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PubMedDate not supplied

Ultrastructural demonstration of the NADPH-diaphorase activity in peritubular myoid cells and fibroblasts of the lamina propria of the mouse seminiferous tubules.

The NOS-related NADPH-diaphorase activity was studied by transmission electron microscopy in the peritubular myoid cells and fibroblasts of normal mouse testis. The reaction product was observed on the membranes of the endoplasmic reticulum, on the Golgi apparatus and nuclear envelope. The peritubular myoid cells and fibroblasts showed similar ultracytochemical features; the intensity of the enzymatic reaction was suggestive of an important role of the NOS/cGMP enzymatic system in these cells. Some hypotheses on the role of NO in the peritubular myoid cells and fibroblasts are proposed.

Animals↗
PubMed1997

Developmental expression of H3.3A variant histone mRNA in mouse.

Cloning and characterization of H3.3A variant histone expression has recently been reported to be associated with meiotic development in mouse testis and ovary. Using Northern analysis and in situ hybridization, the pattern of H3.3A expression was studied during the development of different tissues. In addition to the differential expression detected in male and female meiosis, H3.3A was found to be highly expressed in preantral follicles of adult ovaries and in the basal regions of seminiferous epithelium corresponding to spermatogonia. Different patterns of expression were observed in somatic tissues, which also differed with respect to the developmental stage of the tissue. The lowest expression was detected in adult skeletal muscle. High expressions were found in foetal liver and spinal cord. These different expressions might reflect a possible function of H3.3A in cell differentiation as detected in MEL cells.

Animals↗
PubMedDate not supplied

Review: Pdha-2, past and present.

Pyruvate dehydrogenase (PDH) is a multiunit enzymatic complex essential for the process of generating cellular energy. One of the most important of its subunits is the E1 alpha subunit. Perturbations in the expression of this subunit lead to reduced or lost function of the PDH complex as a whole, resulting in a loss of ATP production. The consequence of such perturbations can lead to neurological abnormalities, lactic acidosis, and in males, death. Pdha-2 codes for the mouse testis isoform of the E1 alpha subunit and maps to chromosome 19 (chromosome 4 in humans). This is a fortuitous evolutionary development because the somatic isoform of the E1 alpha subunit is linked to the X-chromosome, which is not only inactivated early in spermatogenesis but is represented in only half of the haploid spermatid population. Consequently, activation of the testis-specific E1 alpha subunit is essential for the progression of spermatogenesis. Despite its importance, the molecular mechanisms governing the tight tissue- and temporal-specific regulation of Pdha-2 have, until recently, remained poorly understood. In this review, we describe our current understanding of the transcriptional regulation of Pdha-2 and propose potential mechanisms that may play a role in this process.

Adenosine Triphosphate↗
PubMed1998

Stage- and subcellular-specific expression of Id proteins in male germ and Sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, spermatogenesis, and Sertoli cell function.

Immunohistological detection of each of the four Id proteins (Id1-Id4) in sections of mouse testis revealed a unique temporal and spatial expression pattern for each Id protein during spermatogenesis. Furthermore, each Id protein displayed a distinctive, dynamic pattern of subcellular distribution. Id1 was uniquely expressed in MI/MII spermatocytes undergoing meiotic division. Id4 protein was detectable in the cytoplasm of type A1 spermatogonia, as well as in late pachytene and in diplotene spermatocytes. Id2 protein, which was most abundant in Sertoli cell nuclei, was also detectable in pachytene and diplotene spermatocytes, but as with Id4, it was absent from MI/MII cells. In postmeiotic spermatids, Id1, Id2, and Id4 proteins were expressed in a stage- and subcellular-specific manner. Expression of Id3 was restricted to Sertoli cell cytoplasm. In malignant seminoma cells, all four Id proteins were abundantly expressed with accompanying changes in their subcellular distribution. The observed expression of Id proteins in postproliferative Sertoli cells and spermatids and during specific stages of meiosis implies novel functional roles for this class of transcriptional regulator during spermatogenesis.

Animals↗
PubMedDate not supplied

Quantification of apoptotic testicular germ cells in normal and methoxyacetic acid-treated mice as determined by flow cytometry.

