Microarray analysis of Myf5-/-:MyoD-/- hypoplastic mouse lungs reveals a profile of genes involved in pneumocyte differentiation.

Fetal breathing-like movements (FBMs) are important in normal lung growth and pneumocyte differentiation. In amyogenic mouse embryos (designated as Myf5-/-:MyoD-/-, entirely lacking skeletal musculature and FBMs), type II pneumocytes fail to differentiate into type I pneumocytes, the cells responsible for gas exchange, and the fetuses die from asphyxia at birth. Using oligonucleotide microarrays, we compared gene expression in the lungs of Myf5-/-:MyoD-/- embryos to that in normal lungs at term. Nine genes were found to be up-regulated and 54 down-regulated at least 2-fold in the lungs of double-mutant embryos. Since many down-regulated genes are involved in lymphocyte function, immunohistochemistry was employed to study T- and B-cell maturity in the thymus and spleen. Our findings of normal lymphocyte maturity implied that the down-regulation was specific to the double-mutant lung phenotype and not to its immune system. Immunostaining also revealed altered distribution of transcription and growth factors (SATB1, c-Myb, CTGF) from down-regulated genes whose knockouts are now known to undergo embryonic or neonatal death secondary to respiratory failure. Together, it appears that microarray analysis has identified a profile of genes potentially involved in pneumocyte differentiation and therefore in the mechanisms that may be implicated in the mechanochemical signal transduction pathways underlying FBMs-dependent pulmonary hypoplasia.

Fetal ocular movements and retinal cell differentiation: analysis employing DNA microarrays.

As developmental biologists we study the role of fetal movements in providing continuity between prenatal and postnatal life. There are two major categories of fetal motility. The first category consists of movements that have an obvious effect on the survival or development of the fetus (e.g., changes of position, sucking and swallowing). The second category consists of fetal movements that anticipate postnatal functions. For example, fetal ocular movements (FOMs) predict postnatal eye function (e.g., motion vision) of the newborn and therefore represent an important indicator of fetal health. However, while the clinical significance of fetal motility is obvious, its biological significance is elusive. We propose to use retina of genetically modified mouse embryos to study the biological role of FOMs in the genesis of cell diversity and organ functional maturation. Our results have already demonstrated the importance of fetal eye motility in the differentiation of cholinergic amacrine cells (CACs) in the retina (Kablar, 2003). Apparently, these cells are sensitive to motion and also responsible for motion vision. In the current report, we suggest employing the unique opportunity provided by the mouse Myf5-/-:MyoD-/- knock-outs that lack skeletal musculature and FOMs, microarray analysis and the follow-up experiments to identify a group of candidate genes that are essential for the molecular regulation of CAC differentiation and in turn for the functional maturation of the visual system towards its ability to perform motion vision. Finally, the molecules identified via this approach may be important in the mechanochemical signal transduction pathways employed during the process of conversion of a mechanical stimulus into an instruction understandable by the developing retinal neurons and glia cells.

Neurotrophins, airway smooth muscle and the fetal breathing-like movements.

Central nervous system and skeletal muscles secrete a group of polypeptide hormones called neurotrophins (NTs). More recent studies show that NTs and their receptors are also expressed in the lung, suggesting a role for NTs in lung development. To examine the role of NTs during normal and diseased lung organogenesis, we employed wild-type and amyogenic mouse embryos (designated as Myf5-/-:MyoD-/-). Amyogenic embryos completely lacked skeletal muscles and were not viable after birth due to the respiratory failure secondary to lung hypoplasia. To examine the importance of lung-secreted NTs during normal and hypoplastic lung organogenesis, immunohistochemistry was employed. Distribution of NTs and their receptors was indistinguishable between normal and hypoplastic lungs. To further examine the importance of non-lung-secreted NTs (e.g., from the skeletal muscle and CNS) in lung organogenesis, in utero injections of two NTs were performed. The exogenously introduced NTs (i.e., non-lung-secreted) did not appear to improve development of the lung in amyogenic embryos. Moreover, immunohistochemistry showed significantly reduced number of airway smooth muscle cells (ASMCs) in hypoplastic lungs of amyogenic embryos, suggesting that the number of ASMCs is primarily regulated by the fetal breathing-like movements (i.e., mechanical factors).

The role of fetal breathing-like movements in lung organogenesis.

