Early Pachytene Spermatocytes Research Articles

TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis.

Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. Mouse TRIP13 and its ortholog Pch2 are instrumental in remodeling HORMA domain proteins. HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed homologs. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. Trip13-null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These major phenotypes are consistent with reported phenotypes of Trip13 hypomorph alleles. Trip13 heterozygous mice exhibit meiotic defects that are less severe than the Trip13-null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/SYCP3. Terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres. Therefore, the evolutionarily conserved localization of TRIP13/Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon synapsis in diverse organisms.

Radiation dosimetry and repair kinetics of DNA damage foci in mouse pachytene spermatocyte and round spermatid stages.

Accurate quantification of DNA double strand breaks (DSB) in testicular germ cells is difficult because of cellular heterogeneity and the presence of endogenous γH2AX. Here, we used confocal microscopy to quantify DNA damage and repair kinetics following γ-irradiation (0.5-4 Gy) in three major mouse male germ cell stages, early and late pachytene spermatocytes and round spermatids (RSs), following a defined post irradiation time course. Dose-response curves showing linear best fit validated γH2AX focus as a rapid biodosimetric tool in these substages in response to whole body in vivo exposure. Stage specific foci yield/dose and repair kinetics demonstrated differential radiosensitivity and repair efficiency: early pachytenes (EP) repaired most rapidly and completely followed by late pachytene (LP) and RSs. Repair kinetics for all three stages followed 'exponential decay' in response to each radiation dose. In pachytenes immediate colocalisation of γH2AX and 53BP1, which participates in non-homologous end-joining repair pathway, was followed by dissociation from the major focal area of γH2AX by 4 h demonstrating ongoing DSB repair. These results confirm the differential radiosensitivity and repair kinetics of DSBs in male germ cells at different stages. Taken together, our results provide a simple and accurate method for assessing DNA damage and repair kinetics during spermatogenesis.

Uncoupling of transcription and translation of Fanconi anemia (FANC) complex proteins during spermatogenesis

Male germ cell genome integrity is critical for spermatogenesis, fertility and normal development of the offspring. Several DNA repair pathways exist in male germ cells. One such important pathway is the Fanconi anemia (FANC) pathway. Unlike in somatic cells, expression profiles and the role of the FANC pathway in germ cells remain largely unknown. In this study, we undertook an extensive expression analyses at both mRNA and protein levels of key components of the FANC pathway during spermatogenesis in the mouse. Herein we show that Fanc mRNAs and proteins displayed developmental enrichment within particular male germ cell types. Spermatogonia and pre-leptotene spermatocytes contained the majority of the FANC components examined i.e. complex I members FANCB, FANCG and FANCM, complex II members FANCD2 and FANCI, and complex III member FANCJ. Leptotene, zygotene and early pachytene spermatocytes contained FANCB, FANCG, FANCM and FANCD2. With the exception of FANCL, all FANC proteins examined were not detected in round spermatids. Elongating and elongated spermatids contained FANCB, FANCG, FANCL and FANCJ. qPCR analysis on isolated spermatocytes and round spermatids showed that Fancg, Fancl, Fancm, Fancd2, Fanci and Fancj mRNAs were expressed in both of these germ cell types, indicating that some degree of translational repression of these FANC proteins occurs during the transition from meiosis to spermiogenesis. Taken together, our findings raise the possibility that the assembly of FANC protein complexes in each of the male germ cell type is unique and may be distinct from the proposed model in mitotic cells.

Dosage compensation of an aneuploid genome in mouse spermatogenic cells.

