In vivo regulation of the early embryonic cell cycle in Xenopus

We report here the first extensive in vivo study of cell cycle regulation in the Xenopus embryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.     

The lurcher mutation and ionotropic glutamate receptors: contributions to programmed neuronal death in vivo

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34(cdk1) complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.     

Cloning and sequence analysis of two cDNAs encoding cyclin A and cyclin B in the zebra mussel Dreissena polymorpha

Cyclins are key components in the progression of both mitotic and meiotic cell cycle control. Full-length cDNA clones encoding cyclin A and cyclin B were isolated from a zebra mussel testis cDNA library. The clones contained open reading frames of 419 and 434 amino acids, had similarity to cyclins A and B from other species, but also some unique features in their sequences. Cyclin A and B mRNA was expressed in testis, ovary, gill, mantle, muscle, and eggs, as shown by specific polymerase chain reaction.     

Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells

Pituitary tumor-transforming gene (PTTG) is a recently characterized proto-oncogene that is expressed specifically in adult testis. In this study, we have used in situ hybridization and developmental Northern blot assays to demonstrate that PTTG mRNA is expressed stage-specifically in spermatocytes and spermatids during rat spermatogenic cycle. We have used the yeast two-hybrid system to identify proteins that interact with PTTG in testicular cells. Two positive clones were characterized. One of the clones is the ribosomal protein S10, the other encodes a novel human DnaJ homologue designated HSJ2. Northern blot analysis showed that testis contains higher levels of HSJ2 mRNA than other tissues examined, and the expression pattern of HSJ2 mRNA in postnatal rat testis is similar to PTTG. S10 mRNA levels do not vary remarkably among different tissues and remains unchanged during testicular germ cell differentiation. In vitro binding assays demonstrated that both S10 and HSJ2 bind to PTTG specifically and that PTTG can be co-immunoprecipitated with S10 and HSJ2 from transfected cells. Moreover, the binding sites for both proteins were located within the C-terminal 75 amino acids of the PTTG protein. These results suggest that PTTG may play a role in spermatogenesis.     

Ontogeny of estrogen receptor-beta expression in rat testis

The recently discovered estrogen receptor-beta (ERbeta) is expressed in rodent and human testes. To obtain insight in the physiological role of ERbeta we have investigated the cell type-specific expression pattern of ERbeta messenger RNA (mRNA) and protein in the testis of rats of various ages by in situ hybridization and immunohistochemistry. In fetal testes of rats 16 days postcoitum and testes of 4-day-old animals, fetal germ cells (gonocytes) reveal the ERbeta mRNA in their cytoplasm and the ERbeta protein in their nucleus. In testes of 11- and 15-day-old rats, ERbeta mRNA and protein were detected in Sertoli cells and type A spermatogonia. No signal was found in other types of germ cells. In the adult testes, expression of ERbeta mRNA as well as ERbeta protein was found in pachytene spermatocytes from epithelial stages VII-XIV and in round spermatids from stages I-VIII. Low ERbeta expression was observed in all type A spermatogonia, including undifferentiated A spermatogonia, whereas no expression was found in In and type B spermatogonia and early spermatocytes. At all ages, Sertoli cells showed a weak hybridization signal as well as weak immunoreactivity for ERbeta. In adult testes, no ERbeta mRNA or protein was detected in the interstitial tissue, indicating that Leydig cells and peritubular myoid cells do not express ERbeta. The expression of ERbeta in fetal and late male germ cells as well as in Sertoli cells suggests that estrogens directly affect germ cells during testicular development and spermatogenesis.     

Differential responses of the heart and vasculature to chronic blood pressure reduction in essential hypertension

