for urotensins (U) I and II on artificially reared embryos, larvae, and juveniles of this species. Immunoreactivities for UI and UII were first demonstrated in the embryo immediately before hatching, showing labeled perikarya and fibers in the most caudal region of the spinal cord where the presumptive caudal neurosecretory system is located. However, distinct differentiation of the histological neurohemal organ had not yet begun in the embryo. Immunoreactive perikarya and fibers gradually increased in number, and an elaborate urophysis comparable to that of adults was demonstrated in the larvae about 5 months after hatching. At this stage, weak immunoreactivity against UI was detected in the neurohypophysis.     

New data on the immunocytochemical localization of thyrotropin-releasing hormone in the rat central nervous system

An antiserum raised against the synthetic tripeptide pyroglutamyl-histidyl-proline (free acid) was used to localize thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) by immunocytochemistry. The distribution of TRH-immunoreactive structures was similar to that reported earlier; i.e., most of the TRH-containing perikarya were located in the parvicellular part of the hypothalamic paraventricular nucleus, the suprachiasmatic portion of the preoptic nucleus, the dorsomedial nucleus, the lateral basal hypothalamus, and the raphe nuclei. Several new locations for TRH-immunoreactive neurons were also observed, including the glomerular layer of the olfactory bulb, the anterior olfactory nuclei, the diagonal band of Broca, the septal nuclei, the sexually dimorphic nucleus of the preoptic area, the reticular thalamic nucleus, the lateral reticular nucleus of the medulla oblongata, and the central gray matter of the mesencephalon. Immunoreactive fibers were seen in the median eminence, the organum vasculosum of the lamina terminalis, the lateral septal nucleus, the medial habenula, the dorsal and ventral parabrachial nuclei, the nucleus of the solitary tract, around the motor nuclei of the cranial nerves, the dorsal vagal complex, and in the reticular formation of the brainstem. In the spinal cord, no immunoreactive perikarya were observed. Immunoreactive processes were present in the lateral funiculus of the white matter and in laminae V-X in the gray matter. Dense terminal-like structures were seen around spinal motor neurons. The distribution of TRH-immunoreactive structures in the CNS suggests that TRH functions both as a neuroendocrine regulator in the hypothalamus and as a neurotransmitter or neuromodulator throughout the CNS.     

Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos

Hatching in vitro was studied following zona drilling of 507 two-cell mouse embryos using three methods: 1) acidic Tyrode's (AT), 2) partial zona dissection (PZD) using a sharp micronecdle, and 3) zona chiseling (CH), using a large beveled needle. PZD and CH were performed while the embryos were kept in a sucrose/PBS solution. Hatching was compared to 191 umnicromanipulated controls. The incidences of cavitation and completion of hatching did not differ between groups, however more micromanipulated embryos (20–25%) hatched partially than controls (9%). The zona pellucida thinned in 59/59 (100%) control blastocysts during expansion, but in only 3/205 (2%) micromanipulated blastocysts. The hatching gap was wide in all control embryos, but smaller in 96/129 (75%) micromanipulated embryos. Partially hatched blastocysts with a ‘figure-8’ shape were found in 59/129 (46%) micromanipulated embryos and in none of the 39 hatching controls. Hatching usually occurred a day earlier in micromanipulated embryos as 214/218 (98%) had started extruding on day 5 as compared to 20/59 (27%) control blastocysts. Fifty percent of 1-day-old human oocytes were fertilized following PZD and reinsemination and 15/31 (48%) were monospermic. Thirteen monospermic embryos cleaved, six compacted and four cavitatcd—of these, three extruded through the PZD incision upon expansion. The zonae did not thin and one blastocyst twinned spontaneously as it was caught between the thick ridges of the PZD hole. Results indicate that the hatching process is abnormal following zona drilling; more embryos start hatching, extrusion occurs earlier, and many become trapped which may lead to artificial twinning or the formation of trophoblastic vesicles.     

Influence of inbreeding on reproductive performance, ejaculate quality and testicular volume in the dog

The influence of fast freezing and thawing on bovine embryos at different stages of development was investigated. A total of 20 day-7 embryos and 37 day-8 embryos were thawed and classified morphologically before being transferred nonsurgically to synchronized recipients. Ninety percent (18 20 ) of the day 7 embryos (late morulae and blastocysts) were classified as transferable and a pregnancy rate of 52.9% (9 17 ) was obtained with these embryos. Seventy eight percent (29 37 ) of the day 8 embryos (expanded blastocysts) were classified as transferable, but only 24.1% (7 29 ) of these embryos resulted in pregnancy. The best pregnancy rate was obtained with the blastocysts of day 7 (5 8 or 62.5%), which compares favorably with that of freshly collected embryos. It is suggested that the low pregnancy rate found in the day 8 embryos is related to ultrastructural damages of the desmosomes and tonofilaments within the trophoblast layer, which results in a disturbance of the normal hatching process.     

