Meiotic transcriptional reprogramming mediated by cell-cell communications in humans and mice revealed by scATACseq and scRNA-seq

Meiosis is a highly complex process significantly influenced by transcriptional regulation.However,studies on the mechanisms that govern transcriptomic changes during meiosis,especially in prophase I,are limited.Here,we performed single-cell ATAC-seq of human testis tissues and observed reprogramming during the transition from zygotene to pachytene spermatocytes.This event,conserved in mice,involved the deactivation of genes associated with meiosis after reprogramming and the activation of those related to spermatogenesis before their functional onset.Furthermore,we identified 282 transcriptional regulators(TRs)that underwent activation or deactivation subsequent to this process.Evidence suggested that physical contact signals from Sertoli cells may regulate these TRs in spermatocytes,while secreted ENHO signals may alter metabolic patterns in these cells.Our results further indicated that defective transcriptional reprogramming may be associated with non-obstructive azoospermia(NOA).This study revealed the importance of both physical contact and secreted signals between Sertoli cells and germ cells in meiotic progression.

High temperature treatment induced production of unreduced 2n pollen in Camellia oleifera

Unreduced gametes through chromosome doubling play a major role in the process of plant polyploidization.Our previous work confirmed that Camellia oleifera can produce natural 2n pollen,and it is possible to induce the 2n pollen formation by high temperature treatment.This study focused on the optimization of the 2n pollen induction technique and the mechanisms of high temperature-induced2n pollen formation in C.oleifera.We found that the optimal protocol for inducing 2n pollen via high temperature was to perform 45℃with4 h at the prophaseⅠstage of the pollen mother cells(PMCs).Meanwhile,high temperature significantly decreased the yield and fertility of2n pollen.Through the observation of meiosis,abnormal chromosome and cytological behaviour was discovered under high-temperature treatment,and we confirmed that the formation of 2n pollen is caused by abnormal cell plate.Based on weighted gene co-expression network analysis,fifteen hub genes related to cell cycle control were identified.After male flower buds were exposed to heat shock,polygalacturonase gene(CoPGX3)was significantly upregulated.We inferred that high temperature causes the CoPGX3 gene to be overexpressed and that CoPGX3 is redistributed into the cytosol where it degrades cytoplasmic pectin,which leads to an abnormal cell plate.Furthermore,abnormal cytokinesis resulted in the formation of dyads and triads,and PMCs divided to produce 2n pollen.Our findings provide new insights into the mechanism of 2n pollen induced by high temperature in a woody plant and lay a foundation for further ploidy breeding of C.oleifera.

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

Asynchronous meiosis in Cucumis hystrix–cucumber synthetic tetraploids resulting in low male fertility

Interspecific hybridization and allopolyploidization contribute to the improvement of many important crops. Recently, we successfully developed an amphidiploid from an interspecific cross between cucumber(Cucumis sativus, 2n = 2x = 14) and its relative C. hystrix(2n = 2x = 24) followed by chemical induction of chromosome doubling. The resulting allotetraploid plant was self-pollinated for three generations. The fertility and seed set of the amphidiploid plants were very low. In this study, we investigated the meiotic chromosome behavior in pollen mother cells with the aid of fluorescence in situ hybridization, aiming to identify the reasons for the low fertility and seed set in the amphidiploid plants. Homologous chromosome pairing appeared normal, but chromosome laggards were common, owing primarily to asynchronous meiosis of chromosomes from the two donor genomes. We suggest that asynchronous meiotic rhythm between the two parental genomes is the main reason for the low fertility and low seed set of the C. hystrix–cucumber amphidiploid plants.

CENP-A Regulates Chromosome Segregation during the First Meiosis of Mouse Oocytes

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

Defective callose walls and cell plates during abnormal meiosis cause male-sterility in the oat mutant zbs1

During meiosis in flowering plants,degradation of the callose wall in tetrads releases newly produced microspores,which develop into mature pollen grains.In this study,we identified zbs1,a male-sterile mutant of naked oat（Avena nuda L.）that displayed complete spikelet sterility due to inviable mature pollen.The abnormal pollen grains originated from microspores with a defective callose wall and cell plate during meiosis.The defective callose wall and cell plate of the zbs1 mutant were detected by the labeling of cell wall epitopes（β-1,3-glucan） with immunogold during meiosis,and an abnormal chromosome configuration was observed by propidium iodide staining.The mature pollen grains of the zbs1 mutant were irregular in shape,and abnormal germination was observed by scanning electron microscopy.Together,our results indicate that the cause of male sterility in zbs1 is abnormal meiosis.

Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis

Alternative splicing can generate multiple mRNAs that differ in their untranslated regions or coding sequences,and these differences might affect mRNA stability or result in different protein isoforms with diverse functions and/or localizations.In this study,we isolated a sterile mutant in rice with abnormal meiosis of microspore mother cells and megaspore mother cells that carried a point mutation in OsRAD1 gene.Cloning of OsRAD1 cDNAs revealed three transcript variants,named as OsRAD1.1,OsRAD1.2 and OsRAD1.3,respectively,which were derived from alternative splicing of the last intron.Proteins derived from the three transcripts were mostly identical except the difference in the very C-terminal domain.The three transcripts exhibited similar expression patterns in various tissues,but the expression level of OsRAD1.1 was the highest.Specific knockout of OsRAD1.1 led to sterility,while knockout of OsRAD1.2 and OsRAD1.3 together did not change the plant fertility.Overexpression of OsRAD1.2 and OsRAD1.3 cDNAs in OsRAD1.1-specific mutant did not complement the plant fertility.Yeast two-hybrid assay showed that OsRAD1.1,but not OsRAD1.2 and OsRAD1.3,interacted with the three other meiosis proteins OsHUS1,OsRAD9 and OsRAD17,suggesting that the C-terminal domain of OsRAD1.1 is critical for the protein function.

A Three-Dimensional (3D) Environment to Maintain the Integrity of Mouse Testicular Can Cause the Occurrence of Meiosis

Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional （3D） agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum （dpp） mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.

Antioxidant procyanidin B2 protects oocytes against cryoinjuries via mitochondria regulated cortical tension

Background:Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation.Antioxidants were always used to antagonist the oxidative stress caused by vitrification.However,the comprehensive mechanism underlying the protective role of antioxidants has not been studied.Procyanidin B2(PCB2)is a potent natural antioxidant and its functions in response to vitrification are still unknown.In this study,the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored,and the mechanisms underlying the protective role of PCB2 were systematically elucidated.Results:Vitrification induced a marked decline in oocyte quality,while PCB2 could improve oocyte viability and further development after parthenogenetic activation.A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress,rescued mitochondrial dysfunction,and improved cell viability.Moreover,PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties.Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration,ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes.Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation,and PCB2 effectively increased the cortical tension through the electron transfer chain pathway.Additionally,PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality.What's more,targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism.Conclusions:Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.

Characterization of meiotic chromosome behavior in the autopolyploid Saccharum spontaneum reveals preferential chromosome pairing without distinct DNA sequence variation

Autopolyploidy and allopolyploidy may represent an evolutionary advantage and are more common in plants than assumed. However, less attention has been paid to autopolyploidy than to allopolyploidy,and its evolutionary consequences are largely unclear, especially for plants with high ploidy levels. In this study, we developed oligonucleotide(oligo)-based chromosome painting probes to identify individual chromosomes in S. spontaneum. Using fluorescence in situ hybridization(FISH), we investigated chromosome behavior during pachytene, metaphase, anaphase, and telophase of meiosis I(MI) in autotetraploid,autooctoploid, and autodecaploid S. spontaneum clones. All autopolyploid clones showed stable diploidized chromosome behavior;so that homologous chromosomes formed almost exclusively bivalents during MI. Two copies of homologous chromosome 8 with similar sizes in the autotetraploid clone showed preferential pairing with each other with respect to the other copies. However, sequence variation analysis showed no apparent differences among homologs of chromosome 8 and all other chromosomes. We suggest that either the stable diploidized pairing or the preferential pairing between homologous copies of chromosome 8 in the studied autopolyploid sugarcane are accounted for by unknown mechanisms other than DNA sequence similarity. Our results reveal evolutionary consequences of stable meiotic behavior in autopolyploid plants.

Transcriptome Analysis During Tetrasporogenesis of Gracilariopsis lemaneiformis and Preliminary Study of the Expressions of Its Meiotic Genes

The transcriptomes of three different parts of the fertile tetrasporophyte of Gracilariopsis lemaneiformis,including tip(T),middle(M),and subjacent(S)parts,with a gradual tetrasporangium maturity were analyzed and compared to identify the genes involved in the process of tetrasporogenesis.The number of differentially expressed genes(DEGs)for the Gple-S versus Gple-T comparison was 10296,and the numbers of DEGs for the Gple-S versus Gple-M and Gple-T versus Gple-M comparisons were 7435 and 1337,respectively.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed,and the results showed the enrichment of 132 KEGG pathways(corrected P<0.05).A total of 58 DEGs related to meiosis were screened and blasted against 18 meiosis-related genes(dmc1,mlh1,mnd1,msh4,msh2,msh6,mre11,pds5,pms1,rad21,rad50,rad51,smc1,smc2,smc4,smc5,smc6,and spo11),including four meiosis-specific genes.The transcriptome comparison indicated that in the T part,the meiosis,ribosome,and RNA transport-related genes were mostly up-regulated compared with those in the other two groups.In the M part,the genes related to ribosomes and the endoplasmic reticulum were also up-regulated compared with those in the lower part.Finally,in the S part,the genes associated with photosynthesis were mostly up-regulated,which might be helpful to the recovery from spore formation and release.