In this review the recent findings concerning the role of fetal breathing-like movements (FBMs) on lung organogenesis are discussed. We first review the consequences that the lack of FBMs has on lung organogenesis and then we discuss the possible pathways that may be employed in this process. Specifically, we review the data in support of the notion that FBMs are required for the cell cycle kinetics regulation (i.e., cell proliferation and cell death) via the expression of growth factors, such as platelet derived growth factors (PDGFs) and insulin growth factors (IGFs), and thyroid transcription factor 1 (TTF-1). Moreover, the role of FBMs on biochemical differentiation of Clara cells, type I and type II pneumocytes is reviewed. Interestingly, even though type II pneumocytes are able to synthesize surfactant-associated proteins (SPs), in the complete absence of FBMs, they are unable to compile, store and release the surfactant. Similarly, in spite of the expression of some early differentiation markers, in the absence of FBMs, type I pneumocytes are unable to flatten in order to allow the gas exchange in the lung. In fact, we are currently employing the cDNA microarray analysis in search for the molecules that might be specific for the lacking functions in pneumocytes.

Sex-linked genes are not silenced in fetal bovine testes expressing X-inactive specific transcript (XIST).

X-inactive specific transcript (XIST), which is thought to be the central factor for the X-inactivation process in female mammals, is known to be expressed in males during spermatogenesis. Our studies have shown that XIST is not only expressed in adult bovine testis but is also expressed in fetal, newborn, and prepubertal testes long before spermatogenesis is established. To determine whether the XIST expressed in fetal testes is involved in silencing the genes on the X chromosome, we investigated the status of X-linked genes, including glucose-6-phosphate-dehydrogenase (G6PD), hypoxanthine phosphoribosyl transferase (HPRT), and X-linked zinc finger protein gene (ZFX), in fetal bovine gonads at the developmental stage, when meiosis is initiated in fetal ovaries in this species. Reverse transcription and a semiquantitative polymerase chain reaction based on the optical density of each gene-specific band relative to that of the co-amplified Quantum RNA 18S Internal Standard (Ambion, Austin, TX) showed that the XIST gene was expressed in the testes of approximately 90-day-old fetuses and was silent in all their nongonadal organs tested, although at a significantly lower level than that in fetal organs of female fetuses. Our observation that the expression of X-linked genes in the fetal testis was comparable to that in male nongonadal organs, in which X inactivation does not occur, indicates that the low level of XIST, or XIST-like RNA, expressed in the fetal bovine testis is not involved in silencing X-linked genes.

Synaptic pattern of sex complements and sperm head malformation in X-autosome translocation carrier bulls.

Testicular activity and semen characteristics of bulls carrying an X-autosome translocation t(Xp +;23q-) revealed all stages of spermatogenesis although their semen consisted of few and, exclusively, of malformed spermatozoa. Chromosome painting on metaphase spreads of their mother and synaptonemal complex analysis on these and normal bulls were carried out to test whether the location and meiotic pairing behaviour of the rearranged segments could have contributed to the sperm head malformation and oligospermia in our X-autosome translocation (X-AT) carrier bulls. Spermatocytes of X-AT carriers displayed the rearranged chromosomes in a univalent-trivalent association, with 23q- always remaining as a univalent and Xp + in synapsis with normal chromosome 23 and the Y chromosome. Chromosome painting studies to test whether the total absence of meiocytes showing a quadrivalent is due to the non-reciprocal nature of this translocation, identified Xp sequence homology with the distal end of 23q- confirming its relocation to the terminal segment of 23q-. Our synaptonemal complex analyses also confirmed that the bovine pseudo-autosomal region (PAR) is at the distal ends of Xq and Yp and further revealed that over 85% of spermatocytes of X-AT carriers (and up to 13% of spermatocytes of normal bulls) sustain a Y-axis break adjacent to the PAR. Although the exact cause of a Y-axis break in bovine spermatocytes is not known at present, we believe that the break and possible loss of Yq in such high proportions of spermatocytes of X-AT carriers could have contributed to the sperm head malformation and oligospermia in our X-AT carrier bulls.

Meiosis and apoptosis in germ cells of X-autosome translocation carrier boars.

Meiotic features and fate of germ cells were studied using electron microscopy on surface spread spermatocytes and in situ tests for apoptosis on testicular tissues of normal boars and X-autosome translocation (X-AT) carrier boars. Histological sections of the translocation t(Xp+; 14q-) carrier boars showed accumulation of degenerating germ cells including binucleate and multinucleate cells, as well as pyknosis and nuclear fragmentation characteristic of apoptosis. Synaptonemal complex analysis of X-AT carrier boars revealed 19 bivalents including a large complex made up of the altered X (Xp+) and normal chromosome 14, and a smaller element representing the Y chromosome in synapsis with the derived chromosome 14 (14q-) in most (89.3%) of the germ cells. In situ tests for apoptotic DNA fragmentation revealed positive signals exclusively among early spermatocytes and degenerating germ cells. These findings and the absence of stages beyond pachytene suggest that the meiocytes are arrested at pachytene and eliminated through apoptotic process in spite of the complete synapsis displayed by the chromosomes involved in this translocation. Failure of meiotic progress in our X-AT carriers would appear to be the result of the disruption of gene sequence (or function) caused by the involvement of the X chromosome in this rearrangement, rather than the deleterious consequences of abnormal segregation anticipated in reciprocal translocation carriers. We hypothesize that this disruption could have affected the induction of stage-specific gene products in meiosis such as heat shock proteins and caused the excessive release of endonucleases normally produced by early prophase meiocytes, leading to their apoptosis in our X-autosome translocation carrier boars.