Several studies have reported the occurrence and significance of programmed cell death (apoptosis) of testicular germ cells in mammals. In those studies, apoptotic germ cells were identified by morphological criteria or by in situ end labeling (TUNEL) and were enumerated from histological sections by semi-quantitative and time-consuming techniques. In the present study, we have established a flow cytometric technique for quantification of TUNEL-positive cells in the mouse testis. Groups of five adult mice each received 0, 650, or 1300 mg/kg (IP) of methoxyacetic acid (MAA), and testes were collected 24 hours later. MAA is known to induce germ cell apoptosis in rodent testes. MAA induced a significant (P < 0.01) dose-dependent decline in the percentage of pachytene spermatocytes (4C cells). DNA strand breaks generated by the activation of endogenous endonuclease in the apoptotic germ cells were detected by the in situ labeling of the 3'-OH termini with biotinylated dUTP in the presence of terminal deoxynucleotidyl transferase (TUNEL technique). Histologically, TUNEL-positive germ cells were observed in control testes, and the number of these cells was visibly increased following MAA exposure. As determined by flow cytometry, four cell populations contained TUNEL-positive cells: 1C cells (round spermatids), 2C cells (mainly spermatogonia), S-ph cells (spermatogonial cells and preleptotene spermatocytes synthesizing DNA [the S-phase]), and 4C cells (primary spermatocytes). Analysis of the percentages of TUNEL-positive cells within each population yielded values of 1.57+/-0.23% for 1C cells, 1.65+/-0.27% for 2C cells, 6.26+/-1.03% for S-ph cells, and 3.24+/-0.39% for 4C cells. Hence, a substantial proportion of proliferating cells are undergoing apoptosis during normal spermatogenesis. The overall incidence of apoptotic cells among all testicular cells was around 2%. At 650 mg per kilogram of body weight, MAA induced a fourfold to eightfold increase (P < 0.001) in the percentage of TUNEL-positive cells, compared with saline-treated controls, and, overall, 17% of testicular cells were apoptotic. This effect of MAA was most pronounced for S-ph and 4C cells, with 25-30% of cells being affected in each of those populations. At 1300 mg per kilogram of body weight, MAA had no further effect. These quantitative data demonstrate that 1) in the normal testis, it is mainly proliferating cells that undergo apoptosis, and 2) MAA induces primary spermatocyte loss by germ cell apoptosis.

Acetates↗
PubMed1991

Analysis of the patterns of expression of mRNAs for the alpha- and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase in mouse epididymis and testis.

mRNA for the alpha- and beta-subunits of mouse beta-N-acetylhexosaminidase were detected in tissue sections of epididymis and testis using 3H-labelled antisense RNA probes by hybridization in situ. There were clear differences in the intensity of the signals given by the two tissues with alpha-subunit mRNA being more abundant in testis than epididymis, while the opposite was so with beta-subunit mRNA. Differences in the isoenzyme patterns of the two tissues are likely to be due, at least in part, to differences in transcription by the Hexa and Hexb genes.

Animals↗
PubMed2000

Examination of lacZ mutant induction in the liver and testis of Muta Mouse following injection of halogenated aliphatic hydrocarbons classified as human carcinogens.

Possible induction of lacZ mutation was examined in the liver and testis of Muta Mouse following the administration of carcinogenic halogenated compounds, namely 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE), carbon tetrachloride, or 1,2-dibromo-3-chloropropane (DBCP). Slight increases were observed on the mutant frequency in the testis DNA isolated from the mice 14 days after treatment with DBCP at 40 mg/kg or with DBE at 60 mg/kg but not in the liver. Further investigation was necessary to confirm the mutation induction by these chemicals in the testis including experiments with longer sampling intervals. No increase was detected in the frequency following DCE administration of single doses of up to 150 mg/kg or of consecutive injections of up to 280 mg/kg. Marginal but biologically insignificant responses were observed in the liver from the carbon tetrachloride exposed mice. The present results suggest that these carcinogenic chemicals are less efficient for induction of gene mutation in the liver of Muta Mouse.

Animals↗
PubMed2005

Spef1, a conserved novel testis protein found in mouse sperm flagella.

We describe the cloning and characterisation of Spef1, a novel testis-specific gene. Spef1 has evolutionary orthologues in a wide range of species including mammals, other vertebrates, Drosophila, and protozoans with motile cilia or flagella. A second homologue of the gene, Spef2, is also present in several species, suggesting that these genes form part of a novel gene family. The Spef1 protein has two conserved domains, one of which is more strongly conserved in both homologues of the gene. Expression analysis of Spef1 in mice shows that it is expressed predominantly in adult testis, suggesting a role in spermatogenesis. Using an antibody generated to recombinant Spef1, we demonstrate a specific pattern of Spef1 localisation in the seminiferous epithelium of adult mouse testis. Further immunohistochemical analysis using electron microscopy shows Spef1 to be present in the tails of developing and epididymal sperm, internal to the fibrous sheath and around the outer dense fibres of the sperm flagellum.

