Roberta B. Sciurano

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.

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.

Protein immunolocalization supports the presence of identical mechanisms of XY body formation in eutherians and marsupials

The meiotic sex chromosomes of the American marsupials Monodelphis dimidiata and Didelphis albiventris were studied with electron microscopy (EM) and with immunofluorescence localization of meiotic proteins SYCP1 and SYCP3, and proteins essential for meiotic sex chromosome inactivation (MSCI), γ-H2AX and BRCA1. The chromatin of the non-synaptic X and Y chromosomes contains γ-H2AX, first as foci and then as homogeneous staining at late stages. The thick and split X and Y axes are labelled with BRCA1 except at one terminus. The bulgings of the axes contain SYCP1 as well as the inner side of the dense plate. The evenly spaced and highly packed chromatin fibres of the conjoined XY body in these species have the same behaviour and the same components (γ-H2AX in the chromatin, BRCA1 in the axes) as in the XY body of eutherian species. These observations and recent data from the literature suggest that XY body formation is ancestral to the metatherian–eutherian divergence.

Loss of Sertoli-Germ Cell Adhesion Determines the Rapid Germ Cell Elimination During the Seasonal Regression of the Seminiferous Epithelium of the Large Hairy Armadillo Chaetophractus villosus

ABSTRACT The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis.

Ultrastructural and Immunofluorescent Methods for the Study of the XY Body as a Biomarker

Structural and immunohistochemical methods have been extremely useful for the characterization of the XY body (the structure formed by the XY pair during meiotic prophase) in Man and in other mammals. These methods are widely used at the present time for the detection of abnormalities leading to human infertility. The basic ultrastructural methods are spreading of pachytene spermatocytes, thin-sectioning techniques with or without 3-D reconstructions, and the monitoring of all specimens with semi-thin sections. Immunofluorescent techniques also use spreading of meiotic cells for the analysis of the XY body, and they can be combined with the fluorescence in situ hybridization (FISH) technique, in the so-called immuno-FISH. Epitope retrieval techniques are also used.

Androgen Insensitivity Syndrome at Prepuberty: Marked Loss of Spermatogonial Cells at Early Childhood and Presence of Gonocytes up to Puberty

Androgen insensitivity syndrome (AIS) is a hereditary condition in patients with a 46,XY karyotype in which loss-of-function mutations of the androgen receptor (AR) gene are responsible for defects in virilization. The aim of this study was to investigate the consequences of the lack of AR activity on germ cell survival and the degree of testicular development reached by these patients by analyzing gonadal tissue from patients with AIS prior to Sertoli cell maturation at puberty. Twenty-three gonads from 13 patients with AIS were assessed and compared to 18 testes from 17 subjects without endocrine disorders. The study of the gonadal structure using conventional microscopy and the ultrastructural characteristics of remnant germ cells using electron microscopy, combined with the immunohistochemical analysis of specific germ cell markers (MAGE-A4 for premeiotic germ cells and of OCT3/4 for gonocytes), enabled us to carry out a thorough investigation of germ cell life in an androgen-insensitive microenvironment throughout prepuberty until young adulthood. Here, we show that germ cell degeneration starts very early, with a marked decrease in number after only 2 years of life, and we demonstrate the permanence of gonocytes in AIS testis samples until puberty, describing 2 different populations. Additionally, our results provide further evidence for the importance of AR signaling in peritubular myoid cells during prepuberty to maintain Sertoli and spermatogonial cell health and survival.

Protein markers of synaptic behavior and chromatin remodeling of the neo-XY body in phyllostomid bats

The XX/XY system is the rule among mammals. However, many exceptions from this general pattern have been discovered since the last decades. One of these non-conventional sex chromosome mechanisms is the multiple sex chromosome system, which is evolutionary fixed among many bat species of the family Phyllostomidae, and has arisen by a translocation between one original gonosome (X or Y chromosome), and an autosome, giving rise to a “neo-XY body.” The aim of this work is to study the synaptic behavior and the chromatin remodeling of multiple sex chromosomes in different species of phyllostomid bats using electron microscopy and molecular markers. Testicular tissues from adult males of the species Artibeus lituratus, Artibeus planirostris, Uroderma bilobatum, and Vampyrodes caraccioli from the eastern Amazonia were analyzed by optical/electron microscopy and immunofluorescence of meiotic proteins involved in synapsis (SYCP3 and SYCE3), sister-chromatid cohesion (SMC3), and chromatin silencing (BRCA1, γ-H2AX, and RNApol 2). The presence of asynaptic axes—labeled by BRCA1 and γ-H2AX—at meiotic prophase in testes that have a normal development of spermatogenesis, suggests that the basic mechanism that arrests spreading of transcriptional silencing (meiotic sex chromosome inactivation (MSCI)) to the autosomal segments may be per se the formation of a functional synaptonemal complex between homologous or non-homologous regions, and thus, this SC barrier might be probably related to the preservation of fertility in these systems.

The XY Body of the Cat ( Felis catus ): Structural Differentiations and Protein Immunolocalization

The heteromorphic X and Y chromosomes behave in a special way in mammalian spermatocytes; they form the XY body and synapse only partially. The aim of this article was to study the origin and the role of the special differentiations in the XY pair of the domestic cat during pachytene by analyzing its fine structural characteristics and the immunolocalization of the main meiotic proteins SYCP3, SYCP1, SYCE3, SMC3, γ-H2AX, BRCA1, H3K27me3, and MLH1. The cat XY body shows particularly striking structures: an extreme degree of axial fibrillation in late pachynema and a special location of SYCP3-containing fibrils, bridging different regions of the main X axis, as well as one bridge at the inner end of the pairing region that colocalizes with the single mandatory MLH1 focus. There are sequential changes, first bullous expansions, then subdivision into fibrils, all involving axial thickening. The chromatin of the XY body presents the usual features of meiotic sex chromosome inactivation. An analysis of the XY body of many eutherians and metatherians suggests that axial thickenings are primitive features. The sequential changes in the mass and location of SYCP3-containing fibers vary among the clades because of specific processes of axial assembly/disassembly occurring in different species.