Mutant meiotic chromosome core components in mice can cause apparent sexual dimorphic endpoints at prophase or X–Y defective male-specific sterility

Genetic modifications causing germ cell death during meiotic prophase in the mouse frequently have sexually dimorphic phenotypes where oocytes reach more advanced stages than spermatocytes. To determine to what extent these dimorphisms are due to differences in male versus female meiotic prophase development, we compared meiotic chromosome events in the two sexes in both wild-type and mutant mice. We report the abundance and time course of appearance of structural and recombination-related proteins of fetal oocyte nuclei. Oocytes at successive days post coitus show rapid, synchronous meiotic prophase development compared with the continuous spermatocyte development in adult testis. Consequently, a genetic defect requiring 2–3 days from the onset of prophase to reach arrest registers pachytene as the developmental endpoint in oocytes. Pachytene spermatocytes, on the other hand, which normally accumulate during days 4–10 after the onset of prophase, will be rare, giving the appearance of an earlier endpoint than in oocytes. We conclude that these different logistics create apparent sexually dimorphic endpoints. For more pronounced sexual dimorphisms, we examined meiotic prophase of mice with genetic modifications of meiotic chromosome core components that cause male but not female sterility. The correlations between male sterility and alterations in the organization of the sex chromosome cores and X–Y chromatin may indicate that impaired signals from the XY domain (XY chromosome cores, chromatin, dense body and sex body) may interfere with the progression of the spermatocyte through prophase. Oocytes, in the absence of the X–Y pair, do not suffer such defects.     

The novel male meiosis recombination regulator coordinates the progression of meiosis prophase I

Meiosis is a specialized cell division for producing haploid gametes in sexually reproducing organisms. In this study, we have independently identified a novel meiosis protein male meiosis recombination regulator (MAMERR)/4930432K21Rik and showed that it is indispensable for meiosis prophase I progression in male mice. Using super-resolution structured illumination microscopy, we found that MAMERR functions at the same double-strand breaks as the replication protein A and meiosis-specific with OB domains/spermatogenesis associated 22 complex. We generated a Mamerr-deficient mouse model by deleting exons 3–6 and found that most of Mamerr^−/− spermatocytes were arrested at pachynema and failed to progress to diplonema, although they exhibited almost intact synapsis and progression to the pachytene stage along with XY body formation. Further mechanistic studies revealed that the recruitment of DMC1/RAD51 and heat shock factor 2–binding protein in Mamerr^−/− spermatocytes was only mildly impaired with a partial reduction in double-strand break repair, whereas a substantial reduction in ubiquitination on the autosomal axes and on the XY body appeared as a major phenotype in Mamerr^−/− spermatocytes. We propose that MAMERR may participate in meiotic prophase I progression by regulating the ubiquitination of key meiotic proteins on autosomes and XY chromosomes, and in the absence of MAMERR, the repressed ubiquitination of key meiotic proteins leads to pachytene arrest and cell death.     

Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes

The product of the Scmh1 gene, a mammalian homolog of Drosophila Sex comb on midleg, is a constituent of the mammalian Polycomb repressive complexes 1 (Prc1). We have identified Scmh1 as an indispensable component of the Prc1. During progression through pachytene, Scmh1 was shown to be excluded from the XY body at late pachytene, together with other Prc1 components such as Phc1, Phc2, Rnf110 (Pcgf2), Bmi1 and Cbx2. We have identified the role of Scmh1 in mediating the survival of late pachytene spermatocytes. Apoptotic elimination of Scmh1(-/-) spermatocytes is accompanied by the preceding failure of several specific chromatin modifications at the XY body, whereas synapsis of homologous autosomes is not affected. It is therefore suggested that Scmh1 is involved in regulating the sequential changes in chromatin modifications at the XY chromatin domain of the pachytene spermatocytes. Restoration of defects in Scmh1(-/-) spermatocytes by Phc2 mutation indicates that Scmh1 exerts its molecular functions via its interaction with Prc1. Therefore, for the first time, we are able to indicate a functional involvement of Prc1 during the meiotic prophase of male germ cells and a regulatory role of Scmh1 for Prc1, which involves sex chromosomes.     

Developmental control of sumoylation pathway proteins in mouse male germ cells

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.     

