Selective advantage of euploid spermatocytes I in an azoospermic 47,XYY man with gonadal mosaicism.

Although most XYY men have normal sperm counts and are fertile (supposedly due to the loss of the extra Y before meiosis), there is a minority who are infertile. In these cases, the XYY spermatocytes are able to enter meiosis and form different synaptic configurations. With regard to mosaics, there is scarce well-defined information on the presence of the second Y and its meiotic behaviour. In this study, the chromosome constitution and the synaptic behaviour of pachytene spermatocytes from an azoospermic man with testicular hypotrophy and non-mosaic 47,XYY karyotype were analysed. Furthermore, we determined the chromosome constitution of the somatic Sertoli cells. Five karyotypically normal men with obstructive azoospermia, but having complete spermatogenesis, were included as controls. Immuno-FISH using specific protein markers of synapsis and recombination (SYCP3, SYCP1, BRCA1, MLH1, CREST) and a specific Yq12 DNA probe were used. In addition, we used the newly developed Super-Resolution Structured Illumination Microscopy (SR-SIM) to clearly define the synaptic configurations. FISH analysis was also performed on Sertoli cells. The histopathological analysis showed variable degrees of spermatogenesis development in the testicular tissue of the propositus. Immuno-FISH analysis showed that most of the primary spermatocytes were euploid 46, XY. The use of SR-SIM confirmed the existence of this euploidy. Only a few pachytene spermatocytes showed an aneuploid X + YY constitution. Sertoli cells showed two different populations with one or two Y chromosomes, in similar proportions. Thus an abnormal niche of sex-trisomic Sertoli cells should be also considered when searching for the origin of spermatogenesis failure in XYY men.

The role of asynapsis in human spermatocyte failure.

The basic molecular mechanisms by which chromosomal rearrangements in heterozygous state produce spermatogenic disturbances are poorly understood. Testicular biopsies from five patients - one carrier of a Robertsonian translocation rob t(13;14), two carriers of two different Y-autosome translocations, a t(Y;6) and a t(Y;11), one carrier of a reciprocal translocation t(3;13) and one carrier of a heterochromatin duplication in chromosome 9 - were processed for histopathological analysis, electron microscopy and fluorescent immunolocalization of meiotic proteins. In all the patients, the asynaptic regions during pachytene are labelled by BRCA1 and retained RAD51 foci. The variant histone γ-H2AX is located on the chromatin domains of the asynaptic regions and the XY body. In contrast, these meiotic proteins are absent in those chromosomal segments that are non-homologously synapsed. The present observations on five new cases and a review of recent studies show that the common features shared by all these cases are the abnormal location of some meiotic proteins and the presence of transcriptionally silenced chromatin domains on asynaptic regions. The frequent association of these silenced regions with the XY body and the rescue of spermatocyte viability through non-homologous synapsis are also shared by all these carriers. A passive, random mechanism of clustering of asynaptic regions with the XY body is suggested.

Focal spermatogenesis originates in euploid germ cells in classical Klinefelter patients.

BACKGROUND: Klinefelter syndrome is the most frequent chromosome abnormality in human males. This paper aims to investigate the ploidy of meiotic and pre-meiotic germ cells found in spermatogenic foci, and furthermore, the sex chromosome constitution of Sertoli cells which surround these germ cells in non-mosaic Klinefelter patients. METHODS AND RESULTS: A survey of 11 adult patients diagnosed with classical, non-mosaic Klinefelter syndrome who underwent testicular biopsies, showed that six of them had spermatogenesis foci. The topographical study of the biopsies showed that tubuli with germ cells are a minor fraction (8-24%) of all tubuli, although the overwhelming majority is devoid of germ cells. Using fluorescence in situ hybridization (FISH) with probes for the X-centromere and immunolocalization of meiotic proteins, the present work shows that all the 92 meiotic spermatocytes analyzed with FISH were euploid, 46,XY, and thus can form normal, haploid gametes. On the other hand, Sertoli cells show two marks for the X chromosome, meaning that they are 47,XXY. CONCLUSIONS: These results provide a rationale for the high rate of success in the testicular sperm extraction plus ICSI procedures when applied to Klinefelter patients. It is also in agreement with previous studies in the XXY-mouse model. These spermatogenic foci most probably originate from clones of spermatogonia that have randomly lost one of the X chromosomes, probably during periods of life when high spermatogonial mitotic activity occurs.

