Pachytene Stage Research Articles

L3MBTL2 regulates chromatin remodeling during spermatogenesis.

Lethal (3) malignant brain tumor like 2 (L3MBTL2) is a member of the MBT-domain proteins, which are involved in transcriptional repression and implicated in chromatin compaction. Our previous study has shown that L3MBTL2 is highly expressed in the testis, but its role in spermatogenesis remains unclear. In the present study, we found that L3MBTL2 was most highly expressed in pachytene spermatocytes within the testis. Germ cell-specific ablation of L3mbtl2 in the testis led to increased abnormal spermatozoa, progressive decrease of sperm counts and premature testicular failure in mice. RNA-sequencing analysis on L3mbtl2 deficient testes confirmed that L3MBTL2 was a transcriptional repressor but failed to reveal any significant changes in spermatogenesis-associated genes. Interestingly, L3mbtl2 deficiency resulted in increased γH2AX deposition in the leptotene spermatocytes, subsequent inappropriate retention of γH2AX on autosomes, and defective crossing-over and synapsis during the pachytene stage of meiosis I, and more germ cell apoptosis and degeneration in aging mice. L3MBTL2 interacted with the histone ubiquitin ligase RNF8. Inhibition of L3MBTL2 reduced nuclear RNF8 and ubH2A levels in GC2 cells. L3mbtl2 deficiency led to decreases in the levels of the RNF8 and ubH2A pathway and in histone acetylation in elongating spermatids, and in protamine 1 deposition and chromatin condensation in sperm. These results suggest that L3MBTL2 plays important roles in chromatin remodeling during meiosis and spermiogenesis.

Transition from a meiotic to a somatic-like DNA damage response during the pachytene stage in mouse meiosis.

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early “meiotic” repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a “somatic-like” repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase.

Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships

Whole-chromosome painting probes were developed for each of the 10 chromosomes of maize by producing amplifiable libraries of unique sequences of oligonucleotides that can generate labeled probes through transcription reactions. These paints allow identification of individual homologous chromosomes for many applications as demonstrated in somatic root tip metaphase cells, in the pachytene stage of meiosis, and in interphase nuclei. Several chromosomal aberrations were examined as proof of concept for study of various rearrangements using probes that cover the entire chromosome and that label diverse varieties. The relationship of the supernumerary B chromosome and the normal chromosomes was examined with the finding that there is no detectable homology between any of the normal A chromosomes and the B chromosome. Combined with other chromosome-labeling techniques, a complete set of whole-chromosome oligonucleotide paints lays the foundation for future studies of the structure, organization, and evolution of genomes.

Frequencies and cytogenetic mechanisms of gynogenetic reproduction and sterility in artificially produced Cobitis F1 hybrids from parental species with different degree of phylogenetic relatedness

Frequencies and cytogenetic mechanisms of gynogenetic reproduction and sterility in artificially produced Cobitis F1 hybrids from parental species with different degree of phylogenetic relatedness

Triploid production via heat-induced diploidisation of megaspores in Populus pseudo-simonii

Triploid breeding in Populus enhances the commercial properties of cultivated trees. Sexual polyploidization through the production of diploid gametes produces elite triploids with heterosis and ploidy vigour. Populus pseudo-simonii is a species indigenous to China. It is highly adaptable and resistant drought, cold, and saline–alkaline stresses. To improve the properties in growth and pulpwood traits of P. pseudo-simonii, we used heat treatments to induce chromosome doubling in the megaspores as a first step in the production of triploid saplings. The heat treatments were applied during development stages of the female flower buds. We produced 117 triploids via the diploidisation of megaspores. The highest proportion of triploids among the offspring (51.43%) was obtained by applying a heat treatment of 40 °C over 4 h to flower buds in which one-third of the catkins were outside the bract scales. The proportion of pachytene was positively correlated with the average rate of triploid production in each bud developmental stage. Thus, among the phases of meiosis, the pachytene stage may be most susceptible to heat treatments aiming to diploidise the megaspores in P. pseudo-simonii. The megasporocytes in these treated flower buds were mostly in the first meiotic division stages, suggesting that the first meiotic division restitution (FDR) type 2n megaspores may have been induced by our experimental procedures. These triploid hybrids derived from FDR type 2n megaspores will probably contribute significantly to programmes aimed at improving commercial properties of Populus congeners in the section Tacamahaca.

Meiosis in the scorpion Tityus silvestris: new insights into achiasmatic chromosomes.

