Meiosis and spermatogenesis in G-Trisomic males

Summary In 3 adult males with trisomy-G, Down's syndrome, the chromosomes were studied in spermatogonial mitoses and in the first meiotic division. The findings were similar in all cases and are here presented together. Of 8 spermatogonial mitoses of good quality, 4 had 46 chromosomes and 4 had 47 chromosomes. At diakinesis-metaphase I, 21% of the cells had 1 G-bivalent and 1 trivalent, 38% had 2 bivalents and 1 G-univalent and in 41% 2 chromosomal elements composed of G-chromosomes were seen, but it was not possible to determine unequivocally whether 1 of them was a trivalent or not. From their morphology and from the spermatogonial chromosome counts it was tentatively concluded that at least some of them had only 2 G-bivalents. Premeiotic elimination of 1 G-group chromosome is a possible explanation of this phenomenon. The study of larger samples of spermatogonial mitoses should allow definitive conclusions to be made. — Different trivalent and univalent configurations were described. — Spermatogenesis was quantitatively assessed and found to be complete but of lesser magnitude than in a normal male. Spermatogenetic arrest was not noticed. As judged from histological sections of the testes, all 3 G-trisomic males would have to be considered fertile.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Meiosis,II: A modified affinity model in mice

In male mice, observations on MI clustering of nonhomologues in 4-armed tri-bivalent groups considered together with estimates of chiasma frequency in the same clusters, has suggested a modification of theWallce affinity hypothesis. According to the proposed model three classes of nonhomologous linkage would be generated by such a tribivalent figure; two of the three would vary inversely—and the third directly—with ordinary intrabivalent linkage frequencies. The model has the advantage of not requiring the existence of mesocentric chromosomes and furthermore appears attractive because of possible analogy with segregation patterns resulting from non-homologous association ofDrosophila andNeurospora. A possible disadvantage of the model is its dependence on chromatid interference.     

Meiosis and retrotransposon silencing during germ cell development in mice

In mammals, germ cells derive from the pluripotent cells that are present early in embryogenesis, and then differentiate into male sperm or female eggs as development proceeds. Fusion between an egg and a sperm at fertilization allows genetic information from both parents to be transmitted to the next generation, and produces a pluripotent zygote to initiate the next round of embryogenesis. Meiosis is a central event in this self-perpetuating cycle that creates genetic diversity by generating new combinations of existing genetic alleles, and halves the number of chromosomes in the developing male and female germ cells to allow chromosome number to be maintained through successive generations. The developing germ cells also help to maintain genetic and chromosomal stability through the generations by protecting the genome from excessive de novo mutation. Several mouse mutants have recently been characterised whose germ cells exhibit defects in silencing the potentially mutagenic endogenous retroviruses and other retrotransposons that are prevalent in mammalian genomes, and these germ cells also exhibit defects in progression through meiosis. Here we review how mouse germ cells develop and proceed through meiosis, how mouse germ cells silence endogenous retroviruses and other retrotransposons, and discuss why silencing of endogenous retroviruses and other retrotransposons may be required for meiotic progression in mice.     

Rate of homologous chromosome bivalents in spermatocytes may predict completion of spermatogenesis in azoospermic men

Abstract.The rate of bivalent formation during meiosis was correlated with the presence and amount of spermatozoa in the testes of azoospermic men. Four pairs of chromosomes, X-Y, 9, 15, and 18, were evaluated. In addition, left and right testes were compared. Three biopsies from each testis were undertaken to extract spermatozoa for intracytoplasmic sperm injection. In addition, one sample from each testis was used for histological definition, spermatozoa count and detection of chromosome bivalents in spermatocytes. A significantly higher rate of bivalents of all homologous chromosomes was found whenever spermatozoa were detected. The rate of bivalent X-Y was found to be the most sensitive predictor for detection of spermatozoa, with a cut-off value of 47%. The R(2) was 27% ( P=0.003) for the percent of spermatozoa in the minced sample as well as the number of mature spermatids per tubule in the histological section. All pairs of testes were in concord in regard to the likelihood of finding spermatozoa. In the testes where no spermatozoa were found on biopsy, the rate of X-Y bivalent indicated the presence of spermatozoa in the opposite side. Thus, it may be concluded that the rate of X-Y bivalent formation in spermatocytes may predict the presence and amount of spermatozoa in the testicular tissue of azoospermic men. It is suggested that when no spermatozoa are located by testicular fine-needle aspiration, X-Y bivalent evaluation may be conducted if spermatocytes are evinced. A high rate of X-Y bivalents may impel one to continue with testicular open biopsies.     

Meiosis in Sxr male mice

Cytological evidence for the existence of a Y-autosomal rearrangement in meiotic cells of Sxr mice has been sought. At pachytene, in silverstained light microscope spread preparations of XY, Sxr/+ and XO, Sxr/+ spermatocytes, evidence for pairing or association between a possible Y-bearing segment of a specific autosome and a sex chromosome could not, however, be found. Such sex chromosome-autosome associations as were seen were non-specific in nature, and occurred no more often in Sxr mice than in controls.     

Change in length of synapsed bivalents during pachytene

Total pachytene bivalent length was measured in 93 nuclei from plants of an inbred line of maize (KYS). These nuclei are thought to represent samples of substages from the portion of pachytene which begins with the opening of the synizetic knot and ends with the initiation of loss of the synaptonemal complex at diplotene. Pachytene substages were ranked on the basis of the length of the anther in which they occurred, with longer anthers generally expected to contain more advanced stages. The data strongly suggest that maize synapsed bivalents shorten significantly during this period.     

