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Journal of Visualized Experiments : JoVE Oct 2018The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase...
The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell. The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.
Topics: Animals; Chromosomes; Insecta; Male; Micromanipulation; Spermatocytes
PubMed: 30394368
DOI: 10.3791/57359 -
Reproductive Biology Mar 2022D-Aspartate (D-Asp) and its methylated form N-methyl-d-aspartate (NMDA) promote spermatogenesis by stimulating the biosynthesis of sex steroid hormones. d-Asp also...
D-Aspartate (D-Asp) and its methylated form N-methyl-d-aspartate (NMDA) promote spermatogenesis by stimulating the biosynthesis of sex steroid hormones. d-Asp also induces spermatogonia proliferation directly by activating the ERK/Aurora B pathway. In the present study, a mouse spermatocyte-derived cell line (GC-2) which represents a stage between preleptotene spermatocyte and round spermatids was exposed to 200 μM d-Asp or 50 μM NMDA for 30 min, 2 h, and 4 h to explore the influence of these amino acids on cell proliferation and mitochondrial activities occurring during this process. By Western blotting analyses, the expressions of AMPAR (GluA1-GluA2/3 subunits), cell proliferation as well as mitochondria functionality markers were determined at different incubation times. The results revealed that d-Asp or NMDA stimulate proliferation and meiosis in the GC-2 cells via the AMPAR/ERK/Akt pathway, which led to increased levels of the PCNA, p-H3, and SYCP3 proteins. The effects of d-Asp and NMDA on the mitochondrial functionality of the GC-2 cells strongly suggested an active role of these amino acids in germ cell maturation. In both d-Asp- and NMDA-treated GC-2 cells mitochondrial biogenesis as well as mitochondrial fusion are increased while mitochondria fission is inhibited. Finally, the findings showed that NMDA significantly increased the expressions of the CII, CIII, CIV, and CV complexes of oxidative phosphorylation system (OXPHOS), whereas d-Asp induced a significant increase in the expressions only of the CIV and CV complexes. The present study provides novel insights into the mechanisms underlying the role of d-Asp and NMDA in promoting spermatogenesis.
Topics: Animals; D-Aspartic Acid; Male; Mice; N-Methylaspartate; Spermatocytes; Spermatogenesis; Spermatogonia
PubMed: 35032869
DOI: 10.1016/j.repbio.2021.100601 -
The Journal of Reproduction and... Oct 2013Meiosis is a key step for sexual reproduction in which chromosome number is halved by two successive meiotic divisions after a single round of DNA replication. In the... (Review)
Review
Meiosis is a key step for sexual reproduction in which chromosome number is halved by two successive meiotic divisions after a single round of DNA replication. In the first meiotic division (meiosis I), homologous chromosomes pair, synapse, and recombine with their partners in prophase I. As a result, homologous chromosomes are physically connected until metaphase I and then segregated from each other at the onset of anaphase I. In the subsequent second meiotic division (meiosis II), sister chromatids are segregated. Chromosomal abnormality arising during meiosis is one of the major causes of birth defects and congenital disorders in mammals including human and domestic animals. Hence understanding of the mechanism underlying these unique chromosome behavior in meiosis is of great importance. This review focuses on the roles of cohesin and condensin, and their regulation in chromosome dynamics during mammalian meiosis.
Topics: Adenosine Triphosphatases; Animals; Cell Cycle Proteins; Chromatids; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Chromosomes, Mammalian; DNA-Binding Proteins; Female; Humans; Male; Meiosis; Models, Biological; Multiprotein Complexes; Oocytes; Oogenesis; Spermatocytes; Spermatogenesis; Cohesins
PubMed: 24162807
DOI: 10.1262/jrd.2013-068 -
Philosophical Transactions of the Royal... May 2010In the testis, tight junctions (TJs) are found between adjacent Sertoli cells at the level of the blood-testis barrier (BTB) where they coexist with basal ectoplasmic... (Review)
Review
In the testis, tight junctions (TJs) are found between adjacent Sertoli cells at the level of the blood-testis barrier (BTB) where they coexist with basal ectoplasmic specializations and desmosome-gap junctions. The BTB physically divides the seminiferous epithelium into two distinct compartments: a basal compartment where spermatogonia and early spermatocytes are found, and an adluminal compartment where more developed germ cells are sequestered from the systemic circulation. In order for germ cells (i.e. preleptotene spermatocytes) to enter the adluminal compartment, they must cross the BTB, a cellular event requiring the participation of several molecules and signalling pathways. Still, it is not completely understood how preleptotene spermatocytes traverse the BTB at stage VIII of the seminiferous epithelial cycle. In this review, we discuss largely how TJ proteins are exploited by viruses and cancer cells to cross endothelial and epithelial cells. We also discuss how this information may apply to future studies investigating the movement of preleptotene spermatocytes across the BTB.