Autosomal trisomies and monosomies bring serious threats to embryonic development through transcriptional disarray caused primarily by the dosage effect of the aneuploid part of the genome. The present study compared the effect of a mouse-viable 30-Mb segmental trisomy on the genome-wide transcriptional profile of somatic (liver) cells and male germ cells. Although the 1.6-fold change in expression of triplicated genes reflected the gene dosage in liver cells, the extra copy genes were compensated in early pachytene spermatocytes, showing 1.18-fold increase. Although more pronounced, the dosage compensation of trisomic genes was concordant with the incidence of HORMAD2 protein and histone gammaH2AX markers of unsynapsed chromatin. A possible explanation for this includes insufficient sensitivity to detect the meiotic silencing of unsynapsed chromatin markers in the 30-Mb region of the chromosome or an earlier silencing effect of another epigenetic factor. Taken together, our results indicate that the meiotic silencing of unsynapsed chromatin is the major, but most likely not the only, factor driving the dosage compensation of triplicated genes in primary spermatocytes.

Compensatory Upregulation of Myelin Protein Zero-Like 2 Expression in Spermatogenic Cells in Cell Adhesion Molecule-1-Deficient Mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells.

Toxicogenomic investigation on rat testicular toxicity elicited by 1,3-dinitrobenzene

Rats were treated with a single oral dose of 10, 25 and 50 mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli–germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity.

Detection of Four Germ Cell Markers in Rats during Testis Morphogenesis: Differences and Similarities with Mice

Germ cells are the only cells capable of transmitting genetic information from generation to generation. Germ cell development has been widely studied in different species. Among mammals, the mouse is the model used in the majority of studies on germ cell differentiation, sex determination and genetics. In the present study, we suggest that the rat is also a very important model for the investigation of the mechanisms of germ cell development. To study rat germ cell development and compare it with that of mouse, the germ cell markers germ cell nuclear antigen 1 (GCNA1), OCT4, mouse vasa homologue (MVH) and specific surface embryonic antigen 1 (SSEA1) were immunolabeled at different phases of embryonic and postnatal development. SSEA1 and GCNA1 were not detected in rat primordial germ cells and fetal gonocytes. GCNA1 was detected postnatally and was present only in leptotene, zygotene and early pachytene spermatocytes. On the other hand, in mice, these markers were detected in germ cells as soon as 11.5 days postcoitum (dpc). MVH was detected in migrating rat primordial germ cells as well as in those that have already colonized the gonads, whereas in mice, MVH is detected only in germ cells that have reached the gonads. In rats, OCT4-positive germ cells were detected from 13 to 17 dpc, but not at 19 dpc or in postnatal testes. This is in contrast with mice that show OCT4 labeling in both embryonic and adult testes. These data suggest that primordial germ cell development in rats and mice shows considerable differences and that the rat may also be an important model to study the embryonic development of germ cells.

Zmynd15 Encodes a Histone Deacetylase-dependent Transcriptional Repressor Essential for Spermiogenesis and Male Fertility

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Oxidative stress in mature rat testis and its developmental changes

Active oxygen causes various problems including male infertility through the oxidation of DNA, proteins, and lipids. In the present study, we examined the immunohistochemical localization of molecules involved in oxidative stress including 8-hydroxy-2-deoxyguanosine (8-OHdG), superoxide dismutase (SOD), and protein disulfide isomerase (PDI) in mature and developing rat testes. In mature rat testes, 8-OHdG was detected in leptotene, zygotene, and early pachytene spermatocytes, while its expression was weak in late pachytene stage spermatocytes. On the other hand, SOD was detected in late pachytene spermatocytes but not in early pachytene and former spermatocytes, suggesting the efficient removal of active oxygen by SOD in late pachytene spermatocytes. In developing rat testes, 8-OHdG expression peaked at 4 weeks when spermatocytes started to differentiate to the late pachytene stage, while SOD started to be expressed at 4 weeks after birth. These findings suggest that the defense system against oxidative stress by SOD is developed in late pachytene stage spermatocytes at 4 weeks after birth. The present findings aid our understanding of the defensive mechanism against oxidative stress in developing and mature testes.