Inhibitory effect of iloprost on the contractility of lower uterine segment myometrium from rhesus monkeys in normal-term and androstenedione-induced preterm labor.

OBJECTIVE: Iloprost, a combined EP(1) stimulatory and IP inhibitory receptor agonist, was tested in vitro on myometrium from the lower uterine segment of pregnant rhesus monkeys to compare its effects in spontaneous labor and in labor induced by the administration of androstenedione to the mother. METHODS: Pregnant rhesus monkeys carrying fetuses of known gestational age were instrumented under halothane general anesthesia with femoral artery and vein catheters and uterine electromyogram leads. Experimental animals were infused with androstenedione from 139 days' gestation. Control animals were infused with intralipid vehicle from 139 days' gestation. Lower uterine segment myometrium was removed from control animals either before labor began (n = 6) or in spontaneous labor (n = 4) and from animals undergoing premature labor induced by androstenedione (n = 4). Myometrial contractility in response to iloprost was evaluated using a superfusion system in vitro. RESULTS: Iloprost was inhibitory on myometrium obtained from the lower uterine segment from androstenedione-treated animals as well as vehicle-infused animals in spontaneous term labor. In contrast, iloprost had no effect on myometrial strips from control animals not in labor. CONCLUSION: These findings indicate up-regulation of IP receptors which inhibit myometrial contractility and/or down-regulation of EP(1) receptors which stimulate myometrial contractility in the lower uterine segment during labor. A relative increase in inhibitory responses in the lower uterine segment during labor may enable this region to dilate to allow passage of the fetus.

Delay of preterm delivery in sheep by omega-3 long-chain polyunsaturates.

A positive correlation has been shown between dietary intake of long-chain omega-3 fatty acids in late pregnancy and gestation length in pregnant women and experimental animals. To determine whether omega-3 fatty acids have an effect on preterm labor in sheep, a fish oil concentrate emulsion was continuously infused to six pregnant ewes from 124 days gestational age. At 125 days, betamethasone was administered to the fetus to produce preterm labor. Both the onset of labor and the time of delivery were delayed by the fish oil emulsion. Two of the omega-3-infused ewes reverted from contractions to nonlabor, an effect never previously observed for experimental glucocorticoid-induced preterm labor in sheep. Maternal plasma estradiol and maternal and fetal prostaglandin E2 rose in control ewes but not in those infused with omega-3 fatty acid. The ability of omega-3 fatty acids to delay premature delivery in sheep indicates their possible use as tocolytics in humans. Premature labor is the major cause of neonatal death and long-term disability, and these studies present information that may lead to a novel therapeutic regimen for the prevention of preterm delivery in human pregnancy.

Regional variations in contractile responses to prostaglandins and prostanoid receptor messenger ribonucleic acid in pregnant baboon uterus.

OBJECTIVES: The aims of this study were to compare (1) the contractile responses of the lower uterine segment and fundus to prostaglandins, (2) expression of genes encoding prostanoid receptors in myo-metrium from different regions of the uterus, and (3) the distribution of expression of genes encoding prostanoid receptors (P receptors) in key intrauterine tissues. STUDY DESIGN: Cesarean hysterectomy was performed in 8 pregnant baboons, not in labor, in the last third of pregnancy. Contractile responses of fresh tissue were quantified in a superfusion system. Polyadenylated ribonucleic acid was extracted from frozen tissue and gene expression was quantified by Northern blot analysis with complementary deoxyribonucleic acid probes. RESULTS: Prostaglandin E2 contracted strips of myometrium from the fundus but had no significant effect on strips from lower uterine segment. Prostaglandin F2 alpha contracted myometrium from both regions equally. Compared with fundus tissue, lower uterine segment tissue had greater expression of EP2 receptor messenger ribonucleic acid, less expression of EP3 receptor messenger ribonucleic acid, but similar levels of EP4 receptor and FP receptor messenger ribonucleic acid. EP2 receptor, EP3 receptor, and EP4 receptor messenger ribonucleic acids were also detected in cervix, decidua, and chorion. EP2 receptor messenger ribonucleic acid was most abundant in the cervix, EP3 receptor messenger ribonucleic acid was most abundant in the myometrium, and EP4 receptor messenger ribonucleic acid was most abundant in the decidua. CONCLUSIONS: The reduced contractile response of lower uterine segment tissue to prostaglandin E2 is paralleled by greater inhibitory EP2 receptor expression and less contractile EP3 receptor expression, a pattern similar to that seen in the cervix. Drugs with selective activity at prostanoid receptor types and subtypes are likely to allow safer and more effective control of the uterus and cervix than native prostaglandins.

