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PubMed1979

Electron microscopic study on the testicular interstitial cells in the mouse.

The interstitial tissue of the mouse testis contains, besides mature and immature Leydig cells, fibroblasts, occasional plasma cells and a considerable number of macrophages abutting on the Leydig cells. Mature Leydig cells extend filopodia into dilated portions of the intercellular space. Along the cell surface deep hollows are frequently formed which contain numerous filopodia protruded from the plasma membrane limiting the hollow. This unique structure is tentatively designated the "basal infolding-like structure." Abundant SER is classified into the tubular and vesicular forms. The former is composed of interconnected tubules which may form flattened, partially fenestrated cisternae in some areas. The latter form is composed of vesicles of variable sizes. The tubules probably transform into the vesicular form, possibly reflecting the functional phases of Leydig cells. Thus, among vesicles, patches of tubules with smaller calibers may be detectable. Membranous whorls composed of concentric lamellar stacks of flattened, partially fenestrated cisternae of the SER are frequently observed, enclosing a mitochondrion, lipid droplet or microbody in their center. A typical annulate lamellae are occasionally found in the cytoplasm or in the whorl which is continuous to the membranes of the ER. Small foci of the RER are detected and sometimes dilations of its cisternae are observed. Polysomes are distributed not only in the RER foci, but also throughout the cytoplasm intervening among the SER membranes. Mitochondria possess tubular cristae and occasionally contain sparse ribosome-like granules. Out of several Golgi complexes dispersed in the cytoplasm, the juxtanuclear one contains a diplosome and an occasional solitary cilium. Relatively numerous microbodies are scattered in the cytoplasm. Lipid droplet content varied strongly from cell to cell, probably suggesting variable intensities of steroid synthesis. In the peripheral cytoplasm, large vacuoles were found, probably derived from the testosterone-containing SER. These possible secretory vacuoles occurred close to the plasma membrane, suggesting emiocytotic release of the contents. The basal lamina was restricted to certain areas of the plasma membrane.

Animals↗
PubMed2002

RNA amplification results in reproducible microarray data with slight ratio bias.

Microarray expression analysis demands large amounts of RNA that are often not available. RNA amplification techniques have been developed to overcome this prcblem, but limited data are available regarding the reproducibility and maintenance of original transcript ratios. We optimized and validated two amplification techniques: a modified in vitro transcription for the linear amplification of 3 microg total RNA and a SMART PCR-based technique for the exponential amplification of 50 ng total RNA. To determine bias between transcript ratios, we compared the expression profiles in mouse testis versus spleen between the two amplification methods and a standard labeling protocol, using microarrays containing 4596 cDNAs spotted in duplicate. With each method, replicate hybridizations were highly reproducible. However, when comparing the amplification methods to standard labeling, correlation coefficients were lower. Twelve genes that exhibited inconsistent or contradictory expression ratios among the three methods were verified by quantitative RT-PCR. The amplification methods showed slightly more discrepancies in the expression ratios when compared to quantitative RT-PCR results but were more sensitive in terms of detecting expressed genes. In conclusion, although amplification methods introduce slight changes in the transcript ratios compared to standard labeling, they are highly reproducible. For small sample size, in vitro transcription is the preferred method, but one should never combine different labeling strategies within a single study.

Blotting, Southern↗
PubMed2006

Hyalp1 in murine sperm function: evidence for unique and overlapping functions with other reproductive hyaluronidases.

