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1.  Toward a More Precise and Informative Nomenclature Describing Fetal and Neonatal Male Germ Cells in Rodents1 
Biology of Reproduction  2013;89(2):47.
ABSTRACT
The germ cell lineages are among the best characterized of all cell lineages in mammals. This characterization includes precise nomenclature that distinguishes among numerous, often subtle, changes in function or morphology as development and differentiation of germ cells proceed to form the gametes. In male rodents, there are at least 41 distinct cell types that occur during progression through the male germ cell lineage that gives rise to spermatozoa. However, there is one period during male germ cell development—that which occurs immediately following the primordial germ cell stage and prior to the spermatogonial stage—for which the system of precise and informative cell type terminology is not adequate. Often, male germ cells during this period are referred to simply as “gonocytes.” However, this term is inadequate for multiple reasons, and it is suggested here that nomenclature originally proposed in the 1970s by Hilscher et al., which employs the terms M-, T1-, and T2-prospermatogonia, is preferable. In this Minireview, the history, proper utilization, and advantages of this terminology relative to that of the term gonocytes are described.
A case for updating the terminology of male germ cells during the fetal and neonatal stages in male rodents.
doi:10.1095/biolreprod.113.110502
PMCID: PMC4076367  PMID: 23843236
gonocytes; male germ cells; prospermatogonia
2.  A 1.1-Mb Segmental Deletion on the X Chromosome Causes Meiotic Failure in Male Mice1 
Biology of Reproduction  2013;88(6):159.
ABSTRACT
The mammalian X chromosome contains a large number of multicopy genes that are expressed during spermatogenesis. The roles of these genes during germ cell development and the functional significance of gene multiplication remain mostly unexplored, as the presence of multicopy gene families poses a challenge for genetic studies. Here we report the deletion of a 1.1-Mb segment of the mouse X chromosome that is syntenic with the human Xq22.1 region and contains 20 genes that are expressed predominantly in testis and brain, including three members of the nuclear export factor gene family (Nxf2, Nxf3, and Nxf7) and five copies of preferentially expressed antigen in melanoma-like 3 (Pramel3). We have shown that germline-specific Cre/loxP-mediated deletion of this 1.1-Mb segment is efficient and causes defective chromosomal synapsis, meiotic arrest, and sterility in male mice. Our results demonstrate that this 1.1-Mb region contains one or more novel X-linked factors that are essential for male meiosis.
A 1.1-Mb segment between Nxf2 and Nxf3 on the X chromosome is essential for meiosis in male mice.
doi:10.1095/biolreprod.112.106963
PMCID: PMC4070866  PMID: 23677977
male fertility; male infertility; meiosis; meiotic arrest; segmental deletion; spermatogenesis; X chromosome
3.  Meiosis I Arrest Abnormalities Lead to Severe Oligozoospermia in Meiosis 1 Arresting Protein (M1ap)-Deficient Mice1  
Biology of Reproduction  2012;88(3):76.
ABSTRACT
Meiosis 1 arresting protein (M1ap) is a novel vertebrate gene expressed exclusively in germ cells of the embryonic ovary and the adult testis. In male mice, M1ap expression, which is present from spermatogonia to secondary spermatocytes, is evolutionarily conserved and has a specific spatial and temporal pattern suggestive of a role during germ cell development. To test its function, mice deficient in M1ap were created. Whereas females had histologically normal ovaries, males exhibited reduced testicular size and a myriad of tubular defects, which led to severe oligozoospermia and infertility. Although some germ cells arrested at the zygotene/pachytene stages, most cells advanced to metaphase I before arresting and entering apoptosis. Cells that reached metaphase I were unable to properly align their chromosomes at the metaphase plate due to abnormal chromosome synapses and failure to form crossover foci. Depending on the state of tubular degeneration, all germ cells, with the exemption of spermatogonia, disappeared; with further deterioration, tubules displaying only Sertoli cells reminiscent of Sertoli cell-only syndrome in humans were observed. Our results uncovered an essential role for M1ap as a novel germ cell gene not previously implicated in male germ cell development and suggest that mutations in M1AP could account for some cases of nonobstructive oligozoospermia in men.