In vivo administration of nimesulide, a selective PGHS-2 inhibitor, increases in vitro myometrial sensitivity to prostaglandins while lowering sensitivity to oxytocin.

OBJECTIVE: To investigate the action of prostaglandin synthesis inhibitors on uterine sensitivity to established stimulants of myometrial activity, we tested the effect of in vivo administration of nimesulide, a selective cyclooxygenase-2 inhibitor, into ovariectomized nonpregnant sheep on subsequent in vitro myometrial responsiveness to prostaglandin E2 (PGE2), PGE2 alpha, and oxytocin. METHODS: Sixteen ovariectomized ewes were infused intravenously with either estradiol (2 micrograms h, n = 8) or estradiol plus nimesulide (20 mg h, n = 8); for 48 h before necropsy. Longitudinal strips of myometrium were superfused with Krebs buffer solution and their spontaneous baseline contractility recorded after 1 hour. pD2 values and maximum percentage increases in contractile tension were derived from dose-response curves to 10(-9) to 10(-5) mol/L PGE2 and PGE2 alpha, and 10(-11) to 10(-7) mol/L oxytocin. RESULTS: Baseline activity was lower in myometrium from estradiol-treated ewes infused with nimesulide as compared with ewes infused with estradiol alone. Maximum tension increase in response to all three agonists was similar in myometrium from estradiol- and nimesulide-treated ewes, but the pD2 in myometrium from nimesulide-treated ewes was higher in response to PGE2 and PGE2 alpha and lower in response to oxytocin. CONCLUSION: The results show that nimesulide lowers spontaneous myometrial contractility and sensitivity to oxytocin while increasing sensitivity to PGs, indicating a down-regulation of oxytocin receptor and an up-regulation of PG receptors and/or intracellular signaling events.

Differences in the in vitro sensitivity of ovine myometrium and mesometrium to oxytocin and prostaglandins E2 and F2alpha.

We compared the in vitro response to oxytocin, prostaglandin (PG)E2, and PGF2alpha of myometrium and mesometrium from six ovariectomized ewes and 53 ewes at 106-145 days gestational age (dGA), including 14 ewes in spontaneous or betamethasone-induced labor. Myometrial baseline activity increased from 217+/-27 mN/cm2 of cross-sectional area (mean +/-SEM) in ovariectomized ewes to a plateau of 696+/-39 mN/cm2 at 126-135 dGA. No gestation-related changes were observed in mesometrial baseline activity. Myometrial, but not mesometrial, maximum tension in response to agonists increased with gestation to a plateau at 126-135 dGA. The pD2 (negative logarithm of the EC50) values for oxytocin were similar in both tissues and did not change with gestation. During pregnancy, the myometrial pD2 of both PGs was one order of magnitude higher than the mesometrial pD2. The results indicate an increase in myometrial uterotonic receptor-mediated activity that precedes labor with no increase at labor, suggesting that in sheep, activation of the basic mechanisms responsible for strength of myometrial activity at labor occurs by 135 dGA. The greater sensitivity of the myometrium than the mesometrium to PGs supports a major role for intrauterine paracrine factors in regulating myometrial contractility.

Regulation of the switch from myometrial contractures to contractions in late pregnancy: studies in the pregnant sheep and monkey.

Myometrial contractility occurs throughout pregnancy and characteristic patterns of myometrial activity exist according to the endocrine status and the relationship to parturition. These characteristic patterns differ between species, yet certain common features can be observed. Throughout pregnancy, myometrial activity is of the contractures type, long-lasting, low-amplitude epochs of activity switching to contraction-type activity at term. This switch from contractures to contractions tends to occur at night and is related to alteration in maternal plasma oestrogen concentrations, and maternal oxytocin function. Studies in several animal species support the hypothesis that maternal oestrogen prepares the myometrium for a periodic signal that causes the switch from contractures to contractions. Several lines of evidence implicate oxytocin in the switch. These studies show that the detailed preparation for parturition takes longer than previously considered and is brought about by a carefully regulated sequence of events in which oestrogen production by the placenta plays a central role.