Uterine gland formation in mice is a continuous process, requiring the ovary after puberty, but not after parturition

Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition.     

Genetic analysis of the role of gibberellin in the red light inhibition of stem elongation in etiolated seedlings

Red light causes a reduction in the extension growth of dark-grown seedlings. The involvement of gibberellin in this process was tested by screening a number of gibberellin synthesis and gibberellin response mutants of Pisum sativum L. for the kinetic response of stem growth inhibition by red light. Gibberellin deficient dwarfs, produced by mutant alleles at the Le, Na, and Ls loci, and gibberellin response mutants produced by mutant alleles at the La and Cry², Lka, and Lkb loci were tested. Extension growth of expanding third internodes of dark-grown seedlings was recorded with high resolution using angular position transducers. Seedlings were treated with red light at a fluence rate of 4 micromoles per square meter per second either continuously or for 75 seconds, and the response was measured over 9 hours. With certain small exceptions, the response to the red light treatments was similar in all the mutants and wild types examined. The lag time for the response was approximately 1 hour and a minimum in growth rate was reached by 3 to 4 hours after the onset of the light treatment. Growth rate depression at this point was about 80%. Seedlings treated with 75 seconds red light recovered growth to a certain extent. Red/far-red treatments indicated that the response was mediated largely by phytochrome. The similar responses to red light among these wild-type and mutant genotypes suggest that the short-term (i.e. 9 hour) response to red light is not mediated by either a reduction in the level of gibberellin or a reduction in the level or affinity of a gibberellin receptor.     

Genetic interaction of Gsc and Dkk1 in head morphogenesis of the mouse

Mouse embryos lacking Gsc and Dkk1 function display severe deficiencies in craniofacial structures which are not found in either Dkk1 homozygous null or Gsc homozygous null mutant embryos. Loss of Gsc has a dosage-related effect on the severity of head truncation phenotype in Dkk1 heterozygous embryos. The synergistic effect of these mutations in enhancing head truncation provides direct evidence of a genetic interaction between Gsc and Dkk1, which display overlapping expression in the prechordal mesoderm. In the absence of Gsc activity, the expression of Dkk1, WNT genes and a transgenic reporter for WNT signalling are altered. Our results show that Gsc and Dkk1 functions are non-redundant in the anterior mesendoderm for normal anterior development and Gsc may influence Wnt signalling as a negative regulator.     

Simultaneous detection of cytokine and immunophenotype at the single cell level by immunoenzymatic double staining

Summary The goal of this study was to establish a generally applicable immunoenzymatic method for the simultaneous detection of cytokine and immunophenotype at the single cell level. Evaluating various cell preparations and staining protocols, we found that permeabilization by saponin (0.1%) is very efficient, in combination with glutaraldehyde (0.04%) as fixative. Among various staining procedures, sequential immunoperoxidase labelling of the cytokine by use of diaminobenzidine, and detection of the immunophenotype by use of 4-chloronaphthol proved most discriminative. The typical localization of the cytokine reaction product (Golgi staining) within the cell, and the ringlike staining for the immunophenotype on the cell surface, allowed precise identification of double-labelled cells. Primary monoclonal antibodies from the same species could be used without loss of sensitivity and specificity for either or both antigens. This method thus provides the opportunity to study morphology, cytokine and immunophenotype simultaneously at the single cell level with standard equipment. Its application for the analysis of tissue samples is in progress, and may allow us to incorporate the cytokine-type as a new parameter in histopathological diagnostics.     