While Sperm adhesion molecule 1 (SPAM1) is the highly conserved mammalian sperm hyaluronidase (hyase), multiple hyases are present in the mouse testis. In this study we show that one of the murine hyases, Hyalp1, which is predominantly expressed in the testis in a 24-kd isoform has neutral enzymatic activity. On sperm, Hyalp1 is localized on the plasma membrane of the anterior head and was shown to have neutral hyase activity for an isoform of approximately 55-56 kd, contributing modestly to the overall neutral hyase activity. This activity is associated with in vitro cumulus penetration, since antibody inhibition of Hyalp1 significantly (P = .034) retarded the rate of penetration of wild-type (WT) sperm. Antibody-inhibited Spam1 null sperm were more severely retarded (P = 4.2 x 10(-19)), suggesting an up-regulation of Hyalp1 in these mice. A functionality test of the hyaluronic acid (HA) receptor domain identified in the N-terminus by in silico analysis revealed that sperm Hyalp1 is significantly (P = .006) involved in the progesterone-induced HA-enhanced acrosome reaction. Finally, developmental reverse transcription polymerase chain reaction (RT-PCR) shows that testicular transcripts of Hyalp1 are detected as early as 6 days postparturition, similar to transcripts for Spam1, suggesting that the gene might also play a role in the developing testes prior to spermiogenesis. Taken together, the findings reveal that Hyalp1 likely has a unique function in the adult testis, and redundant overlapping ones with Spam1 and may compensate for it in Spam1 null mice.

Amino Acid Sequence↗
PubMedDate not supplied

The suppression effect of DNA sequences within the C4A region on the transcription activity of human CYP21.

Two CYP21 genes, the active CYP21 and the pseudogene, CYP21P, also pairs of duplicated genes including the XA, XB, XB-S; YA, YB; and ZA, ZB are arranged in tandem next to the serum complement C4 genes (C4A and C4B). In this report, we have analyzed the influence of some DNA sequences within the C4A/CYP21P region on the transcription activity of the human CYP21. After transiently transfecting the plasmid constructs into mouse adrenocarcinoma Y1 cells, mouse testis Leydig tumor MA10 cells and human liver tumor HepG2 cells, our results showed that sequences located within the -13943/-13174 and -3278/-2586 regions upstream from the CYP21P had suppression effects on the promoter activity of human CYP21. However, the short sequences spanning from -8415/-8373 and -4511/-4140 upstream from the CYP21P did not alter the basal transcription activity of the CYP21 gene. Our results indicated that specific sequences within the C4A region might function as suppressor-like elements for the transcription of human CYP21.

Adrenal Cortex↗
PubMed2002

Endogenous retrovirus sequences expressed in male mammalian reproductive tissues: a review.

OBJECTIVES: To review the research findings on the expression of endogenous retroviruses and retroviral-related particles in male mammalian reproductive tissues, and to discuss their possible role in normal cellular events and association with disease conditions in male reproductive tissues. DATA SOURCES: Published findings on endogenous retrovirus (ERV) expression in vertebrate reproductive tissues. STUDY SELECTION: Relevant citations on ERVs and male reproduction by research groups worldwide. DATA EXTRACTION: Literature search on Medline and Pubmed upto the year 2000, and retrieval of relevant articles cited from international and local journals. DATA SYNTHESIS: Most of the studies demonstrated integrated retroviruses and retroviral-related sequences in human and mouse testis, epididymis and vas deferens. Endogenous retroviruses in human and mice may be associated with normal cellular differentiation and development, and carcinogenesis. In humans, one ERV family, human endogenous retrovirus-K (HERV-K) is abundantly expressed, and is associated with germ cell tumours, while ERV3 env is expressed in normal human testis. CONCLUSION: The expression of ERVs in male reproductive tissues suggests a possible role in normal and disease conditions involving the testis and epididymis. These speculative functions may include among others spermatogenesis and or sperm maturation or tumour formation. However, further studies need to be carried out to investigate specific roles of ERVs in male reproductive events.

Animals↗
PubMed1998

Expression pattern of the Ets-related transcription factor Elf-1.