Genetic probing of homologous recombination and non-homologous end joining during meiotic prophase in irradiated mouse spermatocytes

This study was designed to obtain a better insight into the relative contribution of homologous recombination (HR) and non-homologous end joining (NHEJ) to the repair of radiation-induced DNA double-strand breaks (DSBs) at first meiotic prophase. Early and late pachytene and early diplotene spermatocytes that had completed crossing over were sampled. We studied the kinetics of γ-H2AX chromatin foci removal after irradiation of mice deficient for HR and mice deficient for NHEJ. Analyzing γ-H2AX signals in unirradiated RAD54/RAD54B deficient spermatocytes indicated incomplete meiotic recombination repair due to the pronounced increase of γ-H2AX foci in late prophase primary spermatocytes. In these mice, 8 h after irradiation, early pachytene spermatocytes showed a reduction of the numbers of γ-H2AX foci by 52% compared to 82% in the wild type, the difference being significant. However, after crossing over (in late pachytene and early diplotene), no effect of RAD54/RAD54B deficiency on the reduction of irradiation-induced foci was observed. In NHEJ deficient SCID mice, repair kinetics in early spermatocytes were similar to those in wild type mice. However, 1 h after irradiation in late pachytene and early diplotene spermatocytes 1.7 times more foci were found than in wild type mice. This difference might be related to the absence of a DNA-PKcs dependent fast repair component in SCID mice. As subsequent repair is normal, HR likely is taking over. Taken together, the results obtained in RAD54/RAD54B deficient mice and in SCID mice indicate that DSB repair in early pachytene spermatocytes is mainly carried out through HR. In late spermatocytes (late pachytenes and early diplotenes) NHEJ is active. However, probably there is an interplay between these repair pathways and when in late spermatocytes the NHEJ pathway is compromised HR may take over.

167. MUSASHI FAMILY OF RNA BINDING PROTEINS: CELL CYCLE REGULATORS IN SPERMATOGENESIS

Mammalian meiosis is a tightly regulated process involving specialized cell cycle progression and morphogenetic changes. We have demonstrated that the Musashi family of RNA binding proteins is implicated in the regulation of spermatogonial stem self renewal and germ cell differentiation. Here we describe the novel mechanism by which the Musashi family proteins, Msi1 and Msi2, act to control exit from spermatogonial mitotic amplification and normal entry into meiosis. Gene and protein analysis indicated overlapping Msi1 and Msi2 profiles in enriched populations of isolated germ cells and reciprocal subcellular expression patterns in spermatogonia and pachytene spermatocytes/ round spermatids in testes sections. Recombinant Msi1 protein-RNA pulldown and microarray analysis coupled with in vitro shRNA knockdown studies in spermatogonial culture and subsequent immunoprecipitation and qPCR established that Msi1 targeted Msi2 mRNA for post transcriptional translational repression. Immunoprecipitation of Msi2 target mRNA and subsequent qPCR together with in vitro shRNA knockdown studies inround spermatidculture identified a cell cycle inhibitor protein CDKN1C (p57kip2) as the principal target of Msi2 translational inhibition. Immunolocalisation of CDKN1C protein indicated that expression of this cell cycle regulator coincided with the nuclear import of Msi1 and the appearance of cytoplasmic Msi2 expression in early pachytene spermatocytes. Using a transgenic Msi1 overexpression mouse model in conjunction with quantitative gene and protein expression, we confirmed Msi1 targeting of Msi2 and subsequent Msi2 targeting of CDKN1C for translational repression in vivo. Ectopic overexpression of Msi1 in germ cellsinduces substantial Msi2 downregulation and aberrant CDKN1C expression, resulting in abnormal spermatogenic differentiation, germ cell apoptosis/arrest and sterility. In conclusion, our results indicate a sophisticated molecular switch encompassing cell cycle protein regulation by Musashi family proteins, is required for normal exit from mitotic division, entry into meiosis and post meiotic germ cell differentiation.

Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis

Endocrine and local secretory factors have long been known to be required for spermatogenesis. Evidence has been accumulating in recent years indicating that direct contact between spermatogenic and Sertoli cells is also required for spermatogenesis. Cell adhesion molecules of various types have been found in the mammalian testis that are expressed in spermatogenic and/or Sertoli cells and involved in homophilic and/or heterophilic binding. We have cloned a novel cell adhesion molecule, cell adhesion molecule-1 (CADM1), also known as immunoglobulin superfamily 4A or spermatogenic immunoglobulin superfamily, from the mouse testis. CADM1 belongs to the immunoglobulin superfamily and is composed of three immunoglobulin-like domains, a transmembrane domain, and a short intracellular domain. In the seminiferous epithelium, CADM1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in elongating spermatids--but not in round spermatids, mature spermatozoa, or Sertoli cells. One of the heterophilic binding partners of CADM1 has proven to be a poliovirus receptor, another member of the immunoglobulin superfamily that is expressed in Sertoli cells. Knockout mice for CADM1 develop male infertility due to defective spermatogenesis. These findings suggest that cell adhesion molecules between spermatogenic and Sertoli cells play essential roles in spermatogenesis.

Small ubiquitin-related modifier (SUMO)-1, SUMO-2/3 and SUMOylation are involved with centromeric heterochromatin of chromosomes 9 and 1 and proteins of the synaptonemal complex during meiosis in men

BACKGROUNDPost-transcriptional modification by SUMOylation is involved in numerous cellular processes including human spermatogenesis. For human male meiosis, we previously showed that the small ubiquitin-related modifier-1 (SUMO-1) protein localizes to chromatin axes in early pachytene spermatocytes, then to kinetochores as meiosis progresses. Here, we delineate possible functional roles based on subcellular localization for SUMO-1 and SUMO-2/3.METHODSWestern and immunoprecipitation analyses were conducted on proteins isolated from the testis of two normal adult fertile men. Combinatorial immunofluorescence and chromosome-specific fluorescence in situ hybridization analyses were performed on male meiocytes obtained during testicular biopsy from four patients undergoing testicular sperm extraction for assisted reproduction technologies.RESULTSThe synaptonemal complex (SC) and SC proteins (SCP)-1 and SCP2, but not SCP3, are SUMOylated by SUMO-1 during the pachytene substage. Likewise, two distinct localization patterns for SUMO-1 are identified: a linear pattern co-localized with autosomal SCs and isolated SUMO-1 near the centromeric heterochromatin of chromosomes 9 and 1. In contrast to SUMO-1, which is not detectable prior to pachytene in normal tissue, SUMO-2/3 is identified as early as leptotene and zygotene and in some, but not all, pachytene cells; no linear patterns were detected. Similar to SUMO-1, SUMO-2/3 localizes in two predominant subnuclear patterns: a single, dense signal near the centromere of human chromosome 9 and small, individual foci co-localized with autosomal centromeres.CONCLUSIONSOur data suggest that SUMO-1 may be involved in maintenance and/or protection of the autosomal SC. SUMO-2/3, though expressed similarly, may function separately and independently during pachytene in men.

Stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and Sertoli cells

Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes, and spermatids suggests the existence of precise programs of gene expression in these cells and in their neighboring somatic Sertoli cells. The objective of this study was to identify the genes that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, and XIII-XIV of the cycle were isolated by microdissection, whereas Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes, and round spermatids were purified from enzymatically dispersed testes. Microarray analysis by using Rat Genome 230 2.0 arrays identified 16,971 probe sets that recognized testicular transcripts, and 398 of these were identified as testis-specific. Expression of 1,286 probe sets were found to differ at least 4-fold between two cell types and also across the stages of the cycle. Pathway and annotated cluster analyses of those probe sets predicted that entire biological pathways and processes are regulated cyclically in specific cells. Important among these are the cell cycle, DNA repair, and embryonic neuron development. Taken together, these data indicate that stage-regulated gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells.