ABSTRACTAchiasmatic male meiosis in scorpions is characterized by a high frequency of gaps, asynaptic regions and multivalent associations. Here, we performed an immunocytogenetic analysis to investigate recombination, and synapsis and chromatin-remodeling events during meiosis of the scorpion Tityus silvestris. Our results demonstrate that the synaptonemal complex (SC) begins its organization in the zygotene stage and persists until metaphase I. The advancement of the synaptic process is related to the epigenetic modification histone H3 lysine 27 trimethylation (H3K27m3). The distribution and dynamics patterns of variant γH2AX and recombinase Rad51 during achiasmatic meiosis suggests formation and repair of DNA double-strand breaks (DSBs) during early stages of prophase I. The epigenetic modifications, histone H3 lysine 4 trimethylation (H3K4m3) and histone H3 lysine 9 acetylation (H3K9ac), showed a dispersed distribution along the bivalents, suggesting that transcriptional activity is maintained constitutively during prophase I. However, H3K9ac modifications are absent in constitutive heterochromatin carrying the 45S rDNA in pachytene and post-pachytene stages. Collectively, our data demonstrate that T. silvestris exhibits adaptations to the achiasmatic mode, and suggest that epigenetic modifications may act in the regulation of these mechanisms to favor the normal continuation of meiosis in this scorpion.

Germ plasm provides clues on meiosis: the concerted action of germ plasm granules and mitochondria in gametogenesis of the clam Ruditapes philippinarum.

SummaryGerm plasm-related structures (GPRS) are known to accompany meiotic cell differentiation but their dynamics are still poorly understood. In this study, we analyzed the ultrastructural mechanisms of GPRS transformation during oogenesis and spermatogenesis of the bivalve mollusc Ruditapes philippinarum (Manila clam), exploring patterns of GPRS activity occurring at meiosis onset, sex-specific difference/similarity of such patterns, and the involvement of mitochondria during GPRS-assigned events. In the two sexes, the zygotene-pachytene stage of meiosis is anticipated by three shared steps. First, the dispersion of germ plasm granules containing the germ line determinant VASA occurs. Second, the VASA protein deriving from germ plasm granules enters neighbouring mitochondria and appears to induce mitochondrial matter release, as supported by cytochrome B localization outside the mitochondria. Third, intranuclear VASA entrance occurs and the protein appears involved in chromatin reorganization, as supported by VASA localization in synaptonemal complexes. In spermatogenesis, these three steps are sufficient for the normal course of meiosis. In oogenesis, these are followed by the action of 'germ plasm granule formation complex', a novel type of structure that appears alternative to the Balbiani body. The possibility of germ plasm involvement in reproductive technologies is also suggested.

C. elegans ZHP-4 is required at multiple distinct steps in the formation of crossovers and their transition to segregation competent chiasmata.

Correct segregation of meiotic chromosomes depends on DNA crossovers (COs) between homologs that culminate into visible physical linkages called chiasmata. COs emerge from a larger population of joint molecules (JM), the remainder of which are repaired as noncrossovers (NCOs) to restore genomic integrity. We present evidence that the RNF212-like C. elegans protein ZHP-4 cooperates with its paralog ZHP-3 to enforce crossover formation at distinct steps during meiotic prophase: in the formation of early JMs and in transition of late CO intermediates into chiasmata. ZHP-3/4 localize to the synaptonemal complex (SC) co-dependently followed by their restriction to sites of designated COs. RING domain mutants revealed a critical function for ZHP-4 in localization of both proteins to the SC and for CO formation. While recombination initiates in zhp-4 mutants, they fail to appropriately acquire pro-crossover factors at abundant early JMs, indicating a function for ZHP-4 in an early step of the CO/NCO decision. At late pachytene stages, hypomorphic mutants exhibit significant levels of crossing over that are accompanied by defects in localization of pro-crossover RMH-1, MSH-5 and COSA-1 to designated crossover sites, and by the appearance of bivalents defective in chromosome remodelling required for segregation. These results reveal a ZHP-4 function at designated CO sites where it is required to stabilize pro-crossover factors at the late crossover intermediate, which in turn are required for the transition to a chiasma that is required for bivalent remodelling. Our study reveals an essential requirement for ZHP-4 in negotiating both the formation of COs and their ability to transition to structures capable of directing accurate chromosome segregation. We propose that ZHP-4 acts in concert with ZHP-3 to propel interhomolog JMs along the crossover pathway by stabilizing pro-CO factors that associate with early and late intermediates, thereby protecting designated crossovers as they transition into the chiasmata required for disjunction.