In the current study, localization of D-aspartic acid (D-Asp) in rat testis was studied by immunohistochemical and biochemical techniques. Immunohistochemical staining of this tissue using specific polyclonal antibody to D-Asp revealed D-Asp immunoreactivity (IR) in the cytoplasm of germ cells, especially around the region rich in elongate spermatids, the most mature of the germ cells. Weak IR was also noted in cytoplasm of spermatocytes and round spermatids; however, it was negligible in interstitial cells and Sertoli cells. The intensity of immunostaining in each seminiferous tubule differed according to its distinct germ cell composition. In testis of young rats, seminiferous tubules lack elongated spermatids, and D-Asp was found to be localized in spermatocytes, the most mature population of germ cells at that age. We used various toxicants to destroy specific testicular cell populations and to confirm the localization of D-Asp in rat testis. Administration of ethane dimethane sulfonate induced a selective destruction of all Leydig cells in this tissue. This resulted in a significant decrease in the D-Asp level, which was probably due to a drop in testosterone brought about by this treatment, and this was followed by a modulation of spermatogenesis. Three days after treatment with methoxyacetic acid (MAA), many seminiferous tubules were found to lack or to have severe depletions of pachytene spermatocytes, but not of elongate spermatids. This caused reductions in protein content and in the total amount of L-Asp, but not that of D-Asp. Twenty days after treatment with MAA, the depleted population of germ cells progressed through the spermatogenic cycle from pachytene spermatocytes to elongate spermatids. At this time, the level of D-Asp decreased significantly, as did that of L-Asp and protein, consistent with D-Asp localization in elongate spermatids. This decrease in the D-Asp level was also seen with immunostaining.     

A novel stage-specific differentiation antigen is expressed on mouse testicular germ cells during early meiotic prophase

A murine cell surface antigen exhibiting stage-specific expression during spermatogenesis was detected with two monoclonal antibodies (mAbs), designated BC7 and CA12. In mouse testis, these mAbs recognized a small population of cells located near the periphery of seminiferous tubules at stages XII and I-VI, and these spermatogenic cells were identified as zygotene and early pachytene spermatocytes. Expression of the antigens was transient and was not detected in germ cells at more advanced stages of spermatogenesis such as late pachytene spermatocytes and round spermatids. Immunoprecipitation and immunoblotting studies showed that both mAbs CA12 and BC7 reacted with the same antigenic molecule, which had an estimated molecular mass of 95 kDa. CA12/BC7 antigen, detected in plasma membrane fraction, was a glycoprotein with sialic acid residues and had affinity with WGA lectin. Furthermore, intraperitoneal injection of mAb BC7 caused an apparent spermatogenic disturbance in prepubertal mice. These results suggested that CA12/BC7 antigen, a novel cell surface glycoprotein, is an essential molecule that plays an important role during early meiotic prophase of spermatogenesis.     

Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation

The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.     

Differential expression of fibronectin, tenascin-C and NCAMs in cultured hippocampal astrocytes activated by kainate, bacterial lipopolysaccharide or basic fibroblast growth factor

We have cloned a mouse cDNA encoding a Mothers-against-dpp (MAD)-related protein, MADR1. Madr1 is ubiquitously expressed in the mouse embryo, indicating a broad function in a variety of tissue during embryogenesis, potentially relaying signals of numerous BMPs. However, its expression in the testis is strictly germ cell-specific and developmentally regulated. Testicular Madr1 expression starts in some seminiferous tubules at 2 weeks of age. After mid-puberty, a stage-specific Madr1 expression is established. During the cycling of the seminiferous epithelium, Madr1 expression initiates in the pachytene spermatocytes of stage V seminiferous tubules, peaks at stage X, then decreases as pachytene spermatocytes differentiate into secondary spermatocytes and then round spermatids. In the testis of adult Bmp8b homozygous mutant males, the Madr1- expressing pachytene spermatocytes are the first cell population to show increased apoptosis. These data suggest that MADR1 serves as a downstream component of the BMP8 signaling pathway during the differentiation of meiotic male germ cells.     

BRCA1 mRNA is expressed highly during meiosis and spermiogenesis but not during mitosis of male germ cells

The 17q-linked breast and ovarian cancer susceptibility gene (BRCA1) is believed to function as a tumor suppressor gene (Miki et al., 1994). In this report BRCA1 RNA expression has been analysed in adult mouse tissues with detailed attention to its expression in prepuberal and adult testis. Measurements of BRCA1 mRNA levels in highly purified somatic cells of the testis and in staged germ cells showed that high level BRCA1 mRNA expression is limited to the germ cells. Within the germ cell lineage, the high level expression was detected in meiotic cells, specifically pachytene spermatocytes and in post-meiotic round spermatids. This is in contrast to premeiotic germ cells which were found to express little or no BRCA1 mRNA. These observations, considered together with recent data on the expression of BRCA1 in breast epithelium, argues against a function for BRACA1 in early progenitor cells in both tissues and cells attention instead to roles intimately associated with terminal differentiation or with final rounds of cell division.     

cDNA cloning and characterization of a rat spermatogenesis-associated protein RSP29