Nucleolar orthophosphate ions. Electron microscope and diffraction studies.

Lead acetate (3-10%, pH between 4.3 and 7.0, alone or containing 2% glutaraldehyde), when used as fixative, has been demonstrated to produce an intracellular microcrystalline precipitate of lead orthophosphate, Pb(5)(PO(4))(3)OH (lead hydroxyapatite). This confirms earlier work with the light microscope (6). In interphase cells the nucleoli are sharply delimited by the massive lead phosphate precipitate. Some diffuse precipitate is found in the nucleoplasm; it is always delimited by the nuclear membrane. Nucleolar localization of this orthophosphate pool is not a diffusion artifact; the pool is probably in a loosely bound state and is not retained by conventional fixatives. In maize root cells in advanced mitotic stages the lead phosphate crystals are seen distributed throughout the cytoplasm and also relatively concentrated on the late anaphase-early telophase chromosomes. This pool of inorganic phosphate anions may be involved in the mitotic cycle of chromatin condensation, and it may be partially responsible for the absence of mature ribosomes in the nucleolus through the chelation of divalent cations. It is evident that the siver-reducing component detected in the nucleoli of fixed cells (6) is a completely different substance.

The three-dimensional reconstruction of the XY chromosomal pair in human spermatocytes.

The spatial reconstruction of the XY pair of chromosomes from human spermatocytes has been made by the study of serial sections 1000 A in thickness. The sex pair during zygotene-pachytene forms a condensed mass of chromatin that has two filamentous, electron-opaque cores: the long and the short core. During early pachytene both cores have a common ending region, about 0.4-0.8 micro long. This common end is a synaptonemal complex, and each of the cores forms a lateral element of that complex. The cores become more convoluted during middle pachytene forming "ringlike bodies." Nucleoli from spermatocytes have three distinct regions: (a) granular; (b) dense fibrillar; and (c) clear intermediate. Occasional association of the XY pair and the heteropycnotic "basal knobs" results in apparent association of nucleoli and the sex pair in a minority of cells. The evidence presented is interpreted as a strong support of the hypothesis of homologous regions in the human XY pair.

The structure of the central region in the synaptonemal complexes of hamster and cricket spermatocytes.

The fine structure of bivalents from golden hamster and house cricket spermatocytes has been studied with a whole mount surface-spreading method combined with negative staining. The elements of the synaptonemal complex show detail of structure which is absent in other preparative procedures. The transverse filaments found in the central region of the synaptonemal complex from both species are straight and have a similar width, 1 6-1 8 nm These filaments occur mainly in bundles The central element differs in architecture in the two species In hamster bivalents it is formed of longitudinal stretches of filaments 1.6-1 8 nm wide and a small amount of an amorphous material similar to that of the lateral elements In the cricket, the central element contains transverse fibrils which are continuous with the transverse filaments of the central region, and an amorphous material lying mainly along the sides of the central element All of the components of the central region of the synaptonemal complex are resistant to pancreatic DNase. The overlapping ends of the transverse filaments, together with additional protein material, make up the central element The widespread occurrence and close morphological and histochemical interspecies similarities of the transverse filaments indicate that they serve an essential role, probably one concerned with holding synapsed bivalents together via the lateral elements. Restrictions placed by the observations reported here on current models of the synaptonemal complex are discussed.

Synaptonemal complexes and XY behavior in two species of Argentinian armadillos: Chaetophractus villosus and Dasypus hybridus (Xenarthra, Dasypodidae).

Spermatocytes from the two armadillo species, C. villosus and D. hybridus were studied in microspreads for synaptonemal complexes (SCs) and in thin sections for electron microscopy (EM). The complete SC karyotype generally agrees with previous reports on mitotic chromosomes, except for the sex chromosomes. The X chromosome is submetacentric in both species and the Y is the shortest one in C. villosus and the second shortest in D. hybridus, and an extremely acrocentric one. A SC is formed along the total length of the Y chromosome, and this SC persists along all the pachytene substages. A single recombination nodule (RN) is located in the region of the SC nearest to the attachment to the nuclear envelope. The lateral element (LE) of the X axis in the SC shows a wavy aspect in most of the SC length distant from the nuclear envelope. Nucleoli are attached to acrocentric or submetacentric bivalents, are visibly double in some cells, and in thin sections show an elaborate nucleolonema. Some differences in the XY are species-specific, as the higher degree of tangling and stronger heteropycnosis in D. hybridus. The effective, single crossover of the XY pair is highly localized, despite the permanence of a long tract of SC.