Alternative Splicing↗
PubMed1999

Novel transcripts of carbonic anhydrase II in mouse and human testis.

Intracellular and extracellular sources of bicarbonate are essential for sperm motility, sperm binding to the zona pellucida and the acrosome reaction. Carbonic anhydrase II, catalysing the synthesis of bicarbonate within spermatozoa, must play a significant role in these mechanisms. We report here the expression of carbonic anhydrase II during mouse spermatogenesis and the primary structure of testicular transcripts coding for carbonic anhydrase II isolated from adult mouse and human testes. The mouse carbonic anhydrase II (Car2) mRNA displays a 5' untranslated region (UTR) larger than the corresponding somatic sequence. The additional 5' sequence contains the 'TATA box' used in somatic tissues and other promoter sequences, suggesting the use of testis-specific promoters further upstream with read-through of downstream promoters. The 3'UTR of the Car2 mRNA is shorter in mature testicular cells than in somatic cells. Polysomal gradient analysis of carbonic anhydrase II transcripts isolated from adult mouse testis and kidney revealed different translation potential: most of the testicular transcripts were present in the non-polysomal fractions, whereas a considerable fraction of kidney transcripts were polysome-associated. These results suggest that specific transcriptional and post-transcriptional mechanisms regulate the expression of carbonic anhydrase II during mammalian spermatogenesis.

5' Untranslated Regions↗
PubMed1987

Developmental activation of phosphoglycerate mutase-2 in the testis of the mouse.

The expression of the phosphoglycerate mutase locus Pgam-2 which synthesizes the muscle-specific PGAM-B subunit was analyzed in the testis of the mouse. No PGAM-B activity was detected in testes of newborn mice, in which only the PGAM-AA isozyme was observed. PGAM-B was first observed between Day 14 and Day 16 of postnatal development. In adult males approximately 50% of total PGAM activity is contributed by the PGAM-B subunit and 50% by the PGAM-A subunit. Immunohistochemical studies show that in the testis PGAM-B is localized exclusively in germ cells. PGAM-B is detected in pachytene spermatocytes and in spermatids, but not in earlier stages of spermatogenesis. The muscle-specific PGAM isozyme was also found in testes of bull, cat, and rat, as well as in human sperm. PGAM-B might thus be useful as a marker for germ cell differentiation, along with other germ cell-specific proteins.

Age Factors↗
PubMed1999

Regulatory role of p27kip1 in the mouse and human testis.

p27kip1 is a cyclin-dependent kinase inhibitor that regulates the G1/S transition of the cell cycle. Immunohistochemical analysis showed that during mouse testicular development p27kip1 is induced when the fetal germ cells, gonocytes, become quiescent on day 16 postcoitum, suggesting that p27kip1 is an important factor for the G1/G0 arrest in gonocytes. In the adult mouse and human testis, in general, spermatogonia are proliferating actively, except for undifferentiated spermatogonia that also go through a long G1/G0 arrest. However, none of the different types of germ cells immunohistochemically stained for p27kip1. During development, Sertoli cells are proliferating actively and only occasionally were lightly p27kip1 stained Sertoli cells observed. In contrast, in the adult testis the terminally differentiated Sertoli cells heavily stain for p27kip1. Twenty to 30% of both fetal and adult type Leydig cells lightly stained for p27kip1, possibly indicating the proportion of terminally differentiated cells in the Leydig cell population. In p27kip1 knockout mice, aberrations in the spermatogenic process were observed. First, an increase in the numbers ofA spermatogonia was found, and second, abnormal (pre)leptotene spermatocytes were observed, some of which seemingly tried to enter a mitotic division instead of entering the meiotic prophase. These observations indicate that p27kip1 has a role in the regulation of spermatogonial proliferation, or apoptosis, and the onset of the meiotic prophase in preleptotene spermatocytes. However, as p27kip1 is only expressed in Sertoli cells, the role of p27kip1 in both spermatogonia and preleptotene spermatocytes must be indirect. Hence, part of the supportive and/or regulatory role of Sertoli cells in the spermatogenic process depends on the expression of p27kip1 in these cells. Finally, we show that the expression of p27kip1 transiently increases by a factor of 3 after x-irradiation in whole testicular lysates. Hence, p27kip1 seems to be involved in the cellular response after DNA damage.

