Expression of activin pathway genes in granulosa cells of dominant and subordinate bovine follicles.

We examined the association between the expression profiles of genes of the activin signalling pathway and ovarian follicular dominance in cattle. In monovular species such as cattle, one ovarian follicle of a cohort is selected to become dominant, whereas all others (i.e. the subordinate follicles) eventually succumb to apoptosis. We showed that Inhibin-betaA, coding for the betaA chain found in the A isoforms of activin, Inh-alpha coding for the inhibin-specific alpha chain, and the activin antagonist follistatin were expressed at higher levels in dominant follicle granulosa cells from Day 3.5 (ovulation=Day 0). Before selection, Inh-betaA but not Inh-alpha was significantly correlated with potential dominant follicles, as defined by high aromatase expression and follicular fluid estrogen concentrations. Follistatin expression marked the largest follicles at Day 1.5, but displayed large variation in levels among cows. The third inhibin gene, Inh-betaB, could only be detected at very low levels from Day 7 and thus was unlikely to play a prominent role in activin/inhibin signalling in cattle during these stages. There was a decrease in activin tone (P=0.07) specifically in the aromatase-high/dominant follicles, as measured by the ratio of Inh-betaA to Inh-alpha plus follistatin transcripts between Days 1.5 and 7. Messenger RNA for both activin type II receptors and the nuclear effector Smad2 were detected in granulosa cells, consistent with an autocrine role for activin signalling. Additionally, expression of the putative activin target genes Smad2 and FSHreceptor were, respectively, either strongly (P<0.001) or weakly (P=0.09) associated with dominant follicles.

Isolation of genes associated with developmental competency of bovine oocytes.

Eggs differ widely in their ability to develop into an embryo. To address this characteristic, the concept of developmental competency has been coined, defined as the ability or potential of an oocyte to undergo maturation, fertilization and development to blastocyst stages or live offspring. Developmental competency is acquired progressively during folliculogenesis and is linked to follicular size. In an effort to understand the molecular changes underlying differences in competency we compared oocytes derived from large follicles (>or=5mm) to those from small follicles (

Regional specificity of RAR gamma isoforms in Xenopus development.

All retinoic acid receptors (RAR alpha, beta, gamma) have two isoforms, whose function is unknown. We now show that at least for RAR gamma, the isoforms are differentially distributed in the embryo. RAR gamma 1 and RAR gamma 2 are detected in the head region, whereas RAR gamma 2 is the sole isoform expressed in the tail region. Specifically, it is expressed in the chordoneural hinge, a region of the tailbud that has organizing properties. Treatment with high doses of retinoic acid (RA) reduces expression in this region. The results are discussed in terms of the known teratogenic effects of RA in the tail region.

Crescent, a novel chick gene encoding a Frizzled-like cysteine-rich domain, is expressed in anterior regions during early embryogenesis.

We describe the isolation of a novel chicken gene that we have termed crescent, based on the most distinctive stage of its highly dynamic expression pattern during early embryogenesis. Crescent encodes a protein that in its N-terminal half shows the characteristic invariant 9 cysteine residues of the cysteine-rich domain (CRD) found in the Frizzled family of proteins, in Smoothened and in Collagen XVIII. The CRD of several Frizzled proteins have recently been shown to bind to Wg. Unlike Frizzled proteins, crescent does not contain a transmembrane domain and thus can not function as a receptor. Crescent expression is first found at stage XII (E-G&K) in the center of the area pellucida. On primitive streak formation, expression is detected in the entire anterior half of the area pellucida in the hypoblast layer. At maximal streak extension, crescent transcripts are localized primarily to the germinal crescent, where the primordial germ cells reside. During head process and head fold stages, crescent labels the anteriormost endodermal cells which will give rise to prospective foregut. With the commencement of somitogenesis, crescent expression rapidly wanes.

Stage- and adult tissue-specific expression of a homeobox gene in embryo and adult Parechinus angulosus sea urchins.

We report the isolation of a gene (PaHbox6), encoding a homeobox-containing protein of the South African sea urchin, Parechinus angulosus. Sequencing identified an Antennapedia-class gene encoding a homeobox that is the homologue of the Hawaiian sea urchin Tripneustes gratilla homeobox gene. Extensive restriction-fragment length polymorphism surrounds the gene. RNase-protection analyses revealed expression of PaHbox6 in mesenchyme blastula embryos at maximal levels of 44 +/- 8 transcripts/embryo. Four adult tissues examined (testes, ovary, intestines, Aristotle's lantern) showed expression of PaHbox6, though at greatly differing levels, with testes highest at eleven transcripts/10 pg RNA. Two transcripts of 5.2 and 5.7 kb were identified in adult tissue.

Embryo loss in cattle between Days 7 and 16 of pregnancy.

Embryo loss between embryonic Days 7 and 16 (Day 0=day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P=0.03) as more embryos were transferred, particularly at later stages (Day 14, P<0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P<0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P<0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P<0.05; winter-spring, P<0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).

Gene expression profiling of individual bovine nuclear transfer blastocysts.

