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PubMed2003

Morphological characterization of the spermatogonial subtypes in the neonatal mouse testis.

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man.

Animals↗
PubMed2002

[Molecular cloning of SRG2, a mouse testis spermatocyte apoptosis-related gene].

A novel mouse gene full-length cDNA sequence-SRG2 were identified (GenBank accession number AF395083), which was significantly changed in cryptorchidism, from a mouse testis cDNA library using a cDNA fragment (GenBank accession number BE644542) as an electronic probe. SRG2 was 1058 bp in length. The putative protein encoded by this gene was 295 amino acids with a theoretical molecular weight of 33 579 and isoelectric point of 9.64. The sequence shared no significant homology with any known protein in databases except TSARG2, with which its homology was 78%. RT-PCR showed that SRG2 was expressed significantly in testis.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of a leucine zipper motif-containing novel cDNA specifically expressed in adult mouse testis.

A novel cDNA clone was isolated from a mouse testis cDNA library. This cDNA is 2,020 nucleotides in size and predicted to encode 527 amino acid residues with a molecular weight of 59.9 kDa. Protein sequence motif analysis revealed that the predicted protein contained one leucine zipper motif in its N-terminal portion and two basic domains in its C-terminal portion. Northern blot analysis of adult ICR mouse organs using the cDNA as probe demonstrated that an approximately 2.0-kb transcript was specifically expressed in testis. We therefore termed this novel cDNA tsec-2, testis-specifically expressed cDNAs-2. Results of Southern blot analysis suggested that the tsec-2 gene may exist as a single copy in the mouse genome.

Amino Acid Sequence↗
PubMed1993

Circular transcripts of the testis-determining gene Sry in adult mouse testis.

Sry is expressed at higher levels in the adult testis, where no function has been determined, than in the genital ridge, its critical site of action. cDNA and 5' RACE clones isolated from testis or from Sry-transfected cell lines have an unusual structure, with 3' sequences located in a 5' position. RNAase protection assays and reverse transcription polymerase chain reactions confirmed that these unusual RNA molecules represent the most abundant transcript in testis. Furthermore, oligonucleotide hybridization and RNAase H digestion proved that these Sry RNA molecules are circular. Similar transcripts were detected in the testes of mice with Mus musculus musculus, Mus musculus domesticus, and Mus spretus Sry genes. The circular RNA is found in the cytoplasm but is not substantially bound to polysomes. We suggest that the circles arise from normal splicing processes as a consequence of the unusual genomic structure surrounding the Sry locus in the mouse.

Animals↗
PubMed2026

Both L-Lactyl and D-Lactyl Enantiomers Modify Histones in Mouse Testis.

Dynamic histone posttranslational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the former may more likely modify histones due to abundant L-lactate produced by glycolysis. Here, we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, despite the fact that L-lactate is much more abundant than D-lactate in mouse testis, we estimated abundance ratios of L-over D-lactylation to lie between 0.4 and 1.6 on seven residues of histones H3 and H4. Next, targeted proteomic analyses were performed on histones extracted from meiotic and postmeiotic male germ cells (spermatocytes and round spermatids, respectively), which are known to use L-lactate as a main source of energy. Nonetheless, residues 18 and 23 of histone H3 (H3K18 and H3K23) were reliably quantified and shown to harbor balanced amounts of both enantiomers. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing about 0.01 to 0.44%: this contrasts with acetylation which exists at up to 25 to 35% relative abundances on some N-terminal lysines. Yet, lactylation appears to be more abundant than acetylation on the C-terminal half of H3 and H4, where the latter modification is scarce. Collectively, our results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, that leads to the modification of histones by L- and D-lactylation.

Animals↗
PubMed1994

Expression of heat shock factor 2 in mouse testis: potential role as a regulator of heat-shock protein gene expression during spermatogenesis.

We have examined the expression and function of heat shock transcription factor 2 (HSF2) in spermatogenic cells of mouse testis. The results of in situ RNA hybridization analysis, RNA filter hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicate that HSF2 mRNA expression in testis is subject to developmental and cell type-dependent, as well as stage-dependent, regulation. Localized expression of HSF2 mRNA in testis first appears between Day 14 and Day 21 of postnatal development. In adult testis, HSF2 mRNA is found at highest levels in spermatocytes and round spermatids. Immunocytochemical staining and gel mobility shift analysis demonstrate that HSF2 protein is localized to the nuclei of spermatocytes and round spermatids and that this transcription factor exists in testis in a constitutively active DNA-binding state. We further demonstrate that the constitutive HSF2 DNA-binding activity present in testis is able to interact with promoter sequences of the hsp70.2 gene, a testis-specific member of the hsp70 gene family. Taken together, our results show that the expression and functional properties of HSF2 are regulated in spermatogenic cell types of the mouse testis, supporting a role for this transcription factor as a regulator of hsp gene expression during spermatogenesis.