Fine structure and meiotic behaviour of the male multiple sex chromosomes in the genus Alouatta.

The meiotic cytology and fine structure of the sex multiples in males from two species of the genus Alouatta are presented and compared with descriptions from other species of this genus. As shown in pachytene by synaptonemal complex analysis and in metaphase I by spreading, there is a quadrivalent in male meiosis in A. caraya, which is formed by an X(1)X(2)Y(1)Y(2) complex, while in A. palliata there is a trivalent formed by an X(1)X(2)Y(1) complex. Chromosome painting with human probes shows that A. caraya sex multiples share the same components as those of A. seniculus sara and A. seniculus arctoidea. However, as shown here for A. palliata and by others in A. fusca, there are differences among the multiples of some species. It is shown that in this genus there are several varieties of sex multiples that share some features, and that the origin of these multiples is most probably a primitive development in the genus Alouatta.

Changes in crossover distribution along a quadrivalent in a man carrier of a reciprocal translocation t(11;14).

A testicular biopsy from an infertile man carrying a heterozygous chromosome translocation t(11;14) was studied with synaptonemal complex analysis and immunolocalization of the protein MLH1 for crossover detection. A full blockage of spermatogenesis at the spermatocyte stage was related to the presence of the translocation quadrivalents at pachytene. Only 2% of the quadrivalents showed full synapsis. Most of the spermatocytes showed asynaptic free ends that frequently mingled with the XY pair. The average number of crossovers per cell was diminished from a mean of 52.7 in controls to a mean of 48 in the patient. The difference between the number of crossovers in the quadrivalent and the normal bivalents was highly significant. The distribution of crossovers over the segment of the quadrivalent corresponding to bivalent #14 was also very different from that of the control. It is concluded that in this translocation, the pattern of crossovers is changed, mainly due to a synaptic hindrance in the quadrivalent, and that the spermatogenesis arrest is mainly due to the quadrivalents that interact with the XY pair.

Synaptonemal complex analysis in human male infertility.

The fine structural features of human spermatocytes from carriers of some of the most frequent chromosomal abnormalities are reviewed on the basis of original data and previous reports from the literature. Special emphasis is given to the Robert-sonian translocations t (13; 14), to one specific reciprocal translocation involving chromosome 21, and to Y disomy in spermatocytes from XYY men. Synaptonemal complex analysis shows that in many carriers of chromosomal aberrations that lead to pachytene configurations having terminal asynaptic segments in autosomes, there is a gradual association of these asynaptic segments with the XY body. This associations with the XY pair is assumed to trigger a process of germ cell deterioration, presumably through the spreading of the X-chromosome inactivation towards autosomal segments. Another different process of germ cell deterioration occurs when the X chromosome becomes an univalent, as in XYY men with persistence of two Y chromosomes in the germ line. The renewed interest in the examination of spermatocytes from human testicular biopsies is commented upon.

Evolutionary sperm morphology and morphometry in armadillos.

Little is known about the evolution of vertebrate spermatozoa. In most eutherian taxa a high degree of uniformity in sperm shapes and dimensions among species was observed. The aim of this work is to trace a possible evolutionary change in sperm morphology and morphometry in dasypodids. The main difference between the spermatozoa of the studied armadillos is the shape of the sperm heads. We have classified the spermatozoa into 4 different groups according with their head shapes. Sperm from group 1 (Dasypus) are considered ancestral and are clearly separated from the others. The remaining sperm types are derivative ones; those from group 2 (Tolypeutes) are farther from those of groups 3 (Priodontes and Cabassous) and 4 (Chaetopractus, Zaedyus and Euphractus) which would have recently differentiated from each other. The sperm shape and size are not constant across taxa in armadillos; an important evolutive differentiation was established on the sperm morphology and morphometry between the different genera in Dasypodidae.

First demonstration of the substructure of recombination nodules.