Germ cell desquamation-based testis regression in a seasonal breeder, the Egyptian long-eared hedgehog, Hemiechinus auritus.

Testes of seasonally breeding species experience a severe functional regression before the non-breeding period, which implies a substantial mass reduction due to massive germ-cell depletion. Two alternative mechanisms of seasonal germ-cell depletion have been described in mammals, apoptosis and desquamation (sloughing), but their prevalence has not been determined yet due to reduced number of species studied. We performed a morphological, hormonal, and molecular study of the mechanism of seasonal testicular regression in males of the Egyptian long eared-hedgehog (Hemiechinus auritus). Our results show that live, non-apoptotic, germ cells are massively depleted by desquamation during the testis regression process. This is concomitant with both decreased levels of serum testosterone and irregular distribution of the cell-adhesion molecules in the seminiferous epithelium. The inactive testes maintain some meiotic activity as meiosis onset is not halted and spermatocytes die by apoptosis at the pachytene stage. Our data support the notion that apoptosis is not the major testis regression effector in mammals. Instead, desquamation appears to be a common mechanism in this class.

Chromosome painting in meiosis reveals pairing of specific chromosomes in polyploid Solanum species.

Analysis of chromosome pairing has been an important tool to assess the genetic similarity of homologous and homoeologous chromosomes in polyploids. However, it is technically challenging to monitor the pairing of specific chromosomes in polyploid species, especially for plant species with a large number of small chromosomes. We developed oligonucleotide-based painting probes for four different potato chromosomes. We demonstrate that these probes are robust enough to monitor a single chromosome throughout the prophase I of meiosis in polyploid Solanum species. Cultivated potato (Solanum tuberosum, 2n = 4x = 48) is an autotetraploid. We demonstrate that the four copies of each potato chromosome pair as a quadrivalent in 66-78% of the meiotic cells at the pachytene stage. Solanum demissum (2n = 6x = 72) is a hexaploid and has been controversial regarding its nature as an autopolyploid or allopolyploid. Interestingly, no hexavalent pairing was observed in meiosis. Instead, we observed three independent bivalents in 83-98% of the meiotic cells at late diakinesis and early metaphase I for the four chromosomes. These results suggest that S. demissum has evolved into a cytologically stable state with predominantly bivalent pairing in meiosis.

Differential Regulation of PIWI-LIKE 2 Expression in Primordial Germ Cell Tumor Cell Lines by Promoter Methylation.

PIWI-LIKE 2, a member of the ARGONAUTE protein family, is exclusively expressed in pre-pachytene and pachytene stages of spermatogenesis. PIWI-LIKE 2 acts in the germ cell development and the silencing of retrotransponsons to maintain the genomic integrity and stem cell character. In the present study we investigated DNA methylation as potential mechanism for the regulation of human PIWI-LIKE 2 expression in cell lines related to spermatozoa precursor cells. We detected a high methylation of the PIWI-LIKE 2 promoter in TCam-2 cells, while in NT2/D1 cells the promoter was hypomethylated. Concordantly, PIWI-LIKE 2 expression is higher in NT2/D1 cells than in TCam-2 cells. By demethylation of the promoter with 5′-Aza-2′-deoxycytidine, PIWI-LIKE 2 expression in TCam-2 was increased, while in NT2/D1 no alterations in PIWI-LIKE 2 expression could be detected. In conclusion, we analyzed the DNA methylation driving PIWI-LIKE 2 expression in undifferentiated germ cell tumors and demonstrated an epigenetic basis for PIWI-LIKE 2 expression in this cell type.

A Role in Apoptosis Regulation for the rad-51 Gene of Caenorhabditis elegans.

The evolutionarily conserved RAD-51 protein is essential for homologous recombination in the germ line as well as homologous repair of DNA double-strand breaks in all eukaryotic cells. In the nematode Caenorhabditis elegans, the rad-51 gene is transcribed into messenger RNAs potentially coding three alternative protein isoforms. Null rad-51 alleles display embryonic lethality, severe defects in chromosome structure, and high levels of germ line apoptosis. To dissect its functions, we genetically modified the C. elegans rad-51 gene by clustered regularly interspaced short palindromic repeats/Cas9 genome-editing technology, obtaining a separation-of-function (sfi-) mutant allele that only disrupts the long-transcript isoform. This mutant shows no defects in an otherwise wild-type meiosis and is able to activate physiological germ cell death, which occurs at the late pachytene stage. However, although the mutant is competent in DNA damage checkpoint activation after exposure to ionizing radiation, it is defective for induction of DNA damage-induced apoptosis in meiotic germ cells. These results suggest that RAD-51 plays a novel role in germ line apoptosis independent of RAD-51-mediated strand invasion for homologous recombination.

