Second messengers mediating the effects of testis ecdysiotropin in testes of the gypsy moth,Lymantria dispar

Exposure of larval and pupal testes of Lymantria dispar to diacyl glycerol mimics, phorbol, 12-myristate, 13-acetate, and 11, 12 dibutyryl phorbol ester, induced or augmented synthesis of immunodetectable ecdysteroids. The non-esterified analog, 4α-phorbol, had little effect. H-7, a protein kinase C inhibitor, inhibited ecdysteroid synthesis. When testis ecdysiotropin and phorbol esters were administered together at the maximum effective dose of each, steroidogenesis was further enhanced. Therefore, diacyl glycerol may be a second messenger for testis ecdysiotropin in testes. In addition, testis ecdysiotropin induced a rapid rise and fall in cAMP titers in both larval and pupal testes. The cyclic AMP analog, dibutyryl cyclic AMP, induced ecdysteroid synthesis in larval testes, but had little steroidogenic effect in pupal testis sheaths. However, dibutyryl cyclic AMP inhibited the steroidogenic effect of testis ecdysiotropin in larval as well as pupal testes. Cyclic AMP may act to modulate the effects of testis ecdysiotropin in inducing ecdysteroid synthesis by testes of L. dispar. © 1993 Wiley-Liss, Inc.     

Direct visualization of the endomitotic cell cycle in living megakaryocytes: Differential patterns in low and high ploidy cells

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (≥ 8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before re-joining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.     

Catalatic capacities in heat-shocked Euglena cells

1. Various heat treatments were applied to the wild strain Z. Klebs. of Euglena gracilis. 2. Samples of cells were taken at day 1 of the culture at 26 degrees C in a 33 mM lactate medium, when the catalatic capacities of the catalase were highest. 3. They were either submitted to heat treatments (36 and 38 degrees C), or heat-shocks (40, 42 degrees C) or non-permissive heat stress (45 degrees C) for 15 min, 1 and 2 hr. 4. After a 2-hr 45 degrees C treatment the cells were unable to recover normal physiological functions. 5. Heat treatments between 36 and 38 degrees C decreased the catalatic capacities of cells, while heat-shocks at 40 and 42 degrees C strongly reinforced these capacities of hydrogen peroxide dismutation. 6. Having been heat-shocked at 42 degrees C for 2 hr, the cells became different from control cells: (a) after several months of culture, they displayed catalatic capacities increased by 65%; (b) they were able from now on to survive a 2 hr heat shock at 45 degrees C.     

Localization of the nuclear matrix protein mitotin in mouse cells with a mitotic or endomitotic cell cycle.

Immunofluorescence staining was used to study the precise subcellular distribution of the nuclear matrix antigen, mitotin, in mouse cells characterized by either a mitotic or an endomitotic organization of the cell cycle. In mitotically dividing cells, mitotin showed a speckled distribution within interphase nuclei. In addition, some interphase cells exhibited a weak, focused signal adjacent to the nucleus, reflecting a possible staining of the centrosome region. Using digital contrast-enhanced immunofluorescence microscopy, a distinct association of mitotin to the centrosome, pole microtubules, and midbody could be revealed in cells at different stages of mitosis. In parallel, trophoblast giant cells characterized by an endomitotic cell cycle were derived from blastocyst outgrowths and analyzed likewise. In all giant cells examined so far, mitotin was restricted to the nuclear compartment alone, although different patterns of intranuclear staining could be detected. The present study provides further information about the precise localization of mitotin in mitotic cells, especially during mitosis. In view of the results, the staining pattern observed in endomitotic cells may allow for a better understanding of the origin and the organization of the endomitotic cell cycle.     