Thyroid hormone response to thyrotropin releasing hormone after cold treatment during pre- and postnatal development in the domestic fowl

The purpose of the present study was to compare the effect of periodic cooling during the establishment of a functional pituitary-thyroid axis at days 11-14 of incubation and at other developmental stages, on the subsequent thyroid hormone response to thyrotropin releasing hormone (TRH). In the first and second experiment chick embryos were cooled for 6 hr/day to 30 degrees C from day 11 till 14 and from day 15 till 18 respectively, whereas control groups were incubated throughout at 37.8 degrees C. In both experiments the thyroxine (T4) response upon TRH in 19 day-old embryos was higher in the previously cold treated embryos, according to the percentages of increase. However, the higher T4 response in the cold treated animals disappeared in 1 or 7 day-old chicks hatched from the 2nd experiment, but remained present in chicks of the same ages in the 1st experiment. In a third experiment the T4 response to TRH injection immediately and 3 and 8 days after a temperature treatment (25 degrees C or 12 degrees C) for one week on four weeks old broiler chickens was found to be similar in both temperature groups. In all experiments there was a concomitant triiodothyronine (T3) increase after TRH injection, but differences between experimental groups were observed at days 15 and 19 of incubation and immediately after the postnatal temperature treatment. As an overall conclusion the results indicate that cold treatment only during the establishment of the hypothalamo-hypophysial control of thyroid function can have a long lasting effect by enhancing the T4 response to TRH injection.     

Sex difference in the neurotensin-immunoreactive cell populations of the preoptic area in quail (Coturnix japonica)

The distribution of neurotensin-immunoreactive cells and fibers was analyzed by immunocytochemistry in the forebrain of male and female Japanese quail (Coturnix japonica) by using an antibody directed against the C-terminal part of the molecule. Immunoreactive perikarya were located almost exclusively in the medial preoptic area with small populations also being present in the nucleus paraventricularis and in the tuberal region. Immunoreactive fibers were observed not only throughout the preoptic area-hypothalamus, but also in the septal region, nucleus intercollicularis, substantia grisea centralis and the classical catecholaminergic areas of the mesencephalon, such as the area ventralis of Tsai and the nucleus tegmenti pedunculo-pontinus, pars compacta. The preoptic neurotensin-immunoreactive cells were exclusively located within the boundaries of the sexually dimorphic medial preoptic nucleus. They were significantly more numerous in females than in males. In females, the number of neurotensin cells varied during the ovulatory cycle: fewer cells were observed in birds that were about to lay an egg (they had a calcified egg in the oviduct) than in those that had already laid or were not going to lay on that day. These data indicate major variations in the expression of neurotensin in response to neurochemical or neuroendocrine changes associated with ovulation.     

Histomorphology of the early yolk-sac larvae of the Atlantic halibut (Hippoglossus hippoglossus L.)—an indication of the timing of functionality

Halibut larvae hatch at a very immature stage, and the duration of the yolk-sac period is very long (up to 50 days). This paper describes the histomorphological development of organs of the yolk-sac larvae (6° C) by use of light and electron microscopy. Rudimentary branchial cavities were open from 2 days after hatching. Kidneys seemed functional 16 days after hatching and onwards, and primitive lamellae on the gill arches were beginning to form at this age. Pancreatic zymogen granules were first observed 20daysafter hatching. The liver was segmented into lobes between 20 and 23 days after hatching, and the gall bladder seemed functional from day 23. The hindgut became extensively folded from day 26, and branchial capillaries were first observed at this stage. The larvae were able to catch food particles 24 days after hatching. Judging from ultrastructural observations, it seemed that halibut larvae were able to digest food particles between day 24 and 26 after hatching (around 150 daydegrees and 50% yolk absorption).     

Development of the cerebrospinal fluid in chick embryos. Physicochemical properties.

The development of the physicochemical properties of the cerebrospinal fluid (CSF) was studied in chick embryos from the 9th day of incubation up to hatching. Some of these properties were compared with the corresponding blood or blood plasma properties. During the second half of incubation the CSF pressure rose from 13.2 plus or minus 0.18 mm H2O in 9-day-old embryos to 80.7 plus or minus 0.48 mm H2O just prior to hatching. The critical stages of this development were the 13th to 15th and the 19th to 21st day of incubation. In 13- and 15-day-old embryos, CSF pressure fell sharply after the intracerebral injection of ouabain, but in 19-day embryos it was unaffected. Except for the 15th and 19th incubation day, the CSF pH was always lower than the plasma pH. From the 11th day of incubation up to hatching, the CSF pH fell from 7.36 plus or minus 0.002 to 7.2 plus or minus 0.005. On the 11th and 13th day, specific CSF resistance was higher than plasma resistance, whereas from the 17th incubation day it was significantly lower than the plasma value. During the second half of incubation, specific CSF resistance fell from 1.059 times 10(6) to 0.824 times 10(6) omega mm.m(-1). A difference between the D.C. potential of the venous blood and the CSF appeared for the first time in 15-day-old embryos, the CSF being negative in relation to the blood. By the end of the incubation period this potential difference rose to 10.82 times 0.07 mv.     