During somatic cell nuclear transfer the gene expression profile of the donor cell has to be changed or reprogrammed extensively to reflect that of a normal embryo. In this study we focused on the switching on of embryonic genes by screening with a microarray consisting of 5000 independent cDNA isolates derived from a bovine blastocyst library which we constructed for this purpose. Expression profiling was performed using linearly amplified RNA from individual day 7 nuclear transfer (NT) and genetically half-identical in vitro produced (IVP) blastocysts. We identified 92 genes expressed at lower levels in NT embryos whereas transcripts of 43 genes were more abundant in NT embryos (P < or = 0.05, > or = 1.5-fold change). A range of functional categories was represented among the identified genes, with a preponderance of constitutively expressed genes required for the maintenance of basal cellular function. Using a stringent quantitative SYBR-green real time RT-PCR based approach we found, when comparing the means of the expression levels of a larger set of individual embryos, that differences were small (< 2-fold) and only significant for two of the seven analysed genes (KRT18, SLC16A1). Notably, examination of transcript levels of a single gene in individual embryos could not distinguish an NT from a control embryo. This unpredictability of individual gene expression on a global background of multiple gene expression changes argues for a predominantly stochastic nature of reprogramming errors.

Pax2 and homeodomain proteins cooperatively regulate a 435 bp enhancer of the mouse Pax5 gene at the midbrain-hindbrain boundary.

Pax and homeodomain transcription factors are essential for the formation of an organizing center at the midbrain-hindbrain boundary (mhb) which controls the genesis of the midbrain and cerebellum in the vertebrate embryo. Pax2 and Pax5 are sequentially activated in this brain region, with Pax2 expression preceding that of Pax5. Using a transgenic reporter assay, we have now identified a conserved 435 bp enhancer in the 5' flanking region of mammalian Pax5 genes which directs lacZ expression in the correct temporal and spatial pattern at the mhb. This minimal enhancer is composed of two distinct elements, as shown by protein-binding assays with mhb-specific extracts. The proximal element contains overlapping consensus binding sites for members of the Pax2/5/8 and POU protein families, whereas a distal element is bound by homeodomain and zinc finger transcription factors. Expression analysis of transgenes carrying specific mutations in these recognition motifs identified the Pax- and homeodomain-binding sites as functional elements which cooperatively control the activity of the mhb enhancer. lacZ genes under the control of either the minimal enhancer or the endogenous Pax5 locus were normally expressed at the mhb in Pax5 mutant embryos, indicating that this enhancer does not depend on autoregulation by Pax5. In Pax2 mutant embryos, expression of the endogenous Pax5 gene was, however, delayed and severely reduced in lateral aspects of the neural plate which, on neural tube closure, becomes the dorsal mhb region. This cross-regulation by Pax2 is mediated by the Pax-binding site of the minimal enhancer which, upon specific mutation, resulted in severely reduced transgene expression in the dorsal part of the mhb. Together these data indicate that Pax2 and homeodomain proteins directly bind to and cooperatively regulate the mhb enhancer of Pax5.

Characterization of three novel members of the zebrafish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function.

The mammalian Pax2, Pax5 and Pax8 genes code for highly related transcription factors, which play important roles in embryonic development and organogenesis. Here we report the characterization of all members of the zebrafish Pax2/5/8 family. These genes have arisen by duplications before or at the onset of vertebrate evolution. Due to an additional genome amplification in the fish lineage, the zebrafish contains two Pax2 genes, the previously known Pax[b] gene (here renamed as Pax2.1) and a novel Pax2.2 gene. The zebrafish Pax2.1 gene most closely resembles the mammalian Pax2 gene in its expression pattern, as it is transcribed first in the midbrain-hindbrain boundary region, then in the optic stalk, otic system, pronephros and nephric ducts, and lastly in specific interneurons of the hindbrain and spinal cord. Pax2.2 differs from Pax2.1 by the absence of expression in the nephric system and by a delayed onset of transcription in other Pax2.1 expession domains. Pax8 is also expressed in the same domains as Pax2.1, but its transcription is already initiated during gastrulation in the primordia of the otic placode and pronephric anlage, thus identifying Pax8 as the earliest developmental marker of these structures. The zebrafish Pax5 gene, in contrast to its mouse orthologue, is transcribed in the otic system in addition to its prominent expression at the midbrain-hindbrain boundary. The no isthmus (noi) mutation is known to inactivate the Pax2.1 gene, thereby affecting the development of the midbrain-hindbrain boundary region, pronephric system, optic stalk and otic region. Although the different members of the Pax2/5/8 family may potentially compensate for the loss of Pax2.1 function, we demonstrate here that only the expression of the Pax2.2 gene remains unaffected in noi mutant embryos. The expression of Pax5 and Pax8 is either not initiated at the midbrain-hindbrain boundary or is later not maintained in other expression domains. Consequently, the noi mutation of zebrafish is equivalent to combined inactivation of the mouse Pax2 and Pax5 genes with regard to the loss of midbrain-hindbrain boundary development.