Naturally occurring analogs of Lymantria testis ecdysiotropin,a gonadotropin isolated from brains of Lymantria dispar pupae1

Lymantria testis ecdysiotropin (LTE) was isolated from the most prominent peptide peak corresponding to an active fraction obtained by high pressure liquid chromatographic (HPLC) separation of a homogenate of 13,000 Lymantria dispar pupal brains. In this work we examined the other active fractions from this separation as well as a second HPLC separation of an additional 2,300 pupal brains. Bioassay of the ecdysteroidogenic effects of each peak on L. dispar testes allowed detection of 20 peptide peaks with testis ecdysiotropic activity in addition to LTE. Of these, ten peptides were purified and sequenced. All of them were comparable to LTE in molecular weight. The amino acid sequences of five of the peptides were similar enough to LTE to be considered to be members of an LTE family. However, the other five peptides had no significant homology with LTE or with each other. A BLAST database search indicated LTE family homology with portions of inhibitory peptides such as those inhibiting cytolysis. In contrast, non-LTE ecdysiotropic peptides, in which undetermined residues designated X were assumed to be cysteine, were strikingly homologous to portions of vertebrate and invertebrate zinc finger peptides and to vertebrate and invertebrate virus proteins. Arch. Insect Biochem. Physiol. 36:37–50, 1997.Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Interactions between Sertoli cells and germ cells in the testis

The production of mature spermatozoa requires a complex interaction between Sertoli cells and germ cells. Sertoli cells regulate aspects of germ cell division and differentiation while germ cells provide signals that modulate Sertoli cell functions. Germ cells can undergo some differentiation independent of Sertoli cells but at certain crucial points the interaction with Sertoli cells is required. There are several means by which this interaction may occur: (1) direct contact of components of the plasma membrane may act as a signal; (2) secondary messengers could be exchanged via gap junctions; (3) the secretion of paracrine factors may facilitate intercellular communication.     

Sertoli-germ cells interactions in the human testis.

In previous histoimmunochemical studies we reported that transferrin (TF) and insulin-like growth factor I (IGF-I) are present in the cytoplasm of the Sertoli cells of the adult human testis. Receptors for TF were found mainly in adluminal germ cells and type I receptors for IGF-I both in Sertoli and germ cells. Using electron microscopy, evidence of transfer of both TF and IGF-I from the Sertoli to the germ cells through a receptor-mediated endocytosis mechanism was also found. In this paper we report the results of the histoimmunochemical localization of alpha inhibin in the human fetal, prepubertal and adult testis. In 8- to 14-week-old fetal testes a positive immunostaining was found mainly in the interstitial cells, whereas no staining was found in the germ cords. In the prepubertal testis the immunostaining was present in the Sertoli cells but not in the interstitial cells. In the adult human testis the immunostaining was present not only in the Sertoli cells but also in the spermatocytes and in several Leydig cells. Using electron microscopy and immunogold labeling the presence of alpha inhibin immunoreactivity was found in the rough endoplasmic reticulum and in the Golgi cisternae of both Sertoli and Leydig cells. Moreover we found evidence of transfer of alpha inhibin from the Sertoli to the germ cells through receptor-mediated endocytosis.     

Phagocytotic and iron-storing capacities of stromal cells in the rat endometrium

Summary Cytoplasmic pigment inclusions of rat endometrial stromal cells were studied by histology, histochemistry, fluorescence microscopy, electron microscopy and X-ray microprobe analysis. It is shown that a number of endometrial perivascular stromal cells contain numerous free cytoplasmic ferritin particles as well as hemosiderin vacuoles. The larger pigment inclusions reveal also positive PAS- and Schmorl reactions indicating that they contain polysaccharide and lipofuscin material, respectively. These pigmentstoring stromal cells also display acid phosphatase activity; they avidly phagocytose instillated latex particles. No pigment-storing cells occur within the surface or glandular epithelium, either in the basal endometrium or in the myometrium.It is demonstrated that the endometrial iron-storing cells function as iron depots; they take part in the phagocytosis and endocytosis of extracellular tissue components and therefore can be named phagocytes. Our data show that fibroblastoid endometrial stromal cells may differentiate into endometrial resident phagocytes, which ensure interstitial proteolysis and hence facilitate the drainage of extracellular fluid into the venous blood capillaries.Supported by a grant from the Nationaal Fonds voor Wetenschappelijk Onderzoek — Fonds voor Geneeskundig Wetenschappelijk Onderzoek, Belgium.     