RSP29, a protein secreted by rat round spermatids, stimulates the secretory function of Sertoli cells in the testis. By making use of the N-terminal sequence homology of RSP29 and a human protein hDP1 that we had previously isolated, we cloned the full length cDNA sequence that encodes RSP29. The entire amino acid sequence of RSP29 showed significant homology with that of hDP1, which was later identified as glyoxalase II. Southern analysis showed that the RSP29 protein sequence is highly conserved in eukaryotes and possibly in prokaryotes. The RSP29 mRNA is expressed in many tissues but has an extremely high abundance in testis. These data suggest that RSP29 may have an important function in most tissues of enkaryotic organisms. The high expression of RSP29 in testis and its stimulatory effects on Sertoli cells suggest that RSP29 could be especially important in the regulation of spermatogenesis.     

Characterization of domains in mice of calnexin-t, a putative molecular chaperone required in sperm fertility, with use of glutathione S-transferase-fusion proteins

Calnexin-t (calmegin) is a male germ cell-specific variant of calnexin, a membrane bound-molecular chaperone in the endoplasmic reticulum (ER). Although it is temporally expressed during spermatogenesis, it has recently been shown to be highly involved in sperm fertility. To investigate the biochemical states of calnexin-t during spermatogenesis, we produced a series of glutathione S-transferase-fusion proteins with several specific coding domains of calnexin-t. Immunostaining and 45Ca2+ overlay assays clearly showed that the internal proline-rich repeat region has Ca2+-binding ability and contains an epitope recognized by monoclonal antibody 1C9. Western blot analysis of protein extracts from the testes of 10-, 18-, 26-, and 60-day-old mice revealed only a single 101-kDa protein during testicular development by 1C9. Anti-C, a cytoplasmic domain-specific antibody generated by immunization with recombinant protein, produced the same results, indicating that the 101-kDa form of calnexin-t is prevalent at all stages of spermatogenesis expressing calnexin-t. In paraffin sections of mouse testis, Anti-C stained spermatocytes and spermatids intensely, whereas 1C9 stained spermatocytes only slightly but spermatids intensely, suggesting that the affinity of 1C9 for its epitope is lower in pachytene spermatocytes than in spermatids. Acid phosphatase treatment of the 101-kDa form generated a 93-kDa band that in turn could be recovered to the 101-kDa form by incubation with HeLa cell S100 fraction, indicating that the 101-kDa form is a phosphorylated type of calnexin-t. The sites of phosphorylation were shown to be restricted to the cytoplasmic domain. Our results suggest that the structure of the ER luminal domain of calnexin-t is likely to differ in middle pachytene versus haploid germ cell phases. In addition, the cytoplasmic domain of calnexin-t was shown to be highly phosphorylated immediately after protein synthesis and constitutively phosphorylated during spermatogenesis.     

Increased cyclin D1 in vulnerable neurons in the hippocampus after ischaemia and epilepsy: a modulator of in vivo programmed cell death?

Several observations suggest that delayed neuronal death in ischaemia, epilepsy and other brain disorders includes an apoptotic component, involving programmed cell death (PCD). PCD is hypothesized to result, in part, from aberrant control of the cell cycle. Because they are instrumental in mitosis, cyclins D are key markers to evaluate whether neurons indeed progress into the cell cycle in situations of pathology. Therefore, we investigated in rat brains, the expression of cyclins D in the delayed neuronal death that occurs following transient global ischaemia and kainate-induced seizures. Following a four-vessel occlusion insult, quantitative in situ hybridization revealed a highly significant and persistent 100% increase of cyclin D1 mRNA in the vulnerable pyramidal neurons of the CA1 hippocampal region. Ischaemia also induced a smaller and transient cyclin D1 mRNA increase in the resistant CA3 area and dentate gyrus. In contrast, the cyclin D2 and D3 mRNAs, expressed constitutively in the adult rat hippocampus, were not upregulated. Following kainate-induced seizures, cyclin D1 mRNA was induced in the vulnerable CA3 region, and to a lesser extent, in non-vulnerable regions. Cyclin D1 immunohistochemistry revealed increased protein levels in the cytoplasm and nucleus of neurons commited to die after ischaemia. Double labelling experiments indicate that cyclin D1 is also expressed in reactive astrocytes but not in microglial cells. Finally, we report that in neurons, cyclin D1 expression peaks before nuclear condensation and the appearance of DNA fragmentation. We propose that cyclin D1, when expressed at high levels in lesioned neurons, may act as a modulator of apoptosis.