Meiosis 1 arresting protein-deficient spermatocytes exhibit faulty synapses and recombination foci formation, leading to pachytene and metaphase I arrest and apoptosis.
doi:10.1095/biolreprod.111.098673
PMCID: PMC4013860  PMID: 23269666
infertility; meiosis; meiotic arrest; oligozoospermia; spermatogenesis; testis; vertebrates
4.  Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) and Maturation of Murine Gonocytes and Spermatogonia Induced by Retinoic Acid In Vitro1 
Biology of reproduction  2007;78(3):537-545.
Vitamin A deficiency in the mouse results in an arrest in the progression of undifferentiated spermatogonia to differentiating spermatogonia. The supplement of retinol to vitamin-A-deficient mice reinitiates spermatogenesis in a synchronous manner throughout the testes. It is unclear whether the effects of retinoids are the result of a direct action on germ cells or are indirectly mediated through Sertoli cells. The expression of Stimulated by retinoic acid gene 8 (Stra8), which is required for spermatogenesis, is directly related to the availability of retinoic acid (RA). Analysis of gene expression by microarrays revealed moderate levels of Stra8 transcript in gonocytes and high levels in A and B spermatogonia. Stra8 mRNA levels were greatly reduced or absent in germ cells once they entered meiosis. This study examined the effect of retinoic acid on cultured neonatal testes and isolated gonocytes/spermatogonia in vitro. THY1+ and KIT+ germ cells were isolated by magnetic-activated cell sorting from the testes of mice of different ages. Isolated germ cells were cultured and treated with either vehicle (ethanol) or RA without feeder cells. We found that 1) Stra8 is predominantly expressed in premeiotic germ cells, 2) RA stimulates gonocyte DNA replication and differentiation in cultured neonatal testes, 3) in the absence of feeder cells, RA directly induces the transition of undifferentiated spermatogonia to differentiating spermatogonia by stimulating Stra8 and Kit gene expression, 4) RA dramatically stimulates Stra8 expression in undifferentiated spermatogonia but has a lesser impact in differentiating spermatogonia, 5) endogenous Stra8 gene expression is higher in differentiating spermatogonia than in undifferentiated spermatogonia and could mediate the RA effects on spermatogonial maturation, and 6) RA stimulates a group of genes involved in the metabolism, storage, transport, and signaling of retinoids.
doi:10.1095/biolreprod.107.064337
PMCID: PMC3208258  PMID: 18032419
differentiation; gonocytes; in vitro; retinoic acid; spermatogenesis; spermatogonia; spermatogonial differentiation; Stra8
5.  Microarray-Based Analysis of Cell-Cycle Gene Expression During Spermatogenesis in the Mouse1 
Biology of Reproduction  2010;83(4):663-675.
Mammalian spermatogenesis is a continuum of cellular differentiation in a lineage that features three principal stages: 1) a mitotically active stage in spermatogonia, 2) a meiotic stage in spermatocytes, and 3) a postreplicative stage in spermatids. We used a microarray-based approach to identify changes in expression of cell-cycle genes that distinguish 1) mitotic type A spermatogonia from meiotic pachytene spermatocytes and 2) pachytene spermatocytes from postreplicative round spermatids. We detected expression of 550 genes related to cell-cycle function in one or more of these cell types. Although a majority of these genes were expressed during all three stages of spermatogenesis, we observed dramatic changes in levels of individual transcripts between mitotic spermatogonia and meiotic spermatocytes and between meiotic spermatocytes and postreplicative spermatids. Our results suggest that distinct cell-cycle gene regulatory networks or subnetworks are associated with each phase of the cell cycle in each spermatogenic cell type. In addition, we observed expression of different members of certain cell-cycle gene families in each of the three spermatogenic cell types investigated. Finally, we report expression of 221 cell-cycle genes that have not previously been annotated as part of the cell cycle network expressed during spermatogenesis, including eight novel genes that appear to be testis-specific.
Distinct networks of cell-cycle genes are associated with the mitotic, meiotic, and postreplicative stages of spermatogenesis.
doi:10.1095/biolreprod.110.084889
PMCID: PMC2957154  PMID: 20631398
cell cycle; gametogenesis; gene regulation; meiosis; microarray; spermatogenesis; testis
6.  Phosphoglycerate Kinase 2 (PGK2) Is Essential for Sperm Function and Male Fertility in Mice1 
Biology of Reproduction  2009;82(1):136-145.
Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2−/− and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs−/− males, the Pgk2−/− males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2−/− mice may provide further insights into the regulation of sperm metabolism.