XY body formation during rat spermatogenesis: an immunocytochemical study using antibodies against XY body-associated proteins

The process of formation of the XY body during meiotic prophase was investigated by immunocytochemistry on cryosections of pubertal rat testes using antibodies against three different XY body-associated proteins. Here we show that these proteins are detectable at only partially overlapping temporal windows. These findings provide the first evidence that the previously described morphological changes in the structure of the XY body that occur during meiotic prophase are accompanied by considerable changes in its protein composition. Received: 22 May 1997 / Accepted: 14 June 1997     

XMR, a dual location protein in the XY pair and in its associated nucleolus in mouse spermatocytes

Xlr and Xmr are sex-specific genes which are expressed during the meiotic prophase I in the mouse. In spermatocytes, XMR concentrates on the asynapsed regions of the XY chromosomes, suggesting that XMR plays a role in sex chromosome condensation and silencing. The present study shows that in the mouse, XMR also concentrates in the nucleolus which is closely associated with the XY chromosome pair. In this species, the formation of a large fibrillo-granular nucleolus signals the activation of the ribosomal genes, but release of pre-ribosomal particles is inhibited. Using laser confocal microscopy we characterized the distribution of XMR in the XY body relative to the XY chromatin and the nucleolus. Immunoelectron microscopy showed that XMR concentrates in the fibrillo-granular component and the granular component (GC) of the nucleolus. In (T[X;16]16H) mouse spermatocytes, the nucleolus displays little or no activity and does not associate with the XY pair. XMR concentrated only on the XY chromosomes in (T[X;16]16H) mouse spermatocytes. These data suggest that XMR could play a role both in the XY pair and the nucleolus associated to the sex chromosomes.     

The meiotic behaviour of the XY pair in Lutreolina crassicaudata (Marsupialia: Didelphoidea)

The meiotic behaviour of the XY pair of the didelphid Lutreolina crassicaudata is analyzed by microspreading of spermatocytes for visualization of chromosomal axes and by three-dimensional reconstruction of spermatocyte nuclei from EM thin sections. The delay in pairing of sex chromosomes compared to autosomes and the absence of a synaptonemal complex between the axes of the X and Y chromosomes, already described for South American marsupials by three-dimensional reconstruction and for Australian species with synaptonemal complex microspreadings, is confirmed for this species. Sections demonstrate that at the diffuse stage and diplotene the dense plate occupies the region of the inner face of the nuclear envelope in contact with the XY body. Spreads show an structure similar in staining to the axes that becomes apparent simultaneously with the dense plate, called a balloon. The mechanism of XY pairing during meiotic prophase appears to be common to American and Australian marsupials as the same morphological pattern is found in all the species described. This mechanism is different from the way of pairing and segregation known for eutherian sex chromosomes.     

Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase IIα localization and chromosome condensation

Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.     

Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.     

Human male meiotic sex chromosome inactivation

In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI), which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.     

Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: Evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro

Summary Dispersed testicular cells prepared from 14-d-old rats were cultured on type 1 collagen gels using a medium composed of a 1∶1 mixture of Ham’s F12 medium and Leibovitz’s L15 medium (F12-L15 medium) containing 10% (vol/vol) fetal bovine serum. The viability of the spermatogenic cells was facilitated by supplementing a rat adrenal extract into the medium. The effective substance(s) (the survival factor) was purified from acid extracts of adrenals by molecular sieve high performance liquid chromatography and identified as epinephrine and norepinephrine. Both epinephrine and norepinephrine promoted the survival of the spermatogenic cells with a half saturating dose of 10 ng/ml. The spermatogenic cells, which could be cultured for 2 wk on a collagen gel by supplementing with the survival factor (epinephrine or norepinephrine), were subjected to Giemsa staining and to DNA flow cytometry. The following results were obtained: a) The spermatogenic cells from 14-d-old rats did not contain spermiogenic cells (lc-cells). b) During a culture period of 2 to 7 d the ratio of meiotic prophase spermatocytes (4c-cells) to premeiotic cells (2c-cells) increased. On Day 7, more than 90% of the surviving cells were meiotic prophase spermatocytes. c) On Day 10, spermatids (lc-cells) appeared for the first time. The time of the first appearance of spermatids in the culture was consistent with that in vivo. These results suggest that both epinephrine and norepinephrine facilitated the viability of meiotic prophase spermatocytes and that a part of the meiotic prophase spermatocytes completed the meiotic divisions in the testicular cell culture.