Genetic and embryological experiments have established the Caenorhabditis elegans adult hermaphrodite gonad as a paradigm for studying the control of germline development and the role of soma-germline interactions. We describe ultrastructural features relating to essential germline events and the soma-germline interactions upon which they depend, as revealed by electron and fluorescence microscopy. Gap junctions were observed between oocytes and proximal gonadal sheath cells that contract to ovulate the oocyte. These gap junctions must be evanescent since individual oocytes lose contact with sheath cells when they are ovulated. In addition, proximal sheath cells are coupled to each other by gap junctions. Within proximal sheath cells, actin/myosin bundles are anchored to the plasma membrane at plaque-like structures we have termed hemi-adherens junctions, which in turn are closely associated with the gonadal basal lamina. Gap junctions and hemi-adherens junctions are likely to function in the coordinated series of contractions required to ovulate the mature oocyte. Proximal sheath cells are fenestrated with multiple small pores forming conduits from the gonadal basal lamina to the surface of the oocyte, passing through the sheath cell. In most instances where pores occur, extracellular yolk particles penetrate the gonadal basal lamina to directly touch the underlying oocytes. Membrane-bounded yolk granules were generally not found in the sheath cytoplasm by either electron microscopy or fluorescence microscopy. Electron microscopic immunocytochemistry was used to confirm and characterize the appearance of yolk protein in cytoplasmic organelles within the oocyte and in free particles in the pseudocoelom. The primary route of yolk transport apparently proceeds from the intestine into the pseudocoelom, then through sheath pores to the surface of the oocyte, where endocytosis occurs. Scanning electron microscopy was used to directly visualize the distal tip cell which extends tentacle-like processes that directly contact distal germ cells. These distal tip cell processes are likely to play a critical role in promoting germline mitosis. Scanning electron microscopy also revealed thin filopodia extending from the distal sheath cells. Distal sheath filopodia were also visualized using a green fluorescent protein reporter gene fusion and confocal microscopy. Distal sheath filopodia may function to stretch the sheath over the distal arm.

In Drosophila, the Dorsal protein establishes the embryonic dorso-ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-kappaB), is vital for the formation of the proximo-distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-kappaB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-kappaB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-kappaB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-kappaB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

A polypeptide of 42 kDa was previously identified in rabbit uterine epithelium during the peri-implantation period as a progesterone-dependent, stage-specific protein. Binding of the lectin RCA-I to the 42-kDa band on Western blots demonstrated that it was a glycoprotein, here designated GP42. With use of a polyclonal antiserum to this glycoprotein, strong immunostaining was present on the surface of epithelial cells in implantation-stage uteri (6-7 days pregnant). Uteri of 4-day pseudopregnant females had only trace reactivity, and estrous uteri were devoid of immunostaining. Comparison of GP42 staining on immunoblots of uterine luminal samples, obtained using buffer with or without the detergent Triton X-100, demonstrated that GP42 is either loosely associated with the epithelial surface or is a secretory product. The N-terminal sequence of GP42 was identical through 13 amino acids with the beta subunit of haptoglobin, an acute-phase protein secreted by the liver. Additional immunoblot analyses were carried out after one- or two-dimensional PAGE separation of polypeptides of rabbit uterine samples and human haptoglobin. These employed anti-GP42 as well as antibodies directed against haptoglobin, and results confirmed the similarity of GP42 with beta-haptoglobin. In nonreducing gels, reactivity with anti-GP42 was present in a band of 110 kDa. This is comparable to the molecular size of serum haptoglobin, which occurs as a tetramer of two alpha (15-kDa) and two beta (38-42-kDa) chains. Cultured epithelial cells, derived from 4-day pseudopregnant uteri, released GP42 into the medium, but stromal cells appeared not to produce the glycoprotein. We conclude that GP42 is a uterine glycoprotein, related or identical to haptoglobin, and produced by rabbit uterine epithelial cells during the peri-implantation period. Possible roles for GP42 in relation to ovo-implantation are discussed in light of known functions for haptoglobin and haptoglobin-related protein.

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominantly localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.

A combination of microscopical methods (TEM, SEM, freeze fracture, confocal, immunoEM) are utilized to demonstrate functional interactions between somatic cells and the germline in the model organism, C. elegans.A variety of signaling mechanisms are required to coordinate activity of maturing oocytes and the somatic sheath of the gonad. Communication via gap junctions allows direct coordination of sheath cell motility and oocyte development. Gap junctions link adjoining sheath cells, and link the sheath to the most mature oocyte. Yolk protein produced in the intestine passes from the pseudocoelom through the basal lamina of the proximal gonad and then through fenestrations (sheath pores) in the gonad sheath before being endocytosed into the most mature oocytes. Myoepithelial specializations anchored by hemi-adherens junctions in the proximal sheath act to squeeze the primary oocyte out of the gonad to the spermatheca for fertilization.