Recombination nodules are submicroscopic structures that are found in all the sexually reproducing, eukaryotic organisms during the pachytene stage of meiotic prophase I. Despite many reports on their number and location, no definite substructure was previously reported in these nodules. The present observations on spread oocytes and spermatocytes of the pigeon, using an improved technique for protein preservation, shown the presence of particulate subunits or "recombinomeres" in late recombination nodules, besides an interparticle matrix. The number of subunits per each nodule ranges from 1 to 5, and this number increases with the advancement of pachytene substages. These subunits are present in recombination nodules of all the other avian species previously studied, and they may be present in other organisms as well. It is suggested that the particulate substructure of recombination nodules mirrors the multiplicity of multienzymatic complexes that are needed for the ordered series of reactions that occur at the molecular level in the sites of meiotic recombination.

The behavior of sex chromosomes in two human X-autosome translocations: failure of extensive X-inactivation spreading.

Two patients, one adult male and one infant girl, bearing different X-autosome translocations, were studied with cytogenetical, ultrastructural and chromosome-painting techniques. The adult male, is a carrier of a reciprocal, balanced translocation involving the X and #2 chromosomes: 46,Y,t(X;2) (q13;p21). This man showed infertility with spermatogenesis arrest at the spermatocyte stage. Synaptonemal complex analysis at pachytene showed the quadrivalent structure and the putative breakage points. Sex-chromatin condensation did not spread towards the autosomal regions of the quadrivalent. The female infant showed diminished body growth and multiple somatic anomalies. She is a 45,Xp-,t(X;21)(p11;p13) carrier, an unbalanced translocation involving chromosomes X and #21, which leads to a monosomy of almost all Xp. The translocated #21 is practically complete, and its centromere is the active one in the rearranged product. The analysis of interphase nuclei with the X-centromere probe shows that the Xq region of the rearranged chromosome is the late -replicating and inactive element. However, X-inactivation does not spread to the attached #21, as shown by the R-banding pattern. Thus, both in the male adult and in the female infant there is a barrier to the spreading effect of X-chromosome inactivation, which is probably due to different mechanisms.

Comparative morphologic placental types in Dasypodidae (Chaetophractus villosus, Cabassous chacoensis, Tolypeutes matacus and Dasypus hybridus).

Information about the morphology of placentas in armadillos is scarce, except for D. novemcinctus. A comparative study of morphologic placental types in armadillos is important in order to have a comprehensive view of the peculiar reproductive physiology in this family. The aim of this paper is to perform a comparative analysis of the morphological features of the placenta in Chaetophractus villosus, Cabassous chacoensis, Tolypeutes matacus and Dasypus hybridus in order to classify them in accordance with Grosser (1909). The placentas were studied macroscopically and histologically (light microscopy in 1 micron thick sections and electron microscopy for fine structure). The macroscopic study in the 4 studied species showed a similar pear-shaped placenta homogeneously villosus in almost all the surface. The histological analysis showed that the 4 studied species had a hemochorial type of placenta. This type of placenta was also found in D. novemcinctus (Dasypodidae), but it is different from those described for other xenarthrans. Hemochorial types of placenta have also been described in more modern mammals. Despite the many primitive features of the armadillos and the different anatomical and physiological features between the genuses of dasypodids, all the studied species share this structural type of placenta.

Synaptonemal complex karyotyping in an oligospermic patient with heterochromatin duplication in chromosome n. 9.

Synaptonemal complex karyotyping has been performed in an oligospermia of unknown etiology in a patient carrying a 9qh+ chromosomal polymorphism. The testicular histology showed hypospermatogenesis at the spermatid level and an abnormal pattern of chromatin condensation. Spermatocytes at early pachytene showed a large, asymmetric loop in SC #9, which disappeared at late pachytene, probably because of synaptic adjustment. The loop was formed by a lateral element 7.02% longer than the average normal one. The loop exceeded the centromere towards the short arm, and it is interpreted as a tandem duplication of about 50% of the paracentromeric heterochromatin. The present observation and the previously reported asynaptic loops in carriers of pericentric inversions and showing severe oligospermia suggest that chromosomal variants producing asynaptic loops may be associated with germ cell loss. Further meiotic studies in infertile carriers of such variants are indicated.