Members of the Ets family of winged helix-loop-helix transcription factors play important roles in the development and function of multiple mammalian cell lineages. Elf-1 is an Ets-related transcription factor that is expressed at high levels in T cells and is known to regulate the expression of several T cell genes, including the granulocyte-macrophage colony stimulating factor (GM-CSF) gene, the interleukin-2 receptor alpha subunit (IL-2Ralpha) gene, and the CD4 gene. In the studies described in this report, we have characterized the pattern of expression of Elf-1 in the developing mouse embryo and in adult mouse tissues as well as in multiple immortalized human and murine cell lines. Elf-1 is expressed at high levels throughout thymocyte development, with equivalent levels of Elf-1 expression seen in all subsets of maturing thymocytes and T cells. Somewhat surprisingly, however, Elf-1 is also expressed at high levels in epithelial cells lining the oral cavity, the lung, the CNS, and the gastrointestinal and urinary tracts as well as in the skin of the developing mouse embryo and at lower levels in the adult mouse testis and liver. Western blot analyses of a large number of immortalized cell lines demonstrated high-level Elf-1 expression in T and B lymphocyte and macrophage cell lines as well as in two prostate carcinoma cell lines. Low-level expression was observed in fibroblasts, embryonic stem cells, and myoblasts. Taken together, our data suggest that in addition to its role in regulating T cell development and function, Elf-1 may regulate gene expression in the B cell and myelomonocytic lineages, as well as in multiple epithelial cell types during murine embryonic development.

Age Factors↗
PubMed2000

Cryptorchidism induces mouse testicular germ cell apoptosis and changes in bcl-2 and bax protein expression.

Up to half of the germ cells die during their development. The mechanism involved in their death is still unclear. We investigated the possibility that the testicular germ cell loss may be due to a change in the testicular temperature and apoptosis through the interplay of bcl-2 and bax proteins. To investigate the effect of temperature on apoptosis and the role of bcl-2 and bax proteins in apoptosis in adult mouse testis, we used the experimental unilateral cryptorchidism model. We found that the weight of the affected testis decreases in 6 to 15 days after operation (p < 0.05). H&E staining showed vacuolization of the seminiferous epithelium and the appearance of multinucleated giant cells with loss of germ cells. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling method (TUNEL) showed positive staining germ cells scattered in normal testicles that increased significantly 6 to 15 days after experimental cryptorchidism (p < 0.05). Positive cells were mainly primary spermatocytes and round spermatids. DNA ladder was seen in 1.5% agarose gel electrophoresis in 6 to 15 days-operated testicles. Biotin-avidin DCS system indirect immunofluorescence technique indicated that the bcl-2 protein was chiefly distributed in the cytoplasm of germ cells in normal testicles, whereas the bax protein was chiefly expressed in the cytoplasm of germ cells in 6 to 15 days-operated testicles. Western blot analysis showed that the bcl-2 protein down-regulated and the bax protein up-regulated apoptosis. Our data indicated that cryptorchidism-induced testicular cell degeneration was mediated by apoptosis probably as the result of increased temperature in the testicle; bcl-2 and bax proteins played important roles in male germ cell apoptosis.

Animals↗
PubMed2001

Localization in situ of specific RNA by electron microscopy.

To better understand the metabolism of RNA in nuclei, the analysis of precise nuclear distribution of specific RNA would be essential. For this purpose, nonradioactive electron microscopic (EM) in situ hybridization may be the most appropriate technique while the details required for the technique have not been fully established. In the present study, we attempted to localize 28S and 18S rRNAs in the nuclei of mouse Sertoli cells by EM in situ hybridization as a model system. After various preliminary experiments we chose the pre-embedding method; fresh-frozen sections of mouse testis were fixed with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde, digested with 10 microg/ml of proteinase K and hybridized with thymine-thymine (T-T) dimerized oligodeoxynucleotides (oligo-DNA) complementary to a part of 28S and 18S rRNAs. Then, the T-T dimers were detected enzyme-immunohistochemically with horseradish peroxidase (HRP) labeled anti-T-T dimer. After osmification of HRP products, the sections were embedded in Epon resin, cut into 100 nm ultra-thin sections and observed under a transmission electron microscope. As a result, we successfully localized both 28S and 18S rRNAs in the dense fibrillar and granular components of the nucleolus, showing the usefulness of nonradioactive EM in situ hybridization in the nuclear localization of specific RNA.

Animals↗
PubMed1999

[Synthesis and distribution of type IV collagen in mice testes].

OBJECTIVE: The variations of the synthesis and distribution type IV collagen in the testes of young, adult, and old mice were studied. METHODS: Biotin-avidin DCS system indirect immunofluorescence technique and in situ hybridization method were used. RESULTS: Sertoli cell could synthesize type IV collagen. Type IV collagen was present in the basement membrane of seminiferous tubules in mouse testis. Type IV collagen mRNA signal was seen in the Sertoli cells. The content of type IV collagen and type IV collagen mRNA peaked at day 15, and then declined successively to adult value and the aged value. CONCLUSIONS: The results suggest that the extracellular matrix of testis might play an important role in the process of spermatogenesis.

