Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment com-bined with high chloroform Carnory’s fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromo-some 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed.Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.

The ZFP541-KCTD19 complex is essential for pachytene progression by activating meiotic genes during mouse spermatogenesis

Meiosis is essential for fertility in sexually reproducing species and this sophisticated process has been extensively studied.Notwithstanding these efforts,key factors involved in meiosis have not been fully characterized.In this study,we investigate the regulatory roles of zinc finger protein 541(ZFP541)and its interacting protein potassium channel tetramerization domain containing 19(KCTD19)in spermatogenesis.ZFP541 is expressed from leptotene to the round spermatid stage,while the expression of KCTD19 is initiated in pachytene.Depletion of Zfp541 or Kctd19 leads to infertility in male mice and delays progression from early to mid/late pachynema.In addition,Zfp541^(-/-)spermatocytes show abnormal programmed DNA double-strand break repair,impaired crossover formation and resolution,and asynapsis of the XY chromosomes.ZFP541 interacts with KCTD19,histone deacetylase 1/2(HDAC1/2),and deoxynucleotidyl transferase terminal-interacting protein 1(DNTTIP1).Moreover,ZFP541 binds to and activates the expression of genes involved in meiosis and post-meiosis including Kctd19;in turn,KCTD19 promotes the transcriptional activation activity of ZFP541.Taken together,our studies reveal that the ZFP541/KCTD19 signaling complex,acting as a key transcription regulator,plays an indispensable role in male fertility by regulating pachytene progression.

Medaka factor in the germline alpha(figα)is indispensable for oogenesis but not for gonadal sex differentiation

A basic helix-loop-helix transcription factor,figα,is one of the earliest marker genes of oocyte differentiation in vertebrates.In the present study,we made figαknockout medaka by CRISPR/Cas9,expecting aborted progress of oogenesis,to see if differentiation of somatic ovarian tissues is affected.Figαknockout male gonads differentiated normally into testes with functional sperm.The females,on the other hand,were sterile;there are oocytes only up to pachytene.No growing oocytes in diplotene were found.The phenotype was already apparent at 10 days after hatching,when diplotene oocytes start to develop in the control.Furthermore,several putative target genes of figαwere not expressed in the mutant female gonads.Previous studies showed that medaka lacking germ cells have morphologically abnormal gonads and female to male sex reversal occurred.Figαknockout female gonads differentiated morphologically into ovaries,suggesting that in medaka,figαknockout prevents oogenesis progress into diplotene stage,and ovarian differentiation do not need developed oocytes beyond pachytene stage.This ovarian phenotype reminded us of"pachytene checkpoint",which blocks meiotic progress into diplotene when chromosomal abnormalities are present.Therefore,we further analyzed phenotype of oocytes in the mutant ovaries.The number of oocytes at each meiotic stage suggested that oogenesis normally proceeded up to pachytene stage.The number of apoptotic oocytes in the knockout ovaries was small.Finally,we did not detect any abnormal pairings or unrepaired double-strand breaks by immunostaining.Taken together,figαknockout likely stop oocyte growth at or around pachytene via unknown mechanisms other than"pachytene checkpoint"and arrested oocytes remained in the ovary.This is in contrast to figαknockout mice where all oocytes underwent apoptosis quickly after birth.

Identification of Chromosomes from Multiple Rice Genomes Using a Universal Molecular Cytogenetic Marker System

To develop reliable techniques for chromosome identification is critical for cytogenetic research, especially for genomes with a large number and smaller-sized chromosomes. An efficient approach using bacterial artificial chromosome （BAC） clones as molecular cytological markers has been developed for many organisms. Herein, we present a set of chromosomal arm-specific molecular cytological markers derived from the gene-enriched regions of the sequenced rice genome. All these markers are able to generate very strong signals on the pachytene chromosomes of Oryza sativa L. （AA genome） when used as fluorescence in situ hybridization （FISH） probes. We further probed those markers to the pachytene chromosomes of O. punctata （BB genome） and O. officinalis （CC genome） and also got very strong signals on the relevant pachytene chromosomes. The signal position of each marker on the related chromosomes from the three different rice genomes was pretty much stable, which enabled us to identify different chromosomes among various rice genomes. We also constructed the karyotype for both O. punctata and O. officinalis with the BB and CC genomes, respectively, by analysis of 10 pachytene cells anchored by these chromosomal arm-specific markers.