Differentiation and regenerative capacities of human odontoma-derived mesenchymal cells

Regenerating human tooth ex vivo and biological repair of dental caries are hampered by non-viable odontogenic stem cells that can regenerate different tooth components. Odontoma is a developmental dental anomaly that may contain putative post-natal stem cells with the ability to differentiate and regenerate in vivo new dental structures that may include enamel, dentin, cementum and pulp tissues. We evaluated odontoma tissues from 14 patients and further isolated and characterized human odontoma-derived mesenchymal cells (HODCs) with neural stem cell and hard tissue regenerative properties from a group of complex odontoma tissues from 1 of 14 patients. Complex odontoma was more common (9 of 14) than compound type and females (9 of 14) were more affected than males in our set of patients. HODCs were highly proliferative like dental pulp stem cells (DPSCs) but demonstrated stronger neural immunophenotype than both DPSCs and mandible bone marrow stromal cells (BMSCs) by expressing higher levels of nestin, Sox 2 and βIII-tubulin. When transplanted with hydroxyapatite/tricalcium phosphate into immunocompromised mice, HODCs differentiated and regenerated calcified hard tissues in vivo that were morphologically and quantitatively comparable to those generated by DPSCs and BMSCs. When transplanted with polycaprolactone (biodegradable carrier), HODCs differentiated to form new predentin on the surface of a dentin platform. Newly formed predentin contained numerous distinct dentinal tubules and an apparent dentin–pulp arrangement. HODCs represent unique odontogenic progenitors that readily commit to formation of dental hard tissues.     

A role for cyclin D3 in the endomitotic cell cycle.

Platelets, essential for thrombosis and hemostasis, develop from polyploid megakaryocytes which undergo endomitosis. During this cell cycle, cells experience abrogated mitosis and reenter a phase of DNA synthesis, thus leading to endomitosis. In the search for regulators of the endomitotic cell cycle, we have identified cyclin D3 as an important regulatory factor. Of the D-type cyclins, cyclin D3 is present at high levels in megakaryocytes undergoing endomitosis and is markedly upregulated following exposure to the proliferation-, maturation-, and ploidy-promoting factor, Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the platelet lineage display a striking increase in endomitosis, similar to changes seen following Mpl ligand administration to normal mice. Electron microscopy analysis revealed that unlike such treated mice, however, D3 transgenic mice show a poor development of demarcation membranes, from which platelets are believed to fragment, and no increase in platelets. Thus, while our model supports a key role for cyclin D3 in the endomitotic cell cycle, it also points to the unique role of Mpl ligand in priming megakaryocytes towards platelet fragmentation. The role of cyclin D3 in promoting endomitosis in other lineages programmed to abrogate mitosis will need further exploration.     

Histochemical study of isolated intratubular cells of the rat testis

Summary The testes of normal white adult rats were studied with histochemical techniques for oxidative enzymes. Smears, imprints and squash preparations were used to obtain isolated cells.LDH, HDH and NADH₂-diaphorase rich granules were found close to the Golgi apparatus of spermatocytes.An increase in some enzymes was described in spermatids during the formation process of the mitochondrial helix. The differences in enzymatic properties of the mitochondrial helix and those of the cytoplasmic mitochondria suggest a process of selection and differentiation.Using a LDH-PMS medium, we found three loci of enzymatic activity, a diffuse cytoplasmic one, a Golgian activity, and a mitochondrial one. Applying Brody's method for the differentiation of LDH isozymes we demonstrated a preponderance of an isozyme non-sensible to urea or excess of lactate. This probably reflected the existence of the X-isozyme in the germinal line.All tested enzymes except MAO were positive in spermatozoa. The great activity of GDH and HDH in the middle piece provides a good link between the energetic metabolism of the spermatozoid and the oxidation of lipids.The enzymes of the Krebs' cycle, SDH, MDH and IDH were all present and we also demonstrated the presence of CO in the midpiece.G-6-PDH was present in moderate amounts.Great concentrations of these enzymes were found in Sertoli cells. Any conclusion about enzymatic distribution in sections must consider this fact.     

