Serotonin receptors are selectively expressed in the avian germ cells and early embryos.

The expression of nine serotonin (5-HT) receptor transcripts was studied using reverse transcription polymerase chain reaction (RT-PCR) in germ cells, cleavage and gastrulation stages of Japanese quail, and qPCR for 5-HT3 and 5-HT4 receptors in oocytes and embryos. We show the presence/absence of nine serotonin transcripts known in birds for receptors 5-HT1A, 5-HT1F, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A in avian germ cells and early embryos. The absence of 5-HT3 and 5-HT5A in primordial germ cells and of 5-HT3 and 5-HT7A in sperm is characteristic.

Molecular aspects of avian oogenesis and fertilisation.

In this paper, we summarise studies which have been carried out on the metabolism of nucleic acids (maternal RNA, DNA, nucleolytic enzymes) in avian oocytes and embryos (Japanese quail, Coturnix coturnix japonica ) within the last 10 years in the Institute of Genetics and Animal Breeding of the Polish Academy of Sciences. The accumulation of maternal RNA in the quail oocyte during oogenesis is shown and discussed. Several individual transcripts were identified in RNA from the germinal disc and some also in extraembryonic RNA under the perivitelline membrane.

Melatonin receptor genes (mel-1a, mel-1b, mel-1c) are differentially expressed in the avian germ line.

The presence of melatonin receptor transcripts (mel-1a, mel-1b and mel-1c) was investigated in primordial germ cells (PGCs), immature and mature oocytes, and sperm of Japanese quail by reverse transcription--polymerase chain reaction (RT-PCR). The mel-1a transcript was detected in as few as in a thousand PGCs. Significant differences in the expression of melatonin receptor genes were found in differentiating germ cells: in PGCs only the mel-1a receptor was expressed, in blastoderms and immature oocytes all three transcripts (mel-1a, mel-1b, mel-1c) were present, while in mature ovulated oocytes the predominant transcript was mel-1c (with sporadic occurrence of mel-1a and mel-1b).

Melatonin and its synthesizing enzymes (arylalkylamine N-acetyltransferase-like and hydroxyindole-O-methyltransferase) in avian eggs and early embryos.

The presence of melatonin and the enzymes (transcripts and activities) involved in its synthesis, i.e. arylalkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), was investigated in the eggs and early embryos of Japanese quail at Hamburger-Hamilton stages 1-10.

Transition from embryonic to adult transcription pattern of serotonin N-acetyltransferase gene in avian pineal gland.

The study reports the change of transcription pattern of serotonin N-acetyltransferase gene and melatonin receptor genes during ontogenesis of the avian pineal gland. The RT-PCR technique was used to investigate the expression of the arylalkylamine N-acetyltransferase (AA-NAT) and melatonin receptor genes during development of the pineal glands isolated from Japanese quail (Coturnix coturnix japonica) embryos incubated from 3 days on until hatching (17 days), and in some organs (pineal, brain hemisphere, eye, leg, heart) of the 3-day-old quail embryo. It was shown that two phases of AA-NAT expression are observed during pineal gland development.

Bidirectional communication between the pineal gland and the immune system.

The pineal gland is a vertebrate neuroendocrine organ converting environmental photoperiodic information into a biochemical message (melatonin) that subsequently regulates the activity of numerous target tissues after its release into the bloodstream. A phylogenetically conserved feature is increased melatonin synthesis during darkness, even though there are differences between mammals and birds in the regulation of rhythmic pinealocyte function. Membrane-bound melatonin receptors are found in many peripheral organs, including lymphoid glands and immune cells, from which melatonin receptor genes have been characterized and cloned.

Presence and developmental regulation of serotonin N-acetyltransferase transcripts in oocytes and early quail embryos (Coturnix coturnix japonica).

By RT-PCR two transcripts for arylalkylamine N-acetyltransferase (AA-NAT; serotonin N-acetyltransferase; EC 2.3.1.

DNase I and II present in avian oocytes: a possible involvement in sperm degradation at polyspermic fertilisation.

During polyspermic fertilisation in birds numerous spermatozoa enter the eggs, in contrast to the situation in mammals where fertilisation is monospermic. However, in birds only one of the spermatozoa which have entered an egg participates in zygote nucleus formation, while the supernumerary spermatozoa degenerate at early embryogenesis. Our previous work has demonstrated the presence in preovulatory quail oocytes of DNase I and II activities able to digest naked lambdaDNA/HindIII substrate in vitro.

Development of embryos from in vitro ovulated and fertilized oocytes of the quail (Coturnix coturnix japonica).

The development of quail embryos obtained after in vitro fertilization of oocytes ovulated in vitro was investigated. About 40% of the specimens, after 18-20 hr of incubation, had undergone cleavage to reach stages IV-VI when viewed under a stereo microscope. However, only 36% of these embryos contained normal, DAPI-stained nuclei when observed under a fluorescent microscope; the other 64% showing a morphologically normal cleavage pattern did not contain nuclei.

