The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.     

HSP70 is part of the synaptonemal complex in Eimeria tenella

In the current study the expression and ultrastructural localization of heat shock protein 70 (HSP70) was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from Eimeria tenella oocysts. The authors used a previously reported method that overcomes the difficulties of the resistance of Eimeria oocysts to disruption and permits the release of intact meiotic chromosomes. HSP70 was localized at the ultrastructural level using an anti-HSP70 monoclonal antibody in combination with a secondary antibody coupled to colloidal gold. Synaptonemal complexes (SCs) were visualized by means of the surface spreading technique to study both HSP70 expression and the consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. For that purpose E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. The results showed a close association of HSP70 with the lateral elements (LEs) of the SCs. That association began at the time that SCs were formed and persisted until disassemble. Comparison between distribution of immunogold label over the SCs from non-treated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased. The current results demonstrated three dose-dependent effects of the quercetin treatment of Eimeria oocysts: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis, and inhibition of sporulation. HSP70, as a structural component of the SCs, may be involved in SC functions such as chromosomal pairing, recombination, or disjunction.     

Mammalian protein SCP1 forms synaptonemal complex-like structures in the absence of meiotic chromosomes

Synaptonemal complexes (SCs) are evolutionary conserved, meiosis-specific structures that play a central role in synapsis of homologous chromosomes, chiasmata distribution, and chromosome segregation. However, it is still for the most part unclear how SCs do assemble during meiotic prophase. Major components of mammalian SCs are the meiosis-specific proteins SCP1, 2, and 3. To investigate the role of SCP1 in SC assembly, we expressed SCP1 in a heterologous system, i.e., in COS-7 cells that normally do not express SC proteins. Notably, under these experimental conditions SCP1 is able to form structures that closely resemble SCs (i.e., polycomplexes). Moreover, we show that mutations that modify the length of the central alpha-helical domain of SCP1 influence the width of polycomplexes. Finally, we demonstrate that deletions of the nonhelical N- or C-termini both affect polycomplex assembly, although in a different manner. We conclude that SCP1 is a primary determinant of SC assembly that plays a key role in synapsis of homologous chromosomes.     

Protein SYCP2 provides a link between transverse filaments and lateral elements of mammalian synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved meiosis-specific nuclear structures critically involved in synapsis, recombination, and segregation of homologous chromosomes. SCs are proteinaceous structures composed of (a) two lateral elements (LEs), to which the chromatin of the homologs is attached, (b) numerous transverse filaments (TFs) that link the LEs, and (c) a central element (CE). Major protein components of mammalian SCs are the TF protein SYCP1 and the LE proteins SYCP2 and SCYP3. How SCs become assembled is presently poorly understood, in particular, it is not known how TFs assemble at the plane of LEs to interconnect the homologous chromosomes. Therefore, we have investigated possible interactions between SYCP1 and other SC proteins. In immunoprecipitation experiments we could find that SYCP1 and SYCP2 interact in extracts of meiotic cells. Using the yeast two-hybrid system, we were able to demonstrate that the C-terminus of SYCP1 directly interacts with SYCP2. These results were confirmed by different interaction traps. Furthermore, we could narrow down the interacting domain of the SYCP2 molecule to its C-terminal region. We propose that SYCP2 acts as a linker between SYCP1 and SYCP3 and therefore would be the missing connecting link between LEs and TFs essential for proper chromosome synapsis.     

SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat.

In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.     

Synaptonemal complexes of the A- and B-chromosomes of the spermatocytes from the East Asian mouse Apodemus peninsulae

The analysis of whole-mount preparations of synaptonemal complexes (SCs) from surface-spread spermatocytes of A. peninsulae (2n = 48A + 1, 2, ... 12 B) had revealed SCs of 23 autosomal bivalents, sex bivalent XY, axial cores and SCs of the B-chromosomes. The intercellular and interindividual variability of the number of B-chromosomes varied from 1 to 12 per cell. The SCs of autosomal bivalents were shown to have a typical structure. The structure and behaviour of SCs of sex bivalent throughout meiotic prophase I appeared to be similar to those observed in other species of this order. Mainly B-univalents and less frequently B-bivalents containing SCs were found to be formed in meiotic prophase I. The full homologues appear to be rarely seen among B-chromosomes of the East-Asiatic mouse. A tendency of forming clusters of B-univalents near the sex bivalent was found, in addition to B-bivalents with lateral elements, having the form of bi- and tri-stranded elements with rare synaptic fragments. Besides this, the SCs of the autosomes of pachytene cells were found to contain structures resembling the recombination nodules.     