Animals↗
PubMed1974

Nucleolar and perichromosomal RNA synthesis during meiotic prophase in the mouse testis.

The transcriptional activity during meiotic prophase in the mouse testis is studied with light microscopy and high-resolution autoradiographic techniques using [(3)H]uridine as a labeled precursor. In the present study, two types of RNA synthesis are detected during meiotic prophase: an extranucleolar RNA synthesis of perichromosomal localization and a nucleolar RNA synthetic activity. In some of the autosomes and close to the basal knobs, the activity of the nucleolar organizers is evidenced by the incorporation of [(3)H]uridine into nucleolar masses from zygotene on and at earlier labeling times. The evolution of nucleoli and the formation of a nucleolus attached to the sex pair are described during the different meiotic stages. Perichromosomal labeling, from leptotene on, reaches a maximum during middle pachytene and falls progressively to a low level at longer incorporation times. Sertoli's cell, the most active RNA synthetic cell in the seminiferous epithelium, rises to a maximum of labeling and drops at earlier times compared with the meiotic prophase cells. The condensed sex chromosomes show some scattered silver grains especially at middle pachytene. The axial chromosome cores and synaptonemal complexes are devoid of silver grains during the meiotic prophase. The observations suggest that a control mechanism operates during meiotic prophase to regulate transcriptional activity in the sex chromosomes and to provide differential RNA synthesis in autosomal bivalents at various stages of prophase and within certain segments of the chromosomes.

Animals↗
PubMedDate not supplied

Thermosensitization by step-down heating in mouse testis.

Step-down heating (SDH) was investigated in mouse testis by giving an initial treatment of 3 min at 43.0 degrees C followed immediately by a treatment in the temperature range 38.0-42.0 degrees C. The dose-response curves for testis weight loss as a function of duration of hyperthermia were compared with those obtained using single-temperature treatments. In all cases the curves were linear, allowing the use of Arrhenius analysis. For single-temperature treatments the Arrhenius relationship showed an inflection at approximately 41 degrees C with a small, but significant, increase in activation energy for hyperthermal temperatures below the transition. SDH increased the thermal sensitivity in this lower range, by approximately 1 degree C, but the activation energy was not significantly altered. The results support the view that in vivo thermosensitization by SDH is not due solely to inhibition of development of thermotolerance.

Animals↗
PubMed2001

PKCtheta II, a new isoform of protein kinase C specifically expressed in the seminiferous tubules of mouse testis.

Protein kinase C (PKC) theta, a Ca(2+)-independent isoform of PKC, has been known to be expressed in skeletal muscle and T cells. In the present study, we isolated and characterized a smaller transcript expressed in the mouse testis, the cDNA of which is referred hereafter as PKCthetaII and the original PKCtheta as PKCthetaI. The cDNA clone of PKCthetaII has 2184 base pairs and 464 amino acids in the possible open reading frame, consisting of the 5' unique sequence of 20 amino acids and the PKCthetaI sequence of 444 amino acids. Genomic DNA analysis revealed that transcription of PKCthetaII is initiated from the PKCthetaII-specific exon, which is located between exons 7 and 8 of the PKCtheta gene, indicating that alternative splicing is the mechanism by which PKCthetaII is generated. PKCthetaII is expressed exclusively in the testis in an age-dependent manner with sexual maturation. In situ hybridization and reverse transcription-polymerase chain reaction of microdissected tissues clearly demonstrated that PKCthetaII is expressed in the seminiferous tubules of the mouse testis. Consistent with its molecular structure lacking the C1 regulatory domain, PKCthetaII is constitutively active as determined by an in vitro kinase assay, being independent of PKC activators, e.g. phosphatidylserine and phorbol ester. PKCthetaII may play a crucial role in spermatogenesis or some related function of the testis.

Alternative Splicing↗
PubMed1999

Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis.

