Kinetics of meiosis in azoospermic males: a joint histological and cytological approach

Abstract

We have developed a protocol for the identification of aberrant chromosome behavior during human male meiosis up to metaphase of the secondary spermatocyte. Histological evaluation by the Johnsen score of a testicular biopsy was combined with immunofluorescence of first meiotic prophase spermatocytes, using antibodies against synaptonemal complex protein 3 (SYCP3) and the product of the ataxia telangiectasia and rad3-related gene (ATR). This combination enables accurate meiotic prophase substaging and the identification of pachytene spermatocytes with asynapsis. Furthermore, we also investigated the competence of late pachytene primary spermatocytes to complete the first meiotic division up to metaphase and of secondary spermatocytes to transform into metaphase by an in vitro challenge with okadaic acid (OA). We tested this protocol on five males with normal Johnsen scores that presented with obstructive azoospermia, five males with low Johnsen scores and non-obstructive azoospermia and six vasectomized control males of proven fertility and normal Johnsen scores. In all azoospermics, the profiling of meiotic prophase stages by immunofluorescence increases the resolving power of the Johnsen score. In both obstructive and non-obstructive azoospermic patients, relatively more leptotene meiotic prophase stages were counted compared to the controls. In non-obstructive azoospermics, a marked heterogeneity in spermatogenesis was found, after combining the results of all three approaches, pointing at functional mosaicism of the germinal epithelium. Asynaptic pachytene spermatocytes were rarely encountered. Also, when first meiotic metaphase could be induced by OA, chiasma counts were normal. In none of the non-obstructive azoospermic males did the pattern of spermatogenesis resemble that of knock-out mouse azoospermics. We conclude that this combined histological and cytological approach enables a detailed phenotypic classification of infertile males, at a level comparable to that applied for male-sterile knock-out mice with a meiotic defect. This may facilitate the identification of candidate genes for human male infertility.

Chromosomal rearrangement interferes with meiotic X chromosome inactivation

“…Later on, its further occurrence was confirmed in mice (de Boer and Branje 1979;Forejt and Gregorova 1977;Forejt 1979Forejt , 1996Forejt et al 1981), in humans (Johannisson et al 1983;Gabriel-Robez and Rumpler 1996;de Boer et al 2004;OliverBonet et al 2005;Sciurano et al 2007), and in other mammals (Power 1991;Jaafar et al 1992;Ansari et al 1993). These cytogenetic studies revealed the coincidence of chromosomal asynapsis, XY-autosomal colocalization, and male-limited sterility.…”

Human Male Meiotic Sex Chromosome Inactivation

“…A first sign of H3.1/H3.2 loss was observed at mid pachytene when the XY axial elements locally start to unravel. (type III, [25], [41] (Figure 1A). In late pachytene XY bodies (types IV and V [25], [41]), when the XY axial elements appeared as a tangled knot of fibrils and DAPI was most intense, a highly variable H3.1/3.2 signal remained (Figure 1B, Table 1).…”

“…In the human XY body, features such as a condensed chromatin domain [23] and staining for γH2AX, BRCA1 [24] and ATR [25], were shown to be present as well. Also an indication for H3.1/3.2 nucleosome eviction was obtained [22].…”

Immunofluorescent characterization of meiotic recombination in human males with variable spermatogenesis

“…When importing, data of the two observers were randomized if differences occurred, which was infrequent. Pachytene substages were determined, based on SYCP3 staining and electron microscopy surface spread images as described before by de Boer et al (Chandley et al, 1984;de Boer et al, 2004). The stages I, II and III were collectively labelled early and the stages IV and V as late pachytene spermatocytes.…”

“…(i) In NOA patients, often a surplus of the early first meiotic prophase stages leptotene and zygotene is noted [(de Boer et al, 2004) and reviewed in (Martin, 2008)] indicating a problem with the homology search. (ii) in previous reports, an increased frequency of synaptic problems at pachytene was found in this category [reviewed in (Martin, 2008;Sun et al, 2007a)], as well as (iii) lower numbers of crossing-over (Gonsalves et al, 2004;Sun et al, 2007a) and (iv) increased levels of meiotic non-disjunction [reviewed by (Harton & Tempest, 2012;Martin, 2008)].…”