Two major components of synaptonemal complexes are specific for meiotic prophase nuclei

Monoclonal antibody II52F10 was elicited against purified synaptonemal complexes (SCs); it recognizes two major components of the lateral elements of SCs, namely an M_r=30 000 and an M_r=33000 protein. We studied the distribution of the antigens of II52F10 within tissues and cells of the male rat by immunoblot analysis and immuno-cytochemical techniques. Nuclear proteins from various cell types, including spermatogonia and spermatids, did not react with antibody II52F10 on immunoblots; the same holds for proteins from isolated mitotic chromosomes. As expected, an M_r=30 000 and an M_r=33 000 protein from spermatocyte nuclei did react with the antibody. In cryostat sections of liver, brain, muscle and gut we could not detect any reaction with II52F10. In the testis the reaction was confined to SCs or SC fragments. Partly on the basis of indirect evidence we identified the antigen-containing cells as zygotene up to and including post-diffuse diplotene spermatocytes. The persistence of some antigen-containing fragments in the earliest stages of spermatids could not be excluded. We conclude that the lateral elements (LEs) of SCs are not assembled by rearrangement of pre-existing components of the nucleus: at least two of their major components are newly synthesized, presumably during zygotene. Furthermore we conclude partly from indirect evidence that the major components of the LEs of SCs are not involved in the chromosome condensation processes that take place during the earliest stages of meiotic prophase.Abbreviations BSA bovine serum albumin - CE central element - FITC fluorescein isothiocyanate - LE lateral element - PBS phosphate-buffered saline (140 mM NaCl, 10 mM sodium phosphate, pH 7.3) - SC synaptonemal complex - TBST Tris-buffered saline with Tween (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.05% Tween-20)     

Mouse SYCP2 is required for synaptonemal complex assembly and chromosomal synapsis during male meiosis

During meiosis, the arrangement of homologous chromosomes is tightly regulated by the synaptonemal complex (SC). Each SC consists of two axial/lateral elements (AEs/LEs), and numerous transverse filaments. SC protein 2 (SYCP2) and SYCP3 are integral components of AEs/LEs in mammals. We find that SYCP2 forms heterodimers with SYCP3 both in vitro and in vivo. An evolutionarily conserved coiled coil domain in SYCP2 is required for binding to SYCP3. We generated a mutant Sycp2 allele in mice that lacks the coiled coil domain. The fertility of homozygous Sycp2 mutant mice is sexually dimorphic; males are sterile because of a block in meiosis, whereas females are subfertile with sharply reduced litter size. Sycp2 mutant spermatocytes exhibit failure in the formation of AEs and chromosomal synapsis. Strikingly, the mutant SYCP2 protein localizes to axial chromosomal cores in both spermatocytes and fetal oocytes, but SYCP3 does not, demonstrating that SYCP2 is a primary determinant of AEs/LEs and, thus, is required for the incorporation of SYCP3 into SCs.     

Synaptonemal complex proteins

Synaptonemal complexes were isolated from rate spermatocytes for the purpose of biochemical and morphological analysis. Several monoclonal antibodies were elicited against purified synaptonemal complexes to study the composition and assembly of these structures. Four classes of antibodies could be discriminated according to the polypeptides that they recognize on Western blots of purified synaptonemal complexes, namely antibodies recognizing (i) a 190-kDa polypeptide; (ii) a 30- and a 33-kDa polypeptide; (iii) two polypeptides with molecular weights of about 120 kDa; and (iv) polypeptides with molecular weights of 66-55 kDa. The localization of these antigens within spermatocytes was analyzed light microscopically, by means of the immunoperoxidase technique and ultrastructurally, by immunogold labelling of surface-spread spermatocytes. The 66- to 55-kDa polypeptides are not confined to synaptonemal complexes; rather, these polypeptides appear to be chromosomal components. The 190-, 30-, and 33-kDa polypeptides make part of the lateral elements of paired as well as unpaired segments of synaptonemal complexes. The 120-kDa polypeptides were localized on the inner edge of the lateral elements, specifically in paired segments of synaptonemal complexes. The distribution of the 190-, 120-, 30-, and 33-kDa polypeptides within the testis was analyzed by immunofluorescence staining of cryostat sections. All these polypeptides turned out to be specific for nuclei of zygotene up to and including diplotene spermatocytes. Only in some early spermatids could the 190-, 30-, and 33-kDa polypeptides be detected, presumably in remnants of synaptonemal complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two major components of the lateral elements of synaptonemal complexes of the rat