Age Factors↗
PubMed1992

Identification and characterization of the regulated pattern of expression of a novel mouse gene, meg1, during the meiotic cell cycle.

The gene designated meg1 (meiosis expressed gene) is a new mouse gene identified during a search for mammalian genes potentially involved in meiotic processes. Two classes of complementary DNAs were isolated from an adult mouse testis complementary DNA library, which shared the same 3' end including the entire putative coding region but differed in their 5' ends. Only one of these complementary DNA classes appeared to correspond to the very abundant 0.75-kilobase testicular transcript of meg1. Sequence analysis predicts a 10.8-kilodalton protein which is highly charged and lysine rich. It is also relatively rich in potential phosphoacceptor amino acids (approximately 17%), several of which are located in phosphorylation consensus sequences. The pattern of expression of meg1 was studied utilizing a combined Northern blot and in situ hybridization analysis. Of the adult tissues examined, meg1 transcripts were detected exclusively in testis. Analysis of mRNA from testes of two germ cell deficient mutant strains did not reveal significant levels of meg1 transcripts. Analysis of RNA from enriched populations of spermatogenic cells from adult testes and localization by in situ hybridization revealed that meg1 transcripts are most abundant in pachytene spermatocytes. These results suggest a role for meg1 during germ cell differentiation, possibly during meiotic prophase.

Amino Acid Sequence↗
PubMed1992

Radiotoxicity of some iodine-123, iodine-125 and iodine-131-labeled compounds in mouse testes: implications for radiopharmaceutical design.

In this work, spermhead survival in mouse testis was used to investigate the radiotoxicity of several intratesticularly localized radioiodinated pharmaceuticals. Radioiodines that decay by electron capture and/or internal conversion (123I, 125I) as well as by beta- decay (131I) were coupled to pharmaceuticals that selectively localize in different cell compartments. Dose response curves yield D37 values of 62 cGy, 75 cGy, 61 cGy and 7.7 cGy for 123IMP (N-isopropyl-p-iodoamphetamine), 131IdU (iododeoxyuridine), H131IPDM (N,N,N'-trimethyl-N'-(2-hydroxyl-3-methyl-5-iodobenzyl)-1,3-propanediami ne) and 125IdC (iododeoxycytidine), respectively. At 37% survival, the relative biological effectiveness (RBE) of these radiochemicals, when compared to the pure gamma-emitting radiochemical 7Be-chloride (D37 = 65 cGy), are 1.0, 0.89, 1.1 and 8.4, respectively. Intratesticular 7Be, with an effective half-life of 430 hr in the organ, was used as the source of reference radiation to determine the RBE values because it solely emits 477 keV gamma rays, and the dose to the testis is delivered chronically, as in the case of the other radiocompounds. Subcellular distribution studies show that all of the cellular activity is localized in the cytoplasm in the cases of 123IMP and H131IPDM, while virtually all of 131IdU and 125IdC were bound to DNA in the cell nucleus. In agreement with our earlier in vivo studies, these data show that subcellular distribution plays a key role in the radiotoxicity of Auger electron emitters such as 123I and 125I, and has no role for beta emitters such as 131I. These findings may have implications in the design of radiopharmaceuticals for both diagnosis (localize Auger emitter in cytoplasm of cell) and therapy (localize Auger emitter in cell nucleus).

Amphetamines↗
PubMedDate not supplied

Ultrastructural analysis of testes from mice fed on genetically modified soybean.