Expression of melatonin receptor transcripts (mel-1a, mel-1b and mel-1c) in Japanese quail oocytes and eggs.

Cloning and sequencing of the cDNA for avian melatonin (MEL) receptors have made it possible to investigate the expression of these receptors in different animal tissues and organs by reverse transcription polymerase chain reaction. Our study demonstrates for the first time, the presence of MEL receptor transcripts in maternal RNA from Japanese quail oocytes and in RNA from the early embryos of the laid eggs. Specific primers permitted discrimination between mel-1a, mel-1b and mel-1c receptor sequences, and special techniques used to obtain the biological material made it possible to avoid accidental contamination with cells of somatic origin.

Detection of deoxyribonuclease I and II activities in Japanese quail oocytes.

Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation.

Accumulation and localisation of maternal RNA in oocytes of Japanese quail.

Accumulation of total RNA and poly(A+)RNA was determined in the oocytes of Japanese quail (Coturnix coturnix japonica) during oogenesis, by a standard spectrophotometric method, after RNA extraction. Intensive RNA accumulation was observed in the oocytes 0.25-2.

Maturation and ovulation of Japanese quail oocytes under in vitro conditions.

1. An in vitro system for ovulation and maturation of Japanese quail oocytes is described. 2.

Characteristics of poly(A)-degrading factor present in the avian oocytes and early embryos.

The presence of poly(A)-degrading activity was studied in vitro in the quail and mouse oocytes and early embryos using 3H-poly(A) as a substrate. The activity was measured by adsorption of the undegraded substrate to DE-81 filter paper discs, by chromatographic separation on Sephadex G-50 column and by agarose gel electrophoresis followed by transfer onto a Zeta-probe membrane (BioRad, Richmond, CA) and autoradiography. High poly(A)-degrading activity was found in the quail previtellogenic and vitellogenic oocytes and lower activity in the early embryos from cleavage stage to gastrulation.

RNase A activity in Japanese quail oocytes.

The presence of RNase A activity was studied in vitro in homogenates of quail oocytes and early embryos using [3H]poly(U) as a substrate. The activity was measured by adsorption of the undegraded substrate onto DE-81 filter paper discs and by chromatographic separation on a Sephadex G-50 column. RNase A activity examined by these methods was almost undetectable in quail previtellogenic, vitellogenic and ovulated oocytes as well as in the embryos from laid eggs.

Maternal RNA content in oocytes of several mammalian and avian species.

The amount of maternal RNA in the oocytes of several mammalian (mouse, rabbit, cow, pig, sheep) and avian (hen, Japanese quail, guinea hen, turkey) species was determined. For mammals these were in the range of 0.47 ng/oocyte (mouse) to 0.

Quantitation of nanogram amounts of nucleic acids in the presence of proteins by the ethidium bromide staining technique.

Modification of the method for determining low amounts of RNA and DNA is proposed. It consists in nucleic acid staining in solution with EtBr (1 microgram/ml) followed by photography of 10 microliters drops on a UV-transparent plate under UV illumination. Densitometric measurements of the Polaroid negatives were used to construct standard concentration curves in the range of 1-16 micrograms/ml of DNA or RNA.

Transcription and polyadenylation processes during early development of quail embryo.

Transcription of tRNA and mRNA occurs in quail as early as during cleavage while rRNA transcription becomes measurable during blastulation. The polyadenylated fraction content is newly synthesized RNA amounting to 6% during cleavage and blastulation decreases to 3% during gastrulation. Up to 3/4 adenylic residues incorporated into RNA during cleavage are accumulated in the polyadenylated molecules mainly in the form of RNAse-resistant tracts.

The effect of transcription inhibitors on early development of the avian embryo.

The effect of transcription [actinomycin D, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), alpha-amanitin] and translation inhibitors (cycloheximide, puromycin) on quail embryo development was investigated under in vitro conditions. The gastrulation process seemed to proceed normally in the presence of transcription inhibitors in the medium but the translation inhibitors stopped development and caused complete degeneration of the embryos.

Simple in vitro system for molecular studies of early avian development in the quail.

1. The development of quail embryos incubated in ovo and cultured in vitro was compared in terms of basic morphological and biochemical criteria. 2.

Incorporation of uridine and leucine by the germinal discs of quail eggs kept cool or warmed periodically during storage.

Nucleic acid and protein biosyntheses are characteristic of the fertilised germinal disc and continue during storage at 20 degrees C though at a reduced rate. There is no comparable activity in unfertilised eggs. 2.

Precursors of cytoplasmic RNA in avian cell nucleus.

In nucleus of chick embryo four pre-rRNA of molecular weight of 3.43, 2.93, 2.