Phosphoglycerate kinase 2, a glycolytic isozyme with restricted expression in the male germline, is required for sperm function and male fertility but not for completion of spermatogenesis.
doi:10.1095/biolreprod.109.079699
PMCID: PMC2802118  PMID: 19759366
gene targeting; glycolysis; male fertility; spermatogenesis; sperm metabolism; sperm motility
7.  Dynamic Nuclear Organization of Constitutive Heterochromatin During Fetal Male Germ Cell Development in Mice1 
Biology of Reproduction  2009;80(4):804-812.
In mice, male germ cells enter mitotic arrest beginning at 13.5 days postcoitum (dpc), and remain suspended in the G0/G1 cell cycle stage until after birth. During this period, male germ cells undergo extensive epigenetic reprogramming, which is essential for their subsequent function as male gametes. A global reorganization and spatial clustering of constitutive heterochromatin has been implicated in epigenetic plasticity during cellular differentiation. Here, we have studied the dynamics of heterochromatin in fetal (12.5–19.5 dpc) and neonatal (4 days postpartum) male germ cells. We monitored constitutive heterochromatin-specific markers, and observed changes in the association of histone H3 trimethylation of lysine 9 (H3K9me3), binding of heterochromatin protein 1, and patterns of 4′,6-diamino-2-phenylindole staining in pericentric regions of chromosomes, along with a coincident loss of chromocenters in fetal prospermatogonia during mitotic arrest. We also observed a transient loss of H3K9me3 associated with major and minor satellite repeat sequences, plus inactivation of histone methyltransferases (Suv39h1 and Suv39h2), and transient activation of histone demethylase (Jmjd2b) in these same cells. These epigenetic changes were correlated with relocation of centromeric regions toward the nuclear periphery in prospermatogonia during mitotic arrest. Taken together, these results show that constitutive heterochromatin undergoes dramatic reorganization during prespermatogenesis. We suggest that these dynamic changes in heterochromatin contribute to normal epigenetic reprogramming of the paternal genome in fetal prospermatogonia suspended in the G0/G1 stage, and that this also represents an epigenomic state that is particularly amenable to reprogramming.
The constitutive heterochromatin undergoes dramatic reorganization during fetal male germ cell development.
doi:10.1095/biolreprod.108.072603
PMCID: PMC2804833  PMID: 19129513
constitutive heterochromatin; early development; epigenetic; gamete biology; gametogenesis; histone modifications; mitotic arrest; prespermatogenesis; spermatogenesis
8.  A Conserved E2F6-Binding Element in Murine Meiosis-Specific Gene Promoters1 
Biology of Reproduction  2008;79(5):921-930.
During gametogenesis, germ cells must undergo meiosis in order to become viable haploid gametes. Successful completion of this process is dependent upon the expression of genes whose protein products function specifically in meiosis. Failure to express these genes in meiotic cells often results in infertility, whereas aberrant expression in somatic cells may lead to mitotic catastrophe. The mechanisms responsible for regulating the timely expression of meiosis-specific genes have not been fully elucidated. Here we demonstrate that E2F6, a member of the E2F family of transcription factors, is essential for the repression of the newly identified meiosis-specific gene, Slc25a31 (also known as Ant4, Aac4), in somatic cells. This discovery, along with previous studies, prompted us to investigate the role of E2F6 in the regulation of meiosis-specific genes in general. Interestingly, the core E2F6-binding element (TCCCGC) was highly conserved in the proximal promoter regions of 19 out of 24 (79.2%) meiosis-specific genes. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%). In the absence of E2F6, only a portion of these meiosis-specific genes was derepressed in somatic cells. However, endogenous E2F6 bound to the promoters of these meiosis-specific genes regardless of whether they required E2F6 for their repression in somatic cells. Further, E2F6 overexpression was capable of reducing their transcription. These findings indicate that E2F6 possesses a broad ability to bind to and regulate the meiosis-specific gene population..
Meiosis-specific genes often harbor an E2F6-binding element in their proximal promoter regions upon which E2F6 binds and reduces expression, indicating that E2F6 may broadly serve as a repressor of meiosis-specific genes in somatic cells.
doi:10.1095/biolreprod.108.067645
PMCID: PMC2715002  PMID: 18667754
E2F6; gene regulation; meiosis; repression; Slc25a31

Results 1-8 (8)