Effect of the sex-linked dwarf gene on thyrotrophic and somatotrophic axes in the chick embryo

Plasma concentrations of thyroxine (T4), triiodothyronine (T3), reversed triiodothyronine (rT3), and insulin-like growth factors I and II (IGF-I, IGF-II) together with peripheral 5'-monodeiodination activity were measured in both normal and sex-linked dwarf embryos between day 14 of incubation and day 1 posthatch. Plasma T4 levels increased gradually during embryonic development while T3 concentrations remained low until day 20, when a sharp increase was observed. rT3 levels also increased from day 14 and dropped on day 20 when T3 levels started to increase. 5'-monodeiodination activity was high on day 14 of incubation, decreased thereafter, and showed an increase at the time of air sac penetration together with increased T3 levels. At this stage, differences between normal and dwarf embryos were observed; the latter had lower nonsignificant 5'-Monodeiodination activity and lower (P less than 0.01) plasma T3 levels. Plasma IGF-II levels were high during the whole embryonic period studied. Dwarf embryos had lower (P less than 0.05) IGF-II levels at the time of hatching. IGF-I levels were high on days 14 and 16, declined afterwards, and started to increase again around hatching. With the exception of T3 and IGF-II levels, introduction of the dwarf gene did not cause major changes in the hormonal parameters studied. This may explain the identical body weight at hatching.     

Selective Degradation of Specific Components of Fertilization Coat and Differentiation of Hatching Gland Cells during the Two Phase Hatching of Bufo japonicus Embryos

The embryonic hatching process in the toad, Bufo japonicus , consists of two phases: rupture of the outer jelly strings at stage 20 (neural tube) and an escape from the inner jelly layers and fertilization coat (FC) of individual embryos at stage 23 (tailbud). SDS-PAGE analyses of FCs revealed that, of the eight major protein bands, two components with 58 K and 62 K in molecular weight gradually decreased from stage 18–19 on and totally disappeared at stage 22. When the FCs were treated with a hatching medium prepared by culturing denuded prehatching embryos, both 58 K and 62 K components of the FCs were solubilized, and in the solubilized materials 18 K and 31 K components appeared. Electron microscopy showed that a meshwork of filament bundles present in the FCs before stage 17 became dissociated at stage 19–20, and completely disappeared at stage 23, just before the hatching of embryos. Hatching gland cells (HGCs), an epidermal cell with numerous secretory granules, were first identified at stage 19, and underwent active secretion of the granules during stage 19–23. These results indicate that the hydrolytic degradation of 58K and 62 K components in FCs effected by the hatching enzyme constitutes the basic mechanism of embryonic hatching during both the first and second phases.     

Development of VIP in the peripheral nervous system of avian embryo

The distribution of the VIP containing structures was studied in the gut and in the paravertebral sympathetic ganglia of the quail and chick embryos by immunocytochemistry. In the gut, development of peptidergic nerves followed a craniocaudal gradient. Immunoreactive fibres were first visible in the oesophagus at day 9 in the quail and day 10 in the chick, at 12 days they extended over the whole length of the gut. Cell bodies were localized at day 9 in the foregut and observed in the mid- and hind-gut just before hatching. Transplantations on the chorioallantoic membrane of fragments of various parts of the digestive tract clearly demonstrated that VIP nerve cell bodies belonged to the intrinsic innervation of the gut. Besides the gut, sympathetic paravertebral ganglia contained cells with VIP immunoreactivity detected at day 9 and 10 in quail and chick respectively. In order to find out whether VIP containing neurons differentiated normally in chick embryos in which quail neural crest cells had been implanted at an early stage of development we looked for the appearance of peptidergic neurones in the following situations: when the quail neural primordium had been grafted orthotopically and isochronically into chick host (1) at the adrenomedullary (somites 18-24) and (2) at the vagal (somites 1-7) levels of the neural axis. In all conditions VIP immunoreactivity was observed in quail cells located either in the sympathetic paravertebral ganglia of the trunk at the level of the graft or in the enteric ganglia according to the graft was made at the adrenomedullary and vagal levels respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of spontaneous motility in chick embryos. Normal development and the activating effect of strychnine and picrotoxin.