Topics: Animals; Humans; Male; Sertoli Cells; Spermatocytes; Spermatogenesis; Testis; Tight Junctions
PubMed: 20403874
DOI: 10.1098/rstb.2010.0010 -
Philosophical Transactions of the Royal... Dec 2011Translational regulation contributes to the control of archetypal and specialized cell cycles, such as the meiotic and early embryonic cycles. Late meiosis and early... (Review)
Review
Translational regulation contributes to the control of archetypal and specialized cell cycles, such as the meiotic and early embryonic cycles. Late meiosis and early embryogenesis unfold in the absence of transcription, so they particularly rely on translational repression and activation of stored maternal mRNAs. Here, we present examples of cell cycle regulators that are translationally controlled during different cell cycle and developmental transitions in model organisms ranging from yeast to mouse. Our focus also is on the RNA-binding proteins that affect cell cycle progression by recognizing special features in untranslated regions of mRNAs. Recent research highlights the significance of the cytoplasmic polyadenylation element-binding protein (CPEB). CPEB determines polyadenylation status, and consequently translational efficiency, of its target mRNAs in both transcriptionally active somatic cells as well as in transcriptionally silent mature Xenopus oocytes and early embryos. We discuss the role of CPEB in mediating the translational timing and in some cases spindle-localized translation of critical regulators of Xenopus oogenesis and early embryogenesis. We conclude by outlining potential directions and approaches that may provide further insights into the translational control of the cell cycle.
Topics: Animals; Cell Cycle; Cyclin B; Embryonic Development; Male; Meiosis; Mitosis; Oocytes; Polyadenylation; Protein Biosynthesis; RNA-Binding Proteins; Repressor Proteins; Spermatocytes; Transcription Factors; Xenopus; Xenopus Proteins; mRNA Cleavage and Polyadenylation Factors
PubMed: 22084390
DOI: 10.1098/rstb.2011.0084 -
Cytogenetic and Genome Research 2017The nuclear organization of spermatocytes in meiotic prophase I is primarily determined by the synaptic organization of the bivalents that are bound by their telomeres... (Review)
Review
The nuclear organization of spermatocytes in meiotic prophase I is primarily determined by the synaptic organization of the bivalents that are bound by their telomeres to the nuclear envelope and described as arc-shaped trajectories through the 3D nuclear space. However, over this basic meiotic organization, a spermatocyte nuclear architecture arises that is based on higher-ordered patterns of spatial associations among chromosomal domains from different bivalents that are conditioned by the individual characteristics of chromosomes and the opportunity for interactions between their domains. Consequently, the nuclear architecture is species-specific and prone to modification by chromosomal rearrangements. This model is valid for the localization of any chromosomal domain in the meiotic prophase nucleus. However, constitutive heterochromatin plays a leading role in shaping nuclear territories. Thus, the nuclear localization of nucleoli depends on the position of NORs in nucleolar bivalents, but the association among nucleolar chromosomes mainly depends on the presence of constitutive heterochromatin that does not affect the expression of the ribosomal genes. Constitutive heterochromatin and nucleoli form complex nuclear territories whose distribution in the nuclear space is nonrandom, supporting the hypothesis regarding the existence of a species-specific nuclear architecture in first meiotic prophase spermatocytes.
Topics: Animals; Cell Nucleolus; Chromosomes; Heterochromatin; Male; Meiotic Prophase I; Mice; Nucleolus Organizer Region; Spermatocytes; Telomere; Translocation, Genetic
PubMed: 28494440
DOI: 10.1159/000460811 -
Sheng Li Xue Bao : [Acta Physiologica... Oct 2014Uchl1 was found to be involved in spermatocyte apoptosis. The aim of the present study was to test whether Uchl1 and its associated proteins Jab1 and p27(kip1) were...