Ontogenetic shifts in resource allocation: colour change and allometric growth of defensive and reproductive structures in the Caribbean spiny lobster Panulirus argus

Resource allocation theory predicts a disproportionately large allocation of resources to defensive structures during early ontogeny in organisms that are subject to more intense predation at smaller than at larger body sizes. We tested this prediction on the Caribbean spiny lobster Panulirus argus, which exhibits a negative relationship between predation risk and body size with a high natural mortality of smaller individuals. Independent allometric growth analyses demonstrated that numerous defensive structures (e.g. orbital horns, segments supporting the antenna, the tail fan) display negative allometric growth throughout ontogeny. We interpret these findings as lobsters investing disproportionately more resources to defensive structures when small to improve survivorship. Similarly, we observed an ontogenetic shift in lobster colour pattern; small individuals (< 23 mm carapace length) that inhabit nursery grounds (preferably among red algae) displayed a disruptive pattern (camouflage), whereas larger juveniles displayed a bicolour pigmentation typical of adult lobsters. This shift in colour pattern further suggests that small lobsters employ cryptic coloration throughout their asocial algal stage. However, this cryptic coloration offers no advantage when lobsters grow larger and start dwelling in crevices. Other structures linked to reproduction (e.g. female pleopods and male pereopods) experienced either isometric or positive allometric growth throughout ontogeny. Our results support one of the main predictions of resource allocation theory and demonstrate ontogenetic shifts in defensive structures and coloration concomitantly with changes in lobster mortality risk mediated by size‐dependent predation risk. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.     

Fgf9 and Wnt4 Act as Antagonistic Signals to Regulate Mammalian Sex Determination

The genes encoding members of the wingless-related MMTV integration site (WNT) and fibroblast growth factor (FGF) families coordinate growth, morphogenesis, and differentiation in many fields of cells during development. In the mouse, Fgf9 and Wnt4 are expressed in gonads of both sexes prior to sex determination. Loss of Fgf9 leads to XY sex reversal, whereas loss of Wnt4 results in partial testis development in XX gonads. However, the relationship between these signals and the male sex-determining gene, Sry, was unknown. We show through gain- and loss-of-function experiments that fibroblast growth factor 9 (FGF9) and WNT4 act as opposing signals to regulate sex determination. In the mouse XY gonad, Sry normally initiates a feed-forward loop between Sox9 and Fgf9, which up-regulates Fgf9 and represses Wnt4 to establish the testis pathway. Surprisingly, loss of Wnt4 in XX gonads is sufficient to up-regulate Fgf9 and Sox9 in the absence of Sry. These data suggest that the fate of the gonad is controlled by antagonism between Fgf9 and Wnt4. The role of the male sex-determining switch— Sry in the case of mammals—is to tip the balance between these underlying patterning signals. In principle, sex determination in other vertebrates may operate through any switch that introduces an imbalance between these two signaling pathways.     

Goosecoid acts cell autonomously in mesenchyme-derived tissues during craniofacial development.

Mice homozygous for a targeted deletion of the homeobox gene Goosecoid (Gsc) have multiple craniofacial defects. To understand the mechanisms responsible for these defects, the behavior of Gsc-null cells was examined in morula aggregation chimeras. In these chimeras, Gsc-null cells were marked with beta-galactosidase (beta-gal) activity using the ROSA26 lacZ allele. In addition, mice with a lacZ gene that had been introduced into the Gsc locus were used as a guide to visualize the location of Gsc-expressing cells. In Gsc-null<->wild-type chimeras, tissues that would normally not express Gsc were composed of both Gsc-null and wild-type cells that were well mixed, reflecting the overall genotypic composition of the chimeras. However, craniofacial tissues that would normally express Gsc were essentially devoid of Gsc-null cells. Furthermore, the nasal capsules and mandibles of the chimeras had defects similar to Gsc-null mice that varied in severity depending upon the proportion of Gsc-null cells. These results combined with the analysis of Gsc-null mice suggest that Gsc functions cell autonomously in mesenchyme-derived tissues of the head. A developmental analysis of the tympanic ring bone, a bone that is always absent in Gsc-null mice because of defects at the cell condensation stage, showed that Gsc-null cells had the capacity to form the tympanic ring condensation in the presence of wild-type cells. However, analysis of the tympanic ring bones of 18.5 d.p.c. chimeras suggests that Gsc-null cells were not maintained. The participation of Gsc-null cells in the tympanic ring condensation of chimeras may be an epigenetic phenomenon that results in a local environment in which more precursor cells are present. Thus, the skeletal defects observed in Gsc-null mice may reflect a regional reduction of precursor cells during embryonic development.     

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.