This paper describes the identification of two major components of the lateral elements of synaptonemal complexes of the rat by immunocytochemical techniques. We prepared monoclonal antibodies against synaptonemal complexes (SCs) by immunization of mice with purified SCs. One of these antibodies, II52F10, reacts with a 30 and a 33 kDa polypeptide, which are major components of purified SCs. Using this antibody, we studied the localization of its antigens light microscopically, by means of the indirect immunoperoxidase technique, as well as ultrastructurally, by means of the immunogold labeling technique. The immunolocalization was carried out on whole-mount preparations of lysed spermatocytes. The antibody reacts with paired as well as unpaired segments of zygotene, pachytene and diplotene SCs. In light microscopic preparations, the attachment plaques, particularly those of late pachytene and diplotene SCs, also appear to react strongly. In electron micrographs the lateral elements in paired as well as unpaired segments could be seen to react. No reaction was observed in the attachment plaques; however, in late pachytene and diplotene SCs the swollen terminal segments of the lateral elements did react with the antibody. Thus, we conclude that a 30 and a 33 kDa polypeptide make part of the lateral elements of synaptonemal complexes of the rat.     

Identification of aberrant chromosomes based on the morphometry of the synaptonemal complexes in a sterile male mouse

The synaptonemal complexes (SCs) of surface-spread spermatocytes of male mouse from the F1 progeny of a male exposed to a mutagen have been examined by electron microscopy. Nonreciprocal translocation was recognised in analysing configuration of SC. Electron microscope analysis revealed translocation in 100% pachytene spermatocytes and light microscope analysis of air-dried metaphase spermatocytes demonstrated this in 58% cells. Different types of association of X-chromosome with aberrant chromosomes were discovered in pachytene spermatocytes. Computer analysis of relative length of SCs permits to detect a nonreciprocal translocation from chromosome 4 to chromosome 16. The length of the translocated fragment was determined to be from 66 to 75% of the length of chromosome 4. It has been impossible to discover a telomere fragment of chromosome 16, because the break point of chromosome 16 is too close to the distal end.     

Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells

The intracellular sites where proteolytic processing of pro-ACTH/endorphin or POMC take place have not yet been reliably identified. We have used affinity-purified antisera that recognize only the products of POMC processing and immunoelectron microscopy to identify the compartments of rat pituitary corticotropes and mouse AtT-20 cells in which cleavage occurs. Immunoperoxidase labeling of cryostat sections and immunogold labeling of ultrathin frozen sections were used for localization of the processing sites. By both procedures we detected processed peptides in Golgi cisternae and secretion granules. Within the Golgi, labeling was limited to the last or transmost cisterna and was most concentrated in its dilated rims which contain condensing secretory protein. No labeling of other Golgi cisternae was seen. All Golgi cisternae were labeled, however, when antisera that recognize unprocessed POMC were used for immunolabeling. We conclude that in AtT-20 and rat pituitary cells: 1) processing of POMC through at least two endo- and exoproteolytic cleavage steps and alpha-amidation of joining peptide begin in the trans Golgi subcompartment; 2) no detectable processing takes place before POMC reaches the trans Golgi cisterna; and 3) this Golgi cisterna as well as secretion granules contain the active enzymes necessary for proteolytic processing and alpha-amidation.     

