Mutagenesis is elevated in male germ cells obtained from DNA polymerase-beta heterozygous mice

Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

The fine structural localization of testicular phosphatases in man: The control testis

Summary Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes—glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase—in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.Supported in part by a grant from the U.S. Atomic Energy Commission (AT-(40-1)-4002).     

Morphometric studies on lipid inclusions in Sertoli cells during the spermatogenic cycle in the rat

Summary The volume and surface area of lipid inclusions often present in the cytoplasm of rat Sertoli cells was measured directly from semi-thin sections of perfusion-fixed testicular tissues using an image analyser linked to a light microscope. Sertoli cell nuclei were used as a reference for comparing any variations in the measured parameters of lipid inclusions during the rat spermatogenic cycle. Volume density of Sertoli cell lipid inclusions was assessed by morphometric analysis of Sertoli cells photographically reconstructed from electron micrographs. Maximum lipid content in Sertoli cells occurred during stages IX–XIV of the spermatogenic cycle, then declined at stages I–III and remained low from stages IV–VIII. The persistence and increase in number of many large Sertoli cell lipid inclusions beyond the stage where spermatid residual bodies are phagocytosed within the Sertoli cells (stage IX) suggests that the synthesis and lipolysis of Sertoli cell lipid inclusions represents an intrinsic functional cycle of the Sertoli cells. Stage-dependent variations in the lipid content of rat Sertoli cells offers morphological evidence that the metabolic duties of the Sertoli cells are synchronised with the spermatogenic cycle to provide local coordination of the proliferation and maturation of the germ cells.     

Concanavalin A-induced attachment of spermatogenic cells to Sertoli cells in vitro

Adhesive contacts between developing germ cells and Sertoli cells may play an important role in mammalian spermatogenesis. Adhesion between isolated spermatogenic cells (pachytene spermatocytes and round spermatids) and Sertoli cells was studied. The attachment of single mouse germ cells to mouse Sertoli cell layers was weak, but the rate of attachment was stimulated by Concanavalin A (conA; 5 μg/ml). ConA-induced attachment was largely stable during a subsequent incubation for 30 min in the presence of 20 mM α-methyl- -mannoside (an inhibitor of conA). The cellular specificity of the stable attachment of germ cells to Sertoli cells was inferred from the observations that a comparable inhibitor-resistant attachment could not be obtained between germ cells and kidney cells, and between mouse myeloma cells and Sertoli cells. Juxtaposition of male germ cells and Sertoli cells through conA bridges may lead to the subsequent formation of strong and specific cell-cell adhesive bonds.     

RAN-Binding Protein 9 is Involved in Alternative Splicing and is Critical for Male Germ Cell Development and Male Fertility

As a member of the large Ran-binding protein family, Ran-binding protein 9 (RANBP9) has been suggested to play a critical role in diverse cellular functions in somatic cell lineages in vitro, and this is further supported by the neonatal lethality phenotype in Ranbp9 global knockout mice. However, the exact molecular actions of RANBP9 remain largely unknown. By inactivation of Ranbp9 specifically in testicular somatic and spermatogenic cells, we discovered that Ranbp9 was dispensable for Sertoli cell development and functions, but critical for male germ cell development and male fertility. RIP-Seq and proteomic analyses revealed that RANBP9 was associated with multiple key splicing factors and directly targeted >2,300 mRNAs in spermatocytes and round spermatids. Many of the RANBP9 target and non-target mRNAs either displayed aberrant splicing patterns or were dysregulated in the absence of Ranbp9. Our data uncovered a novel role of Ranbp9 in regulating alternative splicing in spermatogenic cells, which is critical for normal spermatogenesis and male fertility.     

The differential expression of lamin epitopes during mouse spermatogenesis

The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ～ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc.     

Spontaneous mutagenesis is enhanced in Apex heterozygous mice

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.     

Systematic variation in mRNA 3'-processing signals during mouse spermatogenesis

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.     

A Biological Model of Accelerated Senescence. 3. Histological Characteristics of Age-Related Changes of Spermatogenic Epithelium in SAM (Senescence-Accelerated Mouse) Mice

The data characterizing the age-related morphological changes in the spermatogenic epithelium of SAMP1 (senescence-accelerated prone) and SAMR1 (senescence-accelerated resistant) mice are presented. In many tubules, early spermatogenesis was accompanied by the formation of many morphologically abnormal germ cells on histological sections of the gonads of sexually immature (three–four weeks) mice of both strains. At this stage, destructive processes in the spermatogenic epithelium were more pronounced in SAMR1 mice. In sexually mature (two–three months) SAMP1 and SAMR1 mice, spermatogenesis as a whole proceeded normally. The first signs of regressive changes in the inner structure of most tubules (disintegration, detachment of spermatogenic epithelium from basal membrane) and morphology of germ cells (pycnosis, nuclear and cytoplasmic vacuolization) were found in SAMP1 mice at the age of six–seven months. In the older age groups (9–10 and 12–15 months), all types of spermatogenic cells were represented in both SAMP1 and SAMR1 mice, but most of these cells were atypical. Mitotic figures were recorded in a population of highly differentiated Sertoli cells.     

The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages.     