Caseinolytic mitochondrial matrix peptidase X is essential for homologous chromosome synapsis and recombination during meiosis of male mouse germ cells

Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins.The mitochondrial protease complex(ClpXP),which consists of caseinolytic mitochondrial matrix peptidase X(ClpX)and caseinolytic protease P(ClpP)and mediates the degradation of misfolded,damaged,and oxidized proteins,is essential for maintaining mitochondrial homeostasis.ClpXP has been implicated in meiosis regulation,but its precise role is currently unknown.In this study,we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X(Clpx^(cKO))mouse model to investigate the function of ClpX in meiosis.We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis.Specifically,it caused asynapsis of homologous chromosomes and impaired meiotic recombination,resulting in meiotic arrest in the zygotene-to-pachytene transition phase.The loss of ClpX compromised the double-strand break(DSB)repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1(RAD51)to DSB sites.This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in Clpx^(cKO) spermatocytes,as discerned by electron microscopy.Furthermore,ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in Clpx^(cKO) spermatocytes.Taken together,we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing,synapsis,and recombination in spermatocytes during meiotic prophase I.

Differentiation of murine male germ cells to spermatozoa n a soft agar culture system

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system （SACS）, which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum （FCS）. The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia （Vasa, Dazl, OCT-4, C-Kit, GFR- a-l, CD9 and a-6-integrin）, meiotic cells （LDH, Crem-1 and Boule） and post-meiotic cells （Protamine-1, Acrosin and SP-IO）. Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

Production of aneuhaploid and euhaploid sporocytes by meiotic restitution in fertile hybrids between durum wheat Langdon chromosome substitution lines and Aegilops tauschii

Fertile F1 hybrids were obtained between durum wheat （Triticum durum Desf.） Langdon （LDN） and its 10 disomic substitution （LDN DS） lines with Aegilops tauschii accession AS60 without embryo rescue. Selfed seedset rates for hybrids of LDN with AS60 were 36.87% and 49.45% in 2005 and 2006, respectively. Similar or higher selfed seedset rates were observed in the hybrids of 1D （1A）, 1D （1B）, 3D （3A）, 4D （4B）, 7D （7A）, and 2D （2B） with AS60, while lower in hybrids of 3D （3B） ＋ 3BL, 5D （5A） ＋ 5AL, 5D （5B） ＋ 5B and 6D （6B） ＋ 6BS with AS60 compared with the hybrids of LDN with AS60. Observation of male gametogenesis showed that meiotic restitution, both first-division restitution （FDR） and single-division meiosis （SDM） resulted in the formation of functional unreduced gametes, which in turn produced seeds. Both euhaploid and aneuhaploid gametes were produced in F1 hybrids. This suggested a strategy to simultaneously transfer and locate major genes from the ancestral species T. turgidum or Ae. tauschii. Moreover, there was no significant difference in the aneuhaploid rates between the F1 hybrids of LDN and LDN DS lines with AS60, suggesting that meiotic pairing between the two D chromosomes in the hybrids of LDN DS lines with AS60 did not promote the formation of aneuhaploid gametes.

Analysis of the genetic interactions between Cyclin A1, Atm and p53 during spermatogenesis

Aim： To analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double- strand break repair. Methods： Two lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 （Ccnal）; p53- and Ccnal; Atm-double knockout mice. Results： The block in spermatogenesis observed in Cyclin A1-/- （Ccnal-/-） testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccnal-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccnal-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1. Conclusion： Ccnal-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.