Uchl1 was found to be involved in spermatocyte apoptosis. The aim of the present study was to test whether Uchl1 and its associated proteins Jab1 and p27(kip1) were involved in spermatogenic damages in response to heat-stress in cryptorchidism. Hematoxylin and eosin (HE) staining and DNA end labeling (TUNEL) were used to observe morphological and apoptotic characteristics of spermatogenic cells; Immunohistochemical analysis was used to detect changes of Uchl1 and its associated proteins Jab1 and p27(kip1) in response to heat-stress from cryptorchidism leading to spermatocyte losses; And protein affinity analysis (pull-down) and immunofluorescence co-localization were used to verify the relevance among the three proteins in spermatocytes. The results showed that, Jab1 and p27(kip1), in parallel to Uchl1, increased in spermatocytes of apoptotic appearances in response to heat-stress, but not in multinucleated giant cells; Jab1 bound to Uchl1 in testis protein extracts, and co-localized with Uchl1 and p27(kip1) specifically in spermatocytes with apoptotic appearances. These results suggest that the accumulation of Uchl1 protein is involved in the heat-stress-induced spermatocyte apoptosis through a new pathway related with Jab1 and p27(kip1), but not the formation of multinucleated giant cells.
Topics: Animals; Apoptosis; COP9 Signalosome Complex; Cryptorchidism; Cyclin-Dependent Kinase Inhibitor p27; Hot Temperature; Intracellular Signaling Peptides and Proteins; Male; Mice; Peptide Hydrolases; Spermatocytes; Stress, Physiological; Ubiquitin Thiolesterase
PubMed: 25331998
DOI: No ID Found -
Cell Structure and Function Jul 1984
Review
Topics: Acrosome; Animals; Female; Fertilization; Male; Meiosis; Oocytes; Sperm Motility; Sperm-Ovum Interactions; Spermatocytes; Spermatozoa
PubMed: 6383627
DOI: No ID Found -
Cytometry. Part a : the Journal of the... Jun 2014
Topics: Animals; Flow Cytometry; Male; Meiotic Prophase I; Spermatocytes; Spermatogenesis
PubMed: 24664871
DOI: 10.1002/cyto.a.22468 -
Reproductive Biology and Endocrinology... Jun 2018Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen....
BACKGROUND
Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen. The present study investigates the heat-sensitive transcriptome and miRNAs in the most vulnerable germ cells (spermatocytes and round spermatids) that are primarily targeted at elevated temperature in a bid to identify novel targets for contraception and/or infertility treatment.
METHODS
Testes of adult male rats subjected to surgical cryptorchidism were obtained at 0, 24, 72 and 120 h post-surgery, followed by isolation of primary spermatocytes and round spermatids and purification to > 90% purity using a combination of trypsin digestion, centrifugal elutriation and density gradient centrifugation techniques. RNA isolated from these cells was sequenced by massive parallel sequencing technique to identify the most-heat sensitive mRNAs and miRNAs.
RESULTS
Heat stress altered the expression of a large number of genes by ≥2.0 fold, out of which 594 genes (286↑; 308↓) showed alterations in spermatocytes and 154 genes (105↑; 49↓) showed alterations in spermatids throughout the duration of experiment. 62 heat-sensitive genes were common to both cell types. Similarly, 66 and 60 heat-sensitive miRNAs in spermatocytes and spermatids, respectively, were affected by ≥1.5 fold, out of which 6 were common to both the cell types.
CONCLUSION
The study has identified Acly, selV, SLC16A7(MCT-2), Txnrd1 and Prkar2B as potential heat sensitive targets in germ cells, which may be tightly regulated by heat sensitive miRNAs rno-miR-22-3P, rno-miR-22-5P, rno-miR-129-5P, rno-miR-3560, rno-miR-3560 and rno-miR-466c-5P.
Topics: Animals; Gene Expression; Gene Expression Profiling; Hot Temperature; Male; Rats; Rats, Sprague-Dawley; Spermatocytes; Spermatogenesis
PubMed: 29859541
DOI: 10.1186/s12958-018-0372-8