Adult↗
PubMedDate not supplied

Distribution of mouse sulfated glycoprotein-1 (prosaposin) in the testis and other tissues.

Mouse sulfated glycoprotein-1 (SGP-1) is the homologue of rat SGP-1 and human prosaposin. Rat SGP-1 is one of the major secretory products of rat Sertoli cells in culture. Human prosaposin is the precursor of four lysosomal saposins, termed A, B, C, and D, that are generated by limited proteolysis. Saposins are sphingolipid-binding proteins that function as activators for lysosomal enzymes involved in sphingolipid hydrolysis of the former. Recently, we have generated a cDNA encoding the mouse SGP-1 by polymerase chain reaction amplification of a mouse testicular Uni-Zap XR cDNA library with two synthetic oligonucleotide primers and have used it as a probe for examining the tissue distribution of SGP-1 mRNA. We have also studied the distribution of the translation product of SGP-1 mRNA in the same tissues. The analysis demonstrated that SGP-1 is expressed ubiquitously in all tissues examined. This investigation showed that, in mouse testis, two forms of SGP-1 exist: a 70-kDa secreted protein and a 65-kDa protein corresponding to the lysosomal form of SGP-1, which may be involved in the generation of saposins. Light microscope immunocytochemistry with anti-SGP-1 antibody demonstrated that, in the mouse seminiferous tubules, the translation product of SGP-1 mRNA is expressed in Sertoli cells but not in germinal cells. Electron microscope immunogold labeling with anti-SGP-1 antibody yielded a strong reaction on lysosomes and phagolysosomes containing residual bodies but not on endosomes or luminal residual bodies. These results demonstrate that SGP-1 is not internalized from the lumen but is targeted directly to the lysosomes from the Golgi apparatus. Immunoblotting also confirmed the existence of a secreted form of testicular SGP-1 delivered to the lumen of the seminiferous tubules. The production of a secreted and a lysosomal form of SGP-1 by Sertoli cells indicates that this protein plays a multifunctional role. This study also suggests that the lysosomal form of SGP-1 may be involved in the degradation of membrane glycolipids from residual bodies phagocytosed by Sertoli cells.

Animals↗
PubMed2003

Hypothyroidism induces type 2 iodothyronine deiodinase expression in mouse heart and testis.

In the present study we show the expression profiles of both type 1 and type 2 iodothyronine deiodinases (D1 and D2) in a wide spectrum of mouse tIssues, and D2 regulation by thyroid status. A characteristic tIssue-specific expression for each isoform was observed. D2 transcripts were detected in most tIssues with variable levels of expression. The observed D2 mRNA tIssue distribution was similar to that described in rats and is in agreement with the view of different patterns of expression between rodents and humans. However, it is interesting to note that despite the low levels of D2 transcripts in mouse heart and testis in the euthyroid state, the induction of hypothyroidism caused a significant increase in D2 activity in these tIssues. Similar results were also obtained in adult rats. These results suggest a previously unrecognized role for type 2 deiodinase in controlling intracellular triiodothyronine levels in rodent heart and testis during states of thyroid hormone deficiency.

Animals↗
PubMed1998

Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature.

Physiological scrotal hypothermia is necessary for normal spermatogenesis and fertility in mammals. Cirp is a recently identified cold-inducible RNA-binding protein that is inducible at 32 degrees C in mouse somatic cells in vitro. Cirp is constitutively expressed in the testis of mouse and structurally highly similar to RBM1, a candidate for the human azoospermia factor. To elucidate the role played by Cirp in spermatogenesis, we investigated its expression levels during spermatogenesis and after heat stress. In the mouse testis, cirp mRNA was detected in the germ cells, and the level varied depending on the stage of differentiation. Also, a high level of Cirp protein was detected immunohistochemically in the nucleus of primary spermatocytes. Expression of Cirp was decreased in the GC-2spd(ts) mouse germ cell line when culture temperature was raised from 32 degrees C to 37 degrees C. When mouse testis was exposed to heat stress by experimental cryptorchidism or immersion of the lower abdomen in warm (42 degrees C) water, the expression of Cirp was decreased in the testis within 6 hours after either treatment. In human testis with varicocele analyzed immunohistochemically, germ cells expressed less Cirp protein than those in the testis without varicocele. These results demonstrated that CIRP expression is down-regulated at elevated temperature in male germ cells of mice and humans. Analysis of Cirp expression in the testes will help elucidate the molecular mechanisms leading to male infertility.