Peculiarities of chromosome organization in meiosis

Meiotic and mitotic chromosomes have a complex of differences. (1) At the early prophase I of meiosis, chromosomes acquire protein axial elements (AEs) that were absent in mitosis; in addition to somatic cohesins, AEs contain the meiosis-specific cohesins REC8, SMC1β, and STAG3. (2) At the middle prophase I, protein lateral elements (LEs) of synaptonemal complexes (SCs) are formed on the basis of AEs. The LE proteins are not conserved, but in Saccharomyces cerevisiae and Arabidopsis thaliana they contain functional domains with conserved secondary structures. Among the almost 679 thousand proteins of primitive eukaryotes that we studied by bioinformatics methods, in green and brown algae, some lower fungi, and Coelenterata, we revealed proteins or functional domains similar to SC proteins. (3) During the pachytene and diplotene stages of meiosis, chromosomes of spermatocytes and mother pollen cells acquire a general structure resembling the structure of amphibian and avian lampbrush chromosomes in miniature. Lateral chromatin loops with sizes of 90, 160, and even over 480 Kb were observed in human spermatocytes during the diplotene stage. In combination, all these observations confirm the considerable conservation of the scheme of molecular and ultrastructural organization of meiotic chromosomes in a large variety of eukaryotic organisms.     

肌动蛋白存在于金黄地鼠(Mesocricetus auratus)联会复合体中

以金黄地鼠精母细胞为材料,以抗肌动蛋白抗体为探针,应用免疫荧光和免疫胶体金技术对ＳＣ有无肌动蛋白的问题进行了研究。免疫荧光结果表明：经抗肌动蛋白抗体标记后,减数分裂粗线期标本中ＳＣ发出特异性荧光,说明肌动蛋白存在于ＳＣ中。免疫电镜结果表明：实验组ＳＣ的胶体金颗粒密度远高于对照组的金颗粒密度,说明ＳＣ含有肌动蛋白。观察到,常染色体ＳＣ和性染色体ＳＣ以及偶线期和粗线期ＳＣ中都含有肌动蛋白,肌动蛋白分布于ＳＣ的端部和侧生组分上,代表肌动蛋白的胶体金颗粒在ＳＣ上往往成簇存在。对ＳＣ含有肌动蛋白的意义进行了讨论。     

The sequential appearance of components of the synaptonemal complex during meiosis of the female rat.

This paper describes the light microscopy (LM) and electron microscopy (EM) localization of synaptonemal complex (SC) antigens in oocytes of rats. For this purpose, we used monoclonal antibodies (Mabs) that recognize components of 30 + 33, 125, and 190 kDa antigens of SCs of rat spermatocytes. The LM localization was performed by immunofluorescence and the EM localization by immunogold staining. The reaction of the Mabs with oocytes was similar to the reaction with spermatocytes, but weaker. The 30 + 33 kDa as well as the 190 kDa antigens could always be demonstrated if axial elements of the SC were present, irrespective of whether these were paired or unpaired. Thus, these antigens could be detected from leptotene--early zygotene until diplotene. The 190-kDa antigen appeared in a diffuse manner just before the appearance of the 30 + 33 kDa antigens. The 30 + 33 kDa antigens were not only detected in the axial elements of SCs but also in characteristic aggregates, which appeared in zygotene and persisted until after the SCs had disappeared. Such aggregates had rarely been observed in spermatocytes. The 125 kDa antigen was only present in the tripartite segments of SCs, at the inner edge of the lateral elements. Thus, the reaction of the Mab against the 125 kDa antigen was detectable in zygotene, pachytene, and very early diplotene. It appeared later than 30 + 33 kDa and 190 kDa antigens and it disappeared earlier. We found that several steps of the immunostaining procedure could cause variation in the intensity of the Mab reaction.     

Silver staining of synaptonemal complexes in surface spreads for light and electron microscopy

Synaptonemal complexes (SCs), axes of the X and Y chromosomes, and nucleoli in surface-spread preparations of spermatocytes are selectively stained for both light and electron microscopy by ammoniacal silver. Combined with a simple technique for transferring material from glass slides to grids, sequential light and electron microscopic analysis of full SC complements is possible with no further preparation. This new method has potential for both basic and clinical cytogenetic research.     

小鼠精母细胞联会复合体RNA组分的电镜研究

本文运用常规染色和Bernhard染色方法对切片标本中小鼠粗线期精母细胞联会复合体(SC)的超微结构和电镜细胞化学特点进行了研究。经常规染色后,可见SC由侧生组分(LE)、中央组分(CE)和L-C纤维组成;SC宽约210nm,LE宽约60nm,中央间隔区宽约90nm。在Bernhard染色标本中,SC的LE、CE和L-C纤维着色较深,说明其中含有RNA;SC各结构组分的宽度和形态特点与常规染色标本中的基本一致。本文讨论了SC中存在有RNA等问题。