Characterization of filaments within Leydig cells of the rat testis

Rat Leydig cells were permeabilized and the cytoplasm partially extracted to visualize, describe, and characterize filamentous elements of the cytoskeleton. It was demonstrated by immunofluorescence microscopy that vimentin is abundant within Leydig cells. Ultrastructurally, intermediate filaments in Leydig cells were concentrated at perinuclear sites and comprised bundles that coursed through the cytoplasm. Actin was identified in Leydig cells with the F actin probe, NBD-phallacidin. Fluorescence was strongest at the cortex of the cell. With myosin S-1 subfragments, sparse actin was found positioned almost exclusively in cortical regions of the cell associated with coated pits and in Leydig cell processes.     

In-vitro proliferation of germ cells and supporting cells in the neonatal mouse testis

Summary Testicular cells were prepared from neonatal (48 h after birth) mice by enzymatic dissociation and were cultured in serum-supplemented medium to investigate cell proliferation in vitro. The cultured cells were composed mostly of germ cells, identified by immunocytochemistry using a germ cell-specific antiserum, and supporting (immature Sertoli) cells. After 36 h in culture, the cells were pulse-labeled with ³H-thymidine and fixed at 2-h intervals for 36 h after labeling. Numbers of labeled and unlabeled metaphases of germ cells and supporting cells were counted, and percent labeled metaphases for both cell types were determined for cell-cycle analysis. The results indicate that germ cells, as well as supporting cells, incorporate ³H-thymidine and progress through the cell cycle in vitro. From the curve of the percent labeled metaphases for the supporting cells, the total cell cycle and intervals of DNA synthesis were estimated to be 27.2 h and 13.2 h, respectively.     

Autoantigenic germ cells exist outside the blood testis barrier

Preleptotene spermatocytes and spermatogonia are germ cells located outside the blood-testis barrier provided by the Sertoli cells. These cells have been found to express autoantigens accessible to circulating antibodies. Mice immunized with syngeneic testis with or without bacterial adjuvant had detectable IgG on cells at the periphery of seminiferous tubules. Sera from orchiectomized but not from testes-intact mice immunized with testis and adjuvants readily transferred similar IgG deposits to testes of normal recipients. When testis-specific antisera from orchiectomized mice and testis-intact mice were compared for their reactivity on prepuberal testicular cells, serum from orchiectomized donors had significantly higher reactivity. Ig was eluted from IgG-positive testes with acid buffer and was shown to be highly enriched in antibody to prepuberal testicular cells, confirming the Ag-specific nature of the IgG deposits. The testis IgG deposits reacted with antisera to IgG1 and IgG3 but not IgG2a or IgG2b. This finding can explain lack of association of C3 in the deposits. Only 30 to 40% of seminiferous tubules had IgG deposits and they coincided with stages 7 to 12 of the spermatogenic cycle. Thus, the expression of the autoantigens is stage specific. The in situ formation of immune complexes by circulating autoantibodies demonstrates conclusively that testis autoantigens are not completely sequestered, and the blood-testis barrier as an immunologic barrier is incomplete.     

Functional analysis of stem cells in the adult rat testis

Adult stem cells maintain several self-renewing systems and processes in the body, including the epidermis, hematopoiesis, intestinal epithelium, and spermatogenesis. However, studies on adult stem cells are hampered by their low numbers, lack of information about morphologic or biochemical characteristics, and absence of functional assays, except for hematopoietic and spermatogonial stem cells. We took advantage of the recently developed spermatogonial transplantation technique to analyze germ line stem cells of the rat testis. The results indicate that the stem cell concentration in rat testes is 9.5-fold higher than that in mouse testes, and spermatogenic colonies derived from rat donor testis cells are 2.75 times larger than mouse-derived colonies by 3 mo after transplantation. Therefore, the extent of spermatogenesis from rat stem cells was 26-fold greater than that from mouse stem cells at the time of recipient testis analysis. Attempts to enrich spermatogonial stem cells in rat testis populations using the experimental cryptorchid procedure were not successful, but selection by attachment to laminin-coated plates resulted in 8.5-fold enrichment. Spermatogonial stem cells are unique among adult stem cells because they pass genetic information to the next generation. The high concentration of stem cells in the rat testis and the rapid expansion of spermatogenesis after transplantation will facilitate studies on stem cell biology and the introduction of genetic modifications into the male germ line. The functional differences between spermatogonial stem cells of rat vs. mouse origin after transplantation suggest that the potential of these cells may vary greatly among species.