Cryptic breakpoint identified by whole-genome mate-pair sequencing in a rare paternally inherited complex chromosomal rearrangement

BackgroundPrecise characterization of apparently balanced complex chromosomal rearrangements in non-affected individuals is crucial as they may result in reproductive failure, recurrent miscarriages or affected offspring.Case presentationWe present a family, where the non-affected father and daughter were found, using FISH and karyotyping, to be carriers of a three-way complex chromosomal rearrangement [t(6;7;10)(q16.2;q34;q26.1), de novo in the father]. The family suffered from two stillbirths, one miscarriage, and has a son with severe intellectual disability. In the present study, the family was revisited using whole-genome mate-pair sequencing. Interestingly, whole-genome mate-pair sequencing revealed a cryptic breakpoint on derivative (der) chromosome 6 rendering the rearrangement even more complex. FISH using a chromosome (chr) 6 custom-designed probe and a chr10 control probe confirmed that the interstitial chr6 segment, created by the two chr6 breakpoints, was translocated onto der(10). Breakpoints were successfully validated with Sanger sequencing, and small imbalances as well as microhomology were identified. Finally, the complex chromosomal rearrangement breakpoints disrupted the SIM1, GRIK2, CNTNAP2, and PTPRE genes without causing any phenotype development.ConclusionsIn contrast to the majority of maternally transmitted complex chromosomal rearrangement cases, our study investigated a rare case where a complex chromosomal rearrangement, which most probably resulted from a Type IV hexavalent during the pachytene stage of meiosis I, was stably transmitted from a fertile father to his non-affected daughter. Whole-genome mate-pair sequencing proved highly successful in identifying cryptic complexity, which consequently provided further insight into the meiotic segregation of chromosomes and the increased reproductive risk in individuals carrying the specific complex chromosomal rearrangement. We propose that such complex rearrangements should be characterized in detail using a combination of conventional cytogenetic and NGS-based approaches to aid in better prenatal preimplantation genetic diagnosis and counseling in couples with reproductive problems.

MOF influences meiotic expansion of H2AX phosphorylation and spermatogenesis in mice.

Three waves of H2AX phosphorylation (γH2AX) have been observed in male meiotic prophase I: the first is ATM-dependent and occurs at leptonema, while the second and third are ATR-dependent, occuring at zygonema and pachynema, respectively. The third wave of H2AX phosphorylation marks and silences unsynapsed chromosomes. Little is known about H2AX phosphorylation expands to chromatin-wide regions in spermatocytes. Here, we report that histone acetyltransferase (HAT) MOF is involved in all three waves of H2AX phosphorylation expansion. Germ cell-specific deletion of Mof in spermatocytes by Stra8-Cre (Mof cKO) caused global loss of H4K16ac. In leptotene and zygotene spermatocytes of cKO mice, the γH2AX signals were observed only along the chromosomal axes, and chromatin-wide H2AX phosphorylation was lost. In almost 40% of early-mid pachytene spermatocytes from Mof cKO mice, γH2AX and MDC1 were detected along the unsynapsed axes of the sex chromosomes, but failed to expand, which consequently caused meiotic sex chromosome inactivation (MSCI) failure. Furthermore, though RAD51 was proficiently recruited to double-strand break (DSB) sites, defects in DSB repair and crossover formation were observed in Mof cKO spermatocytes, indicating that MOF facilitates meiotic DSB repair after RAD51 recruitment. We propose that MOF regulates male meiosis and is involved in the expansion of all three waves of H2AX phosphorylation from the leptotene to pachytene stages, initiated by ATM and ATR, respectively.

Chromosome painting and comparative physical mapping of the sex chromosomes in Populus tomentosa and Populus deltoides.