Changes in polyadenylation of lactate dehydrogenase-X mRNA during spermatogenesis in mice

The expression of the mRNA for mouse testicular lactate dehydrogenase (LDH-X) was examined by RNA:cDNA hybridization in situ in the testis and by Northern analyses of meiotic and postmeiotic spermatogenic cell populations. Silver grains accumulated in cells inside the second layer from the periphery of the seminiferous tubule, confirming previous findings that LDH-X mRNA first appears in the spermatocyte and continues to accumulate until the late spermatid stage. Northern analyses showed that meiotic and postmeiotic cells contained 1.2 and 1.3 kb classes of hybridizing mRNA, respectively. RNase H digestion of oligo (dT)-hybridized RNA and poly(U)-Sepharose column chromatography with differential elution by formamide revealed that the difference in size of the two classes of mRNAs was due to the poly(A) tail length of the LDH-X mRNA. When the distribution of the LDH-X mRNA was examined across polysome gradients, both mRNAs were partially associated with polysomes. These results suggest that the changes in the polyadenylation of LDH-X mRNA were associated with the meiotic division during spermatogenesis in the mouse. They raise the possibility that the stable accumulation of the LDH-X mRNAs in the postmeiotic cells is enhanced by poly(A) tails of increased length.     

Meiosis in trisomic female mice with Robertsonian translocations. I. Prophase pairing

The prophase oocytes of two murine Robertsonian translocation (Rb) trisomies of chromosomes 16 and 19 were investigated using electron microscopy and a whole-cell micro-spreading technique after silver staining. About 20% of fetuses of each type were trisomic. They were obtained by mating animals heterozygous for two Rb's, monobrachially homologous for either chromosome 16 or 19, to an entirely acrocentric stock. Because of the almost inevitable prenatal mortality of the trisomic embryos, their fetal ovaries were "rescued" by an in vitro method for prophase studies. Analysis of the recovered oocytes showed frequent, close pairing associations of the three trisomic axes and evidence suggesting that the closely apposed axes coincided with the side-by-side formation of parallel, complete, true synaptonemal complexes; hence, the cytogenetic dogma that pairing is always two-by-two was contradicted. The presence of two parallel complexes has implications for crossing-over recombination. Triple associations of axes were found in almost half the trisomy 19 (Ts19) and in about 70% of the trisomy 16 (Ts16) prophases. The extent of triple associations varied and was greater in Ts16 than in Ts19 oocytes. Other relevant observations concerned the proportions of univalents and of univalence of the trisomic axes (21% in Ts16 and 46% in Ts19) and the distinctive, thickened appearance of all univalent axes. The pairing behaviour observed in balanced heterozygotes confirms what appears to be nonhomologous pairing and synaptic adjustment within the short-arm axes of the Rb trivalents.     

Gene expression,X-inactivation,and methylation during spermatogenesis: The case of Zfa,Zfx, and Zfy in mice

While it has become clear that X-inactivation in the female soma is complete in mouse (in contrast to being “patchy” in man), the degree of X-inactivation in the testes has not been ascertained. We have compared autosomal and X-linked zinc finger homolog expression and X-linked and Y-linked zinc finger homolog methylation in an attempt to elucidate this question. Using RTPCR, we have extended earlier studies of Zfx and Zfa expression in developing testes and find that Zfa expression starts at the time of X-inactivation while Zfx expression is continuous. Cell separation studies did not preclude continued expression of Zfx in adult germ cells. The methylation status of four CCGG residues in the Zfx promoter was studied using PCR bridging this region before and after DNA digestion with the isoschizomers Msp I and Hpa II, the latter being methylation sensitive. Hpa II resistant Zfx promoter DNA was found in all female tissues, but not in male tissues, including the testes. Previous studies have shown that Zfy is expressed at meiosis (like Zfa and unlike Zfx). Despite its expression, the Zfy gene is adjacent to, or contains, highly methylated CCGG sites since hybridization after Msp I digestion detected multiple small fragments that were not released after DNA digestion with Hpa II. Thus, Zfx is not methylated in sperm, while Zfy is, in contrast to their apparent patterns of expression. © 1993 Wiley-Liss, Inc.     

Atg7 is required for acrosome biogenesis during spermatogenesis in mice

The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in the process of fertilization. The molecular mechanism underlying the biogenesis of this lysosome-related organelle (LRO) is still largely unknown. Here, we show that germ cell-specific Atg7-knockout mice were infertile due to a defect in acrosome biogenesis and displayed a phenotype similar to human globozoospermia; this reproductive defect was successfully rescued by intracytoplasmic sperm injections. Furthermore, the depletion of Atg7 in germ cells did not affect the early stages of development of germ cells, but at later stages of spermatogenesis, the proacrosomal vesicles failed to fuse into a single acrosomal vesicle during the Golgi phase, which finally resulted in irregular or nearly round-headed spermatozoa. Autophagic flux was disrupted in Atg7-depleted germ cells, finally leading to the failure of LC3 conjugation to Golgi apparatus-derived vesicles. In addition, Atg7 partially regulated another globozoospermia-related protein, Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), during acrosome biogenesis. Finally, the injection of either autophagy or lysosome inhibitors into testis resulted in a similar phenotype to that of germ cell-specific Atg7-knockout mice. Altogether, our results uncover a new role for Atg7 in the biogenesis of the acrosome, and we provide evidence to support the autolysosome origination hypothesis for the acrosome.