Single-nucleotide polymorphisms in HORMAD1 may be a risk factor for azoospermia caused by meiotic arrest in Japanese patients

Genetic mechanisms are implicated as a cause of some male infertility, yet are poorly understood. Meiosis is unique to germ cells and essential for reproduction. The synaptonemal complex is a critical component for chromosome pairing, segregation and recombination. Hormadl is essential for mammalian gametogenesis as knockout male mice are infertile. Hormadl-deficient testes exhibit meiotic arrest in the early pachytene stage and synaptonema! complexes cannot be visualized. To analyze the hypothesis that the human HORMAD1 gene defects are associated with human azoospermia caused by meiotic arrest, mutational analysis was performed in all coding regions by direct sequence analysis of 30 Japanese men diagnosed with azoospermia resulting from meiotic arrest. By the sequence analysis, three polymorphism sites, Single Nucleotide Polymorphism 1 （c. 163A〉G）, SNP2 （c. 501T〉G） and SNP3 （c. 918C〉T）, were found in exons 3, 8 and 10. The 30 patients with azoospermia and 80 normal pregnancy-proven, fertile men were analyzed for HORMAD1 polymorphisms. Both SNP1 and SNP2 were associated with human azoospermia caused by complete early meiotic arrest （P〈0.0S）. We suggest that the HORMAD1 has an essential meiotic function in human spermatogenesis.

Gene expression dynamics during the gonocyte to spermatogonia transition and spermatogenesis in the domestic yak

Background:Spermatogenesis is a cellular differentiation process that includes three major events:mitosis of spermatogonia,meiosis of spermatocytes and spermiogenesis.Steady-state spermatogenesis relies on functions of spermatogonial stem cells(SSCs).Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals.Currently,our knowledge about SSC and spermatogenesis is severely limited in domestic animals.Results:In the present study,we examined transcriptomes of testes from domestic yaks at four different stages(3,5,8 and 24 months of age)and attempted to identify genes that are associated with key developmental events of spermatogenesis.Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3,5,8 and 24 months old yaks were gonocytes,spermatogonia,spermatocytes and elongated spermatids,respectively.RNA-sequencing(RNA-seq)analyses revealed that 11904,4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition,the mitosis to meiosis transition and the meiosis to post-meiosis transition.Further analyses identified a list of candidate genes than may regulate these important cellular processes.CXCR4,a previously identified SSC niche factor in mouse,was one of the up-regulated genes in the 5 months old yak testis.Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis.Conclusions:Together,these findings demonstrated histological changes of postnatal testis development in the domestic yak.During development of spermatogonial lineage,meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression.Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.

SPATA17的单核苷酸多态性可能是日本减速分裂阻滞患者的遗传危险因素

Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, 10 novel genes involved in human spermatogenesis were identified by microarray analysis of human testicular tissue. One of these is spermatogenesis-associated 17 （SPATA17）. To investigate whether defects in the SPATA17 gene are associated with azoospermia due to meiotic arrest, a mutational analysis was conducted, in which the SPATA17 coding regions of 18 Japanese patients with this condition were sequenced. A statistical analysis was carried out that included 18 patients with meiotic arrest, 20 patients with Sertoli-cell-only syndrome （SCOS） and 96 healthy control men. No mutations were found in SPA TA17. However, three coding single nucleotide polymorphisms （cSNPs： SNP 1-SNP3） were detected in the patients with meiotic arrest. No significant differences in the genotype or allele frequencies of SNP1 and SNP2 were found between patients with meiotic arrest and the others. However, the frequency of the SNP3 allele was significantly elevated in the meiotic arrest group （P 〈 0.05）. This study suggests that SPATA17 may play a critical role in human spermatogenesis, especially in meiosis.

Pollen development and multi-nucleate microspores of Populus bolleana Lauche

Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.

DRP1 deficiency induces mitochondrial dysfunction and oxidative stress-mediated apoptosis during porcine oocyte maturation

Background: Environmental pollution induces oxidative stress and apoptosis in mammalian oocytes, which can cause defects in reproduction;however, the molecular regulation of oxidative stress in oocytes is still largely unknown. In the present study, we identified that dynamin-related protein 1(DRP1) is an important molecule regulating oocyte mitochondrial function and preventing oxidative stress/apoptosis. DRP1 is a member of the dynamin GTPase superfamily localized at the mitochondrial-endoplasmic reticulum interaction site, where it regulates the fission of mitochondria and other related cellular processes.Results: Our results show that DRP1 was stably expressed during different stages of porcine oocyte meiosis, and might have a potential relationship with mitochondria as it exhibited similar localization. Loss of DRP1 activity caused failed porcine oocyte maturation and cumulus cell expansion, as well as defects in polar body extrusion.Further analysis indicated that a DRP1 deficiency caused mitochondrial dysfunction and induced oxidative stress,which was confirmed by increased reactive oxygen species levels. Moreover, the incidence of early apoptosis increased as detected by positive Annexin-V signaling.Conclusions: Taken together, our results indicate that DRP1 is essential for porcine oocyte maturation and that a DRP1 deficiency could induce mitochondrial dysfunction, oxidative stress, and apoptosis.