Somatic Donor Cell Type Correlates with Embryonic,but Not Extra-Embryonic,Gene Expression in Postimplantation Cloned Embryos

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10^–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.     

Base excision repair efficiency and mechanism in nuclear extracts are influenced by the ratio between volume of nuclear extraction buffer and nuclei-implications for comparative studies

The base excision repair (BER) pathway corrects many different DNA base lesions and is important for genomic stability. The mechanism of BER cannot easily be investigated in intact cells and therefore in vitro methods that reflect the in vivo processes are in high demand. Reconstitution of BER using purified proteins essentially mirror properties of the proteins used, and does not necessarily reflect the mechanism as it occurs in the cell. Nuclear extracts from cultured cells have the capacity to carry out complete BER and can give important information on the mechanism. Furthermore, candidate proteins in extracts can be inhibited or depleted in a controlled way, making defined extracts an important source for mechanistic studies. The major drawback is that there is no standardized method of preparing nuclear extract for BER studies, and it does not appear to be a topic given much attention. Here we have examined BER activity of nuclear cell extracts from HeLa cells, using as substrate a circular DNA molecule with either uracil or an AP-site in a defined position. We show that BER activity of nuclear extracts from the same batch of cells varies inversely with the volume of nuclear extraction buffer relative to nuclei volume, in spite of identical protein concentrations in the BER assay mixture. Surprisingly, the uracil-DNA glycosylase activity (mainly UNG2), but not amount of UNG2, also correlated negatively with the volume of extraction buffer. These studies demonstrate that the method for preparation of nuclear extract is an important factor to consider for in vitro BER analysis and conditions used in comparative studies must be carefully worked out.     

In vivo role of alpha-mannosidase IIx: ineffective spermatogenesis resulting from targeted disruption of the Man2a2 in the mouse

Alpha-mannosidase IIx (MX) is an enzyme closely related to the Golgi N-glycan processing enzyme alpha-mannosidase II (MII). The enzymatic activity of MX in vitro is minimal. Therefore, the in vivo role of MX in N-glycan processing is as yet unclear. The targeted disruption of the gene encoding MX in the mouse resulted in an obvious phenotype, i.e., MX-deficient males were found to be infertile. Testes from homozygous mutant male mice are smaller than those from wild-type or heterozygous littermates. Histology of the MX null mouse testis showed significant reduction of spermatogenic cells in the seminiferous tubules. Electron microscopy showed that prominent intercellular spaces surround MX-deficient spermatogenic cells, suggesting a failure of germ cell adhesion to Sertoli cells. Quantitative structural analyses of N-glycans from wild-type and MX-deficient mouse testis showed that wild-type testes contain GlcNAc-terminated complex type N-glycans, while they are significantly reduced in MX-deficient mutant testis. An in vitro assay for adhesion of spermatogenic cells to Sertoli cells was carried out. By testing the effect of each purified N-glycan oligosaccharide, it was demonstrated that a GlcNAc-terminated tri-antennary, fucosylated N-glycan has an activity on the adhesion between germ cells and Sertoli cells. Thus, the targeted disruption of the gene encoding MX uncovered a novel carbohydrate recognition system in a biologically important process, spermatogenesis.     

IGSF11 is required for pericentric heterochromatin dissociation during meiotic diplotene

Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic and germ cells are required for this regulation. Our results show that deficiency for adhesion molecule IGSF11, which is expressed in both Sertoli cells and germ cells, leads to male infertility in mice. Combining a new meiotic fluorescent reporter system with testicular cell transplantation, we demonstrated that IGSF11 is required in both somatic cells and spermatogenic cells for primary spermatocyte development. In the absence of IGSF11, spermatocytes proceed through pachytene, but the pericentric heterochromatin of nonhomologous chromosomes remains inappropriately clustered from late pachytene onward, resulting in undissolved interchromosomal interactions. Hi-C analysis reveals elevated levels of interchromosomal interactions occurring mostly at the chromosome ends. Collectively, our data elucidates that IGSF11 in somatic cells and germ cells is required for pericentric heterochromatin dissociation during diplotene in mouse primary spermatocytes.     

Mapping the testicular interstitial fluid proteome from normal rats

Communication between the testicular somatic (Sertoli, Leydig, peritubular myoid, macrophage) and germ cell types is essential for sperm production (spermatogenesis), but the communicating factors are poorly understood. We reasoned that identification of proteins in the testicular interstitial fluid (TIF) that bathes these cells could provide a new means to explore spermatogenic function. The aim of this study was to map the proteome of TIF from normal adult rats. Low‐abundance proteins in TIF were enriched using ProteoMiner beads and identified by MALDI‐MS/MS, recognizing 276 proteins. Comparison with proteomic and genomic databases showed these proteins originated from germ cells, somatic cells (Sertoli, peritubular myoid, Leydig), and blood plasma. In silico analysis revealed homologues of >80% TIF proteins in the human plasma proteome, suggesting ready exchange between these fluids. Only 36% of TIF proteins were common with seminiferous tubule fluid that transports mature spermatids to the epididymis, indicating these two fluids are quite different. This TIF proteome provides an important new resource for the study of intercellular communication in the testis.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.