236. Effects of long term recombinant rat follicle-stimulating hormone replacement on the restoration of spermatogenesis after chronic suppression of gonadotrophins in adult rats

Follicle stimulating hormone (FSH) in short-term rat studies supports spermatogenesis at multiple levels, notably spermatogonial development. The role of FSH in supporting full spermatogenesis in rats is still in question as long-term studies have not been possible due the development of neutralising antibodies to heterologous FSH preparations. This study sought to assess the effects of a homologous recombinant rat FSH (rr-FSH) preparation on the long-term restoration of spermatogenesis. Adult rats were GnRH-immunised (GnRH-im) for 12 weeks then, administered an anti-androgen; flutamide (flut), alone or together with rr-FSH (8µg/rat/daily) for 56 days (1 spermatogenic cycle). Germ and Sertoli cell numbers were quantified using an optical disector stereological method. Testis weight, serum FSH and inhibin B and Sertoli cell nuclear volume were significantly reduced to 15%, 13%, 25% and 57% of controls respectively, following GnRH-im+flut treatment. GnRH-im+flut treatment reduced A/I spermatogonial, type B spermatogonial+preleptotene, leptotene+zygotene and early pachytene spermatocyte numbers to 28%, 68%, 50% and 19% (P < 0.001) of controls respectively, with later germ cells rarely observed. After FSH treatment, no significant affect on testis weight, serum FSH and inhibin B or Sertoli cell number were observed. However, rr-FSH treatment significantly increased numbers of A/I spermatogonia, leptotene+zygotene and early pachytene spermatocytes from 28 = >42%, 50 = >69% and 19 = >27% of controls, respectively, while no differences were observed in later germ cell types. rr-FSH also increased (P < 0.05) the volume of Sertoli cell nuclei from 57 = >66% of control. In conclusion, FSH is unable to support full rat spermatogenesis; however, FSH can partially support germ cells notably spermatogonia through to early pachytene spermatocytes, despite the absence of androgenic support.

Identification of Testis‐Specific Male Contraceptive Targets

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

Evolutionarily Conserved Mammalian Adenine Nucleotide Translocase 4 Is Essential for Spermatogenesis

The adenine nucleotide translocases (Ant) facilitate the transport of ADP and ATP by an antiport mechanism across the inner mitochondrial membrane, thus playing an essential role in cellular energy metabolism. We recently identified a novel member of the Ant family in mouse, Ant4, of which gene configuration as well as amino acid homology is well conserved among mammals. The conservation of Ant4 in mammals, along with the absence of Ant4 in nonmammalian species, suggests a unique and indispensable role for this ADP/ATP carrier in mammalian development. Of interest, in contrast to its paralog Ant2, which is encoded by the X chromosome and ubiquitously expressed in somatic cells, Ant4 is encoded by an autosome and selectively expressed in testicular germ cells. Immunohistochemical examination as well as RNA expression analysis using separated spermatogenic cell types revealed that Ant4 expression was particularly high in spermatocytes. When we generated Ant4-deficient mice by targeted disruption, a significant reduction in testicular size was observed without any other distinguishable abnormalities in the mice. Histological examination as well as stage-specific gene expression analysis in adult and neonatal testes revealed a severe reduction of spermatocytes accompanied by increased apoptosis. Subsequently, the Ant4-deficient male mice were infertile. Taken together, these data elucidated the indispensable role of Ant4 in murine spermatogenesis. Considering the unique conservation and chromosomal location of the Ant family genes in mammals, the Ant4 gene may have arisen in mammalian ancestors and been conserved in mammals to serve as the sole and essential mitochondrial ADP/ATP carrier during spermatogenesis where the sex chromosome-linked Ant2 gene is inactivated.

Differential expression of members of the E2F family of transcription factors in rodent testes