The longitudinal development of spontaneous motility in chick embryos was studied by Kovach's method (Kovach 1970) from the 10th day of incubation up to hatching, in completely intact eggs. From the 10th to 12th day of incubation, very low amplitude movements of a burst character predominated in spontaneous motility. From the 13th day, both low and high amplitude movements could be distinguished. From the 18th day, high amplitude movements alternating with intervals of motor inactivity preponderated. This discontinuous motility, which was most pronounced on the 20th day of incubation, changed to periodic strong hatching movements. Reduction of spontaneous motility after the 17th day of incubation was not confirmed. Strychnine already activated spontaneous motility in 11-day embryos, but typical convulsions did not appear until the 15th incubation day. With picrotoxin, motility was likewise stimulated in 11-day embryos and paroxysmal activation did not occur until the 15th incubation day. In older embryos, convulsions were gradually succeeded by a continuous increase in spontaneous motility. The effect of picrotoxin had a much longer latent period than the effect of strychnine.     

The ossification centers in the lower end of tibiotarsus in domestic fowl.

The morphogenesis of lower end of tibia in chick is studied commonly but the process of ossification of the same has received very little attention so far. The present study is directed to throw some light on the appearance of ossification centers in the lower end of tibiotarsus of chick. The histology of lower end of tibiotarsus was studied by procuring developing tibiotarsi from chick embryos (20) of 6th day incubation till hatching and 3 post hatched chicks. The transparancies of chick embryos at different incubation periods and post hatched chicks were prepared by Dawson's Alizarin staining method. Three cartilage center (tibial, fibulare and intermedium) appeared in 6...9 days of incubation period in the tarsal region. These gradually fused with the lower end of tibia. Three ossification centres developed in the lower end of tibiotarsus. One for intermedium appeared on 16th day and two fotibial and fibulare on 20th day. All these three centres could be located in the transparancies of the chick embryos in tarsal region. The present study proves that the three cartilages centres maintain their individuality during the ossification process even though those fuse completely with the lower end of tibia in chick. The centers for tibial and fibulare are similar to epiphyseal centres of mammals in histological details.     

鲤鱼发育早期HPG轴和GH/IGF轴相关因子的转录起始分析

采用RT-PCR的方法,以不同发育时期的鲤鱼胚胎和幼鱼为材料,研究了与鱼类生殖相关的HPG轴以及与生长相关的GH/IGF轴中GnRH、GtH以及GH、GHR和IGF重要信号分子的转录起始特征.结果显示,sGnRH、cGnRH、GtH-Ⅰβ卢亚基和GHR于鲤鱼胚胎受精后20h开始转录,IGF-1于受精后23h开始转录,GtH-Ⅱβ亚基于受精后26h开始转录,GtH α亚基于受精后46h开始转录,GH于1dph(孵出后第1天)开始转录.其中,GHR和IGF-1均早于GH开始转录,GtH α亚基和β亚基的转录起始时间不同步.研究结果为揭示鲤鱼生殖与生长间的调控机制积累了重要的科学依据.     

卤虫（Artemia salina）孵化腺细胞分化时相的免疫细胞化学研究（简报）

动物孵化酶(hatching enzyme HE)是早期胚胎在特定发育阶段由孵化腺细胞产生和分泌的,在动物早期胚胎孵化中具有关键性作用^[4]。孵化腺细胞(hatching gland cell,HGC)一般为单细胞腺体,是从胚胎发育到特定阶段(孵化前)出现、至胚胎孵出后的特定时期消失的一时性细胞(transient type of     

Long-term measurement of heart rate in chicken eggs

Taking advantage of acoustocardiogram (ACG), we measured the heart rate (HR) of chick embryos continuously from day 12 until hatching and then investigated the development of HR irregularities (HRI), HR variability (HRV), and the existence of a circadian rhythm in mean HR (MHR). HRI comprised transient bradycardia and tachycardia, which first developed on day 14 and 16 in most embryos, respectively. Transient bradycardia increased in frequency and magnitude with embryonic development and occurred over periods of up to 30 min in some embryos. MHR was maximal on around days 14-15 and thereafter decreased to about 250-260 bpm on days 16-18. Baseline HRV, which is an oscillation of the MHR baseline, occurred as HR decreased from days 15-16 and became predominant on days 17-18. The magnitude of the baseline oscillations reached up to 50 bpm in some embryos and the period ranged between about 40-90 min (ultradian rhythm). A circadian rhythm of MHR was not found in late chick embryos. On days 18-19, embryonic activities were augmented and then breathing movements began to occur, disturbing ACG signals and thus making it difficult to measure the HR. Instead, the development of breathing activities was recorded. Breathing frequency was irregular at first and then increased to a maximum of about 1.5 Hz prior to hatching.