We have considered the possible effects of a diet containing genetically modified (GM) soybean on mouse testis. This organ, in fact, is a well known bioindicator and it has already been utilized, for instance, to monitor pollution by heavy metals. In this preliminary study, we have focussed our attention on Sertoli cells, spermatogonia and spermatocytes by means of immunoelectron microscopy. Our results point out that the immunolabelling for Sm antigen, hnRNPs, SC35 and RNA Polymerase II is decreased in 2 and 5 month-old GM-fed mice, and is restored to normal at 8 months. In GM-fed mice of all ages considered, the number of perichromatin granules is higher and the nuclear pore density lower. Moreover, we found enlargements in the smooth endoplasmic reticulum in GM-fed mice Sertoli cells. A possible role played by traces of the herbicide to which the soybean is resistant is discussed.

Animals↗
PubMed1992

A 35-kDa protein binding to a cytosine-rich strand of hypervariable minisatellite DNA.

A minisatellite-binding protein, Msbp-4, with a molecular mass of 35 kDa has been purified from mouse tumor cells that binds to hypervariable Pc-1 and Pc-2 minisatellites. The binding is much more efficient than that to genetically stable minisatellite homologues. As assayed by Southwestern analysis, Msbp-4 favors multiple copies of the Pc-2 repeat sequence GGCAGGA and requires the cytosine-rich single strand for the binding. The activity is also present in extracts from mouse testis but not from liver. The phosphatase treatment revealed that Msbp-4 is phosphorylated and may have a regulatory function, because dephosphorylation affects the activity and specificity of the binding. Sequence preference is demonstrated by a competition experiment using single-base substitution mutants. Thus, the binding properties of Msbp-4 observed here lead to an implication that the protein-DNA complexes result in formation of a single-stranded DNA loop of the G-rich strand in the minisatellite which may enhance the ability of the minisatellite to undergo recombination.

Animals↗
PubMed2006

[The origin and nature of embryonic stem cells].

The inner cell mass (ICM), blastomeres, epiblasts and primordial germ cells (PGCs) are usually used as primary materials for the establishment of embryonic stem cell (ESC) lines. ES-like cells have even been isolated from neonatal mouse testis. ESC are traditionally regarded as ICM cells, though some scholars believe they more closely resemble cells from the epiblast. However, recent evidence of ESC molecular markers indicate that the characteristics of ESC resemble those of early germ cells. The unknown origin and nature of ESC may limit the successful establishment of ESC lines from many different species. Here we review the progress of research regarding embryonic pluripotent cells, early germ cells and ESC. We find ESC can be derived from many cell types. Future study should elucidate the origin of ESC by comparing different ESC lines so as to determine the nature of ESC and improve the efficiency of ESC derivation.

Animals↗
PubMed1990

Increased levels of junB and c-jun mRNAs in male germ cells following testicular cell dissociation. Maximal stimulation in prepuberal animals.

We have examined the relative transcript levels of the junB and c-jun proto-oncogenes during development of the mouse testis. junB and c-jun mRNA levels are low in total RNA from intact immature or mature testes. Dissociation of testicular cells, however, increases the levels of junB and c-jun mRNAs, with higher increases in the dissociated cells from testes of 8-day-old mice than from 17-day-old or sexually mature mice. These differences in junB and c-jun mRNA levels localize to specific cell types. In testes from 8-day-old mice, the mRNA levels for both proto-oncogenes are higher in type B spermatogonia and in the interstitial cell fraction than in type A spermatogonia. In testes of 17-day-old mice, the highest mRNA levels for both proto-oncogenes are seen in preleptotene spermatocytes and interstitial cells, with decreasing levels in leptotene/zygotene spermatocytes and prepuberal pachytene spermatocytes. junB and c-jun mRNAs are nearly undetectable in pachytene spermatocytes, round spermatids, and residual bodies/cytoplasts. The increased junB mRNA levels originate not only from the expected 2.1-kilobase transcript but from a more slowly migrating transcript of about 2.3 kilobases. RNase H analysis demonstrates that this migration change was due to an increase in mRNA polyadenylation. The low levels of junB and c-jun mRNAs in intact testes and the much higher levels in isolated cells from identical testes suggest that the disruption of cell-to-cell contact increases the amount of junB and c-jun transcripts in specific cells of the testis. Coupled with this increase, structural changes are seen with the junB mRNA.

Animals↗
PubMed2007

Genetic and epigenetic changes induced by chronic low dose exposure to arsenic of mouse testicular Leydig cells.

Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. Genotoxicity of arsenic has been a topic of controversy. Both genetic alterations (mutations) and epigenetic changes (methylation) have been shown to play a crucial role in environmental carcinogenesis. Chronic exposure to arsenic has been shown to induce malignant transformation of mammalian cells. However, the genetic aberrations induced by arsenic in this process are unclear. The purpose of this study was to determine if both lower (1 pg/ml) and higher concentrations (100 ng/ml) of arsenic induces either mutations or methylation changes that could lead to the development of genomic instability in TM3 cells, immortalized Leydig cells derived from normal mouse testis. Two independent exposure times were used in this study which resulted in cells of 33 and 100 generations in age. Arsenic-induced genetic and epigenetic changes were screened at a genome-wide level by random amplified polymorphic DNA (RAPD), also known as AP-PCR method with undigested DNA as well as DNA digested by the methylation sensitive isosizomeric restriction enzymes MSPI and HpaII and untreated controls. Changes in the DNA fingerprint of both, the restriction enzyme digested DNA (indicating methylation changes) as well as undigested DNA (indicating mutations) from arsenic-treated (low as well as high dose) samples were observed as compared to their controls. Thus, this study provides the first evidence at DNA sequence level for mutagenic potential of arsenic. Further characterization of these altered genomic regions is underway. The understanding of these genetic and epigenetic changes in arsenic-induced carcinogenesis will provide a basis for better interventional approaches in both the treatment and prevention of arsenic-induced cancer.

Animals↗
PubMed1990

Biological consequence of nuclear versus cytoplasmic decays of 125I: cysteamine as a radioprotector against Auger cascades in vivo.

When the radionuclide 125I is localized in mouse testis as 125I-iododeoxyuridine (an analogue of thymidine) and incorporated into the DNA of spermatogonial cells, the cytocidal effects are as severe as those due to densely ionizing alpha particles. In contrast, 125I confined to the cytoplasm of these cells is much less radiotoxic, the efficacy being the same as for selective irradiation of the testis with sparsely ionizing external X rays. The biological effects, in both cases, are strongly mitigated upon pretreatment of the testes with very small amounts (0.75 microgram) of cysteamine, a radioprotector. These findings suggest an important role for such chemical agents in radiation protection and in understanding the mechanisms of radiation damage involving radionuclides incorporated in tissue.

Animals↗
PubMed1990

Cloning and analysis of cDNA encoding bovine butyrophilin, an apical glycoprotein expressed in mammary tissue and secreted in association with the milk-fat globule membrane during lactation.

Butyrophilin is a glycoprotein expressed on the apical surfaces of secretory cells in lactating mammary tissue, which may function in the secretion of milk-fat droplets (Franke, W. W., Heid, H. W., Grund, C., Winter, S., Freudenstein, C., Schmid, E., Jarasch, E.-D., and Keenan, T. W. (1981) J. Cell Biol. 89, 485-494). A cDNA clone encoding bovine butyrophilin was isolated and the primary structure of the protein deduced from the DNA sequence. Bovine butyrophilin contains 526 amino acids with a putative signal peptide of 26 amino acids. Hydropathy analysis predicts the existence of a single membrane-spanning region with the amino terminus facing the exoplasmic space. Butyrophilin cDNA hybridized to mRNA of 2.9 kilobases in bovine mammary tissue taken from pregnant or lactating animals, but specific mRNA was not detected in a 2-year-old virgin cow. The amount of message was maximal in lactating tissue. Butyrophilin mRNA could not be detected in bovine heart, intestine, kidney, liver, ovary, or uterus. A search of the GenBank and EMBL data banks showed closest homology was between the C termini of butyrophilin and "ret finger protein" (Takahashi, M., Inaguma, Y., Hiai, H., and Hirose, F. (1988) Mol. Cell. Biol. 8, 1853-1856). The ret-finger protein gene is expressed in a variety of tumor cell lines, mouse testis and embryonic tissue, which like the mammary gland undergo periods of rapid cell division and development. The possible significance of this homology and the possible function of butyrophilin in milk-lipid secretion are discussed.

Amino Acid Sequence↗