Dioecious species accounted for 6% of all plant species, including a number of crops and economically important species, such as poplar. However, sex determination and sex chromosome evolution have been studied only in few dioecious species. In poplar, the sex-determining locus was mapped to chromosome 19. Interestingly, this locus was mapped to either a peritelomeric or a centromeric region among different poplar species. We developed an oligonucleotide (oligo)-based chromosome painting probe based on the sequence of chromosome 19 from Populus trichocarpa. We performed chromosome painting in P. tomentosa and P. deltoides. Surprisingly, the distal end on the short arm of chromosome 19, which corresponds to the location of the sex-determining locus reported in several species, was not painted in both species. Thus, the DNA sequences associated with this region have not been anchored to the current chromosome 19 pseudomolecule, which was confirmed by painting of somatic metaphase chromosome 19 of P. trichocarpa. Interestingly, the unpainted distal ends of the two chromosome 19 did not pair at the pachytene stage in 22-24% of the meiotic cells in the two species, suggest that these regions from the sex chromosomes have structurally diverged from each other, resulting in the reduced pairing frequency. These results shed light on divergence of a pair of young sex chromosomes in poplar.

A Comparative Chromosome Mapping Study in Japanese Podismini Grasshoppers (Orthoptera: Acrididae: Melanoplinae)

In the present paper, karyotypes of 7 Japanese Podismini species, Anapodisma beybienkoi, Fruhstorferiola okinawaensis, Parapodisma caelestis, P. mikado, P. setouchiensis, P. tenryuensis, and Sinopodisma punctata (2n♂ = 21, all acrocentric), are described and compared on the basis of conventional (C-banding, DAPI/CMA₃-staining, Ag-NOR) and molecular (FISH with 18S rDNA and telomeric probes) cytogenetic staining methods. This is the first study to report karyotypes of A. beybienkoi and P. caelestis. Differential staining techniques showed karyotypic diversity in these species. The number of 18S rDNA signals ranged from 2 to 6, and the signals were located on the autosomes or sex chromosomes. In all species, clusters of rDNA coincided with Ag-NORs. Telomeric signals occurred at the chromosome ends at the pachytene stage and seldom at other stages of meiosis. Paracentromeric and some distal and interstitial blocks of constitutive heterochromatin were detected in the chromosomes of Anapodisma, Fruhstorferiola, and Parapodisma species. Staining with DAPI and CMA₃ revealed 2 groups of heterochromatin composition. In addition, intraspecific differences in the number of rDNA clusters and C-bands were observed within Parapodisma species. Based on the evidence of cytogenetic characteristics, the monophyly of Tonkinacridina cannot be supported.

Induction of fetal primary oocytes and the meiotic prophase from mouse pluripotent stem cells.

Meiosis is a key mechanism that ensures sexual reproduction and creates genetic diversity. Here we describe a method that induces fetal oocytes and the prophase of the first meiotic division from mouse pluripotent stem cells (PSCs) under defined conditions. PSCs are induced into epiblast-like cells (EpiLCs), which are in turn induced into primordial germ cell-like cells (PGCLCs). PGCLCs are expanded robustly in the presence of forskolin and rolipram, which elevate intracellular cyclic AMP levels. The expanded PGCLCs comprehensively erase their DNA methylome in a manner that recapitulates genome-wide DNA demethylation in germ cells in vivo, and are in turn induced efficiently into the oogenic pathway and the prophase of the first meiotic division up to the pachytene stage in response to bone morphogenetic protein and retinoic acid. This in vitro strategy provides a powerful foundation for exploring the mechanisms of initiation and progression of mammalian oogenesis and meiosis.

Telomeric TERB1-TRF1 interaction is crucial for male meiosis.

During meiotic prophase, the meiosis-specific telomere-binding protein TERB1 regulates chromosome movement required for homologous pairing and recombination by interacting with the telomeric shelterin subunit TRF1. Here, we report the crystal structure of the TRF1-binding motif of human TERB1 in complex with the TRFH domain of TRF1. Notably, specific disruption of the TERB1-TRF1 interaction by a point mutation in the mouse Terb1 gene results in infertility only in males. We find that this mutation causes an arrest in the zygotene-early pachytene stage and mild telomere abnormalities of autosomes but unpaired X and Y chromosomes in pachytene, leading to massive spermatocyte apoptosis. We propose that the loss of telomere structure mediated by the TERB1-TRF1 interaction significantly affects homologous pairing of the telomere-adjacent pseudoautosomal region (PAR) of the X and Y chromosomes in mouse spermatocytes. Our findings uncover a specific mechanism of telomeres that surmounts the unique challenges of mammalian X-Y pairing in meiosis.

Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells invitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression invivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.