BackgroundThe E2F family of transcription factors is required for the activation or repression of differentially expressed gene programs during the cell cycle in normal and abnormal development of tissues. We previously determined that members of the retinoblastoma protein family that interacts with the E2F family are differentially expressed and localized in almost all the different cell types and tissues of the testis and in response to known endocrine disruptors. In this study, the cell-specific and stage-specific expression of members of the E2F proteins has been elucidated.MethodsWe used immunohistochemical (IHC) analysis of tissue sections and Western blot analysis of proteins, from whole testis and microdissected stages of seminiferous tubules to study the differential expression of the E2F proteins.ResultsFor most of the five E2F family members studied, the localizations appear conserved in the two most commonly studied rodent models, mice and rats, with some notable differences. Comparisons between wild type and E2F-1 knockout mice revealed that the level of E2F-1 protein is stage-specific and most abundant in leptotene to early pachytene spermatocytes of stages IX to XI of mouse while strong staining of E2F-1 in some cells close to the basal lamina of rat tubules suggest that it may also be expressed in undifferentiated spermatogonia. The age-dependent development of a Sertoli-cell-only phenotype in seminiferous tubules of E2F-1 knockout males corroborates this, and indicates that E2F-1 is required for spermatogonial stem cell renewal. Interestingly, E2F-3 appears in both terminally differentiated Sertoli cells, as well as spermatogonial cells in the differentiative pathway, while the remaining member of the activating E2Fs, E2F-2 is most concentrated in spermatocytes of mid to late prophase of meiosis. Comparisons between wildtype and E2F-4 knockout mice demonstrated that the level of E2F-4 protein displays a distinct profile of stage-specificity compared to E2F-1, which is probably related to its prevalence and role in Sertoli cells. IHC of rat testis indicates that localization of E2F-5 is distinct from that of E2F-4 and overlaps those of E2F-1 and E2F-2.ConclusionThe E2F-1 represents the subfamily of transcription factors required during stages of DNA replication and gene expression for development of germ cells and the E2F-4 represents the subfamily of transcription factors that help maintain gene expression for a terminally differentiated state within the testis.

Disruption of Spermatogenic Cell Adhesion and Male Infertility in Mice Lacking TSLC1/IGSF4, an Immunoglobulin Superfamily Cell Adhesion Molecule

TSLC1/IGSF4, an immunoglobulin superfamily molecule, is predominantly expressed in the brain, lungs, and testes and plays important roles in epithelial cell adhesion, cancer invasion, and synapse formation. We generated Tslc1/Igsf4-deficient mice by disrupting exon 1 of the gene and found that Tslc1(-/-) mice were born with the expected Mendelian ratio but that Tslc1(-/-) male mice were infertile. In 11-week-old adult Tslc1(-/-) mice, the weight of a testis was 88% that in Tslc1(+/+) mice, and the number of sperm in the semen was approximately 0.01% that in Tslc1(+/+) mice. Histological analysis revealed that the round spermatids and the pachytene spermatocytes failed to attach to the Sertoli cells in the seminiferous tubules and sloughed off into the lumen with apoptosis in the Tslc1(-/-) mice. On the other hand, the spermatogonia and the interstitial cells, including Leydig cells, were essentially unaffected. In the Tslc1(+/+) mice, TSLC1/IGSF4 expression was observed in the spermatogenic cells from the intermediate spermatogonia to the early pachytene spermatocytes and from spermatids at step 7 or later. These findings suggest that TSLC1/IGSF4 expression is indispensable for the adhesion of spermatocytes and spermatids to Sertoli cells and for their normal differentiation into mature spermatozoa.

Quantification of PERF 15 mRNA in Tissue Sections from Rat Testes

We previously conducted basic research to quantify in situ hybridization (ISH) signals in rat testes. In this experimental model, we selected ribosomal RNA (rRNA) as the hybridizable RNA in paraffin sections, since it allowed us to easily analyze ISH signals expressed with digoxygenin (DIG)-labeled probes quantitatively through “posterization” of the images. We applied this method to analyze the quantification of transcript, PERF 15 mRNA. PERF 15 is expressed specifically in the testes and localized in the rigid cytoskeletal structure of the sperm head, and has been considered to be involved in the apoptotic process of spermatogenic cells. Quantification of the signals may help to clarify the detailed function of PERF 15. We further analyzed the signals concomitant with a confocal laser scanning microscope. The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA was greatest in late pachytene and diplotene spermatocytes and early spermatids, followed by early pachytene spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved. The present study may help to determine the concentration of mRNA in tissue sections.