Adult↗
PubMedDate not supplied

Construction and preliminary characterization of a series of mouse and rat testis cDNA libraries.

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18-day-old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6-, 8-, 17-, and 18-20-day-old mice, and peritubular cells from 18-20 day old mice, all recovered from outbred white Swiss (CD-1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 Bl6/J, C3 HEB, BDF-1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20-day-old Sprague-Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85-98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni-Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 x 10(5) to 2.9 x 10(8) plaque-forming units, and insert sizes averaged 1.0-1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided.

Animals↗
PubMed1995

Expression of Siah-2, a vertebrate homologue of Drosophila sina, in germ cells of the mouse ovary and testis.

Siah-2 is one of three murine homologues of the Drosophila gene seven in absentia (sina). The sina protein is nuclear localizing and required downstream of Ras1, Raf and the tyrosine kinase sevenless in Drosophila. Recent results have demonstrated a high degree of functional conservation between vertebrate and insect tyrosine kinase signalling pathways, involving such proteins as Son of sevenless, Grb2 and GAP. These findings, together with the high degree of sequence conservation between the Siah proteins and sina, suggest that the mammalian Siah proteins may also participate in signal transduction by some tyrosine kinases. Here, we report a high level of expression of Siah-2 in a specific population of germ cells within both the mouse ovary and testis. Siah-2 expression was absent in primordial oocytes but was detected in all growing oocytes, coincident with their recruitment from the pool of quiescent cells. The level of Siah-2 mRNA increased as the oocytes matured and was readily detectable in Graafian follicles and in fertilized zygotes up until the two cell stage, a time of extensive maternal transcript degradation and zygotic gene activation. The expression of Siah-2 in the testis was first detected in postmeiotic spermatids. These represented a comparable stage of germ cell development to those cells first expressing Siah-2 in the ovary. The expression pattern of Siah-2 in germ cells was similar to that described for the proto-oncogene c-mos, and the possibility that Siah-2 lies downstream of p39mos in signal transduction within the mouse oocyte requires further investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Comparison of a pump-around, a diffusion-driven, and a shear-driven system for the hybridization of mouse lung and testis total RNA on microarrays.

In the present study, we demonstrate the benefits of a shear-driven rotating microchamber system for the enhancement of microarray hybridizations, by comparing the system with two commonly used hybridization techniques: purely diffusion-driven hybridization under coverslip and hybridization using a fully automated hybridization station, in which the sample is pumped in an oscillating manner. Starting from the same amount of DNA for the three different methods, a series of hybridization experiments using mouse lung and testis DNA is presented to demonstrate these benefits. The gain observed using the rotating microchamber is large: both in terms of analysis speed (up to tenfold increase) and in final spot intensity (up to sixfold increase). The gain is due to the combined effect of the hybridization chamber miniaturization (leading to a sample concentration increase if comparing iso-mass conditions) and the transport enhancement originating from the rotational shear-driven flow induced by the rotation of the chamber bottom wall.

Animals↗
PubMed1999

Influence of gonadotropins on adrenalectomy induced changes in the testis of albino mouse.

Effects of adrenalectomy and administration of gonadotropins on cell counts of different cell types of spermatogenesis and morphology of the Leydig cells were studied in 30 day old mice. Adrenalectomy (duration, 12 days; age at autopsy 42 days) caused a significant decrease in the diameters of seminiferous tubules and Leydig cell nucleus and, cell counts of intermediate spermatogonia, round and elongated spermatids. Administration of FSH (75 micrograms/0.1 ml saline) + LH (25 micrograms/0.1 ml saline) everyday for 12 days to adrenalectomized mice restored testicular activity as revealed by significant increases in mean diameter of the Leydig cell nuclei and cell counts of intermediate spermatogonia and elongated spermatids over those of adrenalectomized mice. The results indicate that (i) testis of adrenalectomized mouse responds to gonadotropin treatment and (ii) impairment in gonadotropin secretion is possibly a major factor in inducing testicular regression following adrenalectomy.

Adrenalectomy↗