1. One of the features of weaver mutant mice is male infertility, which suggests that Kir3.2, a G-protein-gated inwardly rectifying K+ channel subunit, may be involved in spermatogenesis. Therefore, we have characterized the Kir3.2 isoform in mouse testis using immunological, molecular biological and electrophysiological techniques. 2. Testicular membrane contained a protein that was recognized by the antibody specific to the C-terminus of Kir3.2c (aG2C-3). Its molecular mass was approximately 45 kDa, which was smaller than that of Kir3.2c ( approximately 48 kDa). The immunoprecipitant obtained from testis with aG2C-3 contained a single band of the 45 kDa protein, which could not be detected by the antibody to the N-terminus common to the known Kir3.2 isoforms (aG2N-2). 3. A novel alternative splicing variant of Kir3.2, designated Kir3.2d, was isolated from a mouse testis cDNA library. The cDNA had an open reading frame encoding 407 amino acids, whose molecular mass was calculated to be approximately 45 kDa. Kir3.2d was 18 amino acids shorter than Kir3.2c at its N-terminal end, which was the only difference between the two clones. The 18 amino acid region possesses the epitope for aG2N-2. 4. In heterologous expression systems of both Xenopus oocytes and mammalian cells (HEK 293T), Kir3.2d either alone or with Kir3.1 exhibited G-protein-gated inwardly rectifying K+ channel activity. 5. Prominent Kir3.2d immunoreactivity in the testis was detected exclusively in the acrosomal vesicles of spermatids, while Kir3.1 immunoreactivity was diffuse in the spermatogonia and spermatocytes. These results indicate the possibility that the testicular variant of Kir3.2, Kir3. 2d, may assemble to form a homomultimeric G-protein-gated K+ channel and be involved in the development of the acrosome during spermiogenesis.

Alternative Splicing↗
PubMed1997

Expression pattern of the mitochondrial capsule selenoprotein mRNA in the mouse testis after puberty; in situ hybridization study.

Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatozoa. In this study, to determine the expression pattern of MCS mRNA in the mouse testis after puberty, in situ hybridization using digoxigenin-labeled RNA probes for MCS was performed in the testes of 8- and 20-week-old ICR mice. In the testes of both ages, MCS mRNA first appeared in step 3 round spermatids, gradually increased during early spermiogenesis, and persisted a high level until step 14 spermatids. After the step 14 spermatids, the signal began to decline and was weakly detected in steps 15-16 spermatids. On the other hand, compared with that in the testes of 8-week-old mice, MCS mRNA level in the testes of 20-week-old mice increased over 2-fold at stages VI-III, while it slightly increased at stages IV-V. These findings suggest that MCS gene transcription may be up-regulated after puberty in the mouse testis.

Aging↗
PubMed2004

CatSper gene expression in postnatal development of mouse testis and in subfertile men with deficient sperm motility.

BACKGROUND: The search for Ca2+ channels residing in sperm has led to the recent cloning and characterization of a novel gene, named CatSper, which codes for a unique Ca2+ channel expressed exclusively in the testis. It plays an essential role in sperm motility, penetration into the oocyte, and ultimately in male fertility. In this study, we assessed the temporal profile of CatSper gene expression during mouse testis development and performed a semi-quantitative evaluation of expression levels in a group of subfertile men which lack sperm motility. METHODS: A small piece of testicular tissue obtained by either multi-site testicular biopsy or orchidectomy was used for semi-quantitative RT-PCR of CatSper and beta2-microglobulin (beta2m, as an internal control) genes. RESULTS: Our results reveal that: (i) the expression of mouse CatSper is developmentally regulated with a direct correlation between CatSper expression and mouse sexual maturation. CatSper gene expression is first detected at 3 weeks of age and coincides with the appearance of round spermatids in the developing mouse testis. (ii) There is a significant reduction in the level of CatSper gene expression (up to 3.5-fold difference) among patients which lack sperm motility as compared with patients whose infertility cannot be ascribed to a deficiency in motility (used as a control). CONCLUSIONS: The data obtained in this study support a potential role for CatSper in sperm motility and fertility in mouse and human. CatSper is therefore implicated as a potential target to explore the molecular mechanisms of male infertility.

Adult↗
PubMedDate not supplied

Expression of claudin-1 in mouse testis.

In testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of the blood-testis barrier (BTB) and crucial for spermatogenesis. The present study aimed to find postnatal changes in the expression of claudin-1, one of the TJ genes in mouse testis. By semiquantitative RT-PCR, it was found that claudin-1 expression in testis increased up to a peak at 10 days after birth and decreased thereafter. Western blot analysis showed abundant expression of 21-kDa protein in testis, lung, and brain from the adult mouse. The developmental change in the expression of claudin-1 protein in testis coincided with that from the RT-PCR. Testosterone treatment significantly increased claudin-1 expression in immature Sertoli cells, suggesting the possible regulation of claudin-1 expression by androgen in mouse Sertoli cells. Claudin-1 expression appears to be developmentally regulated in the mouse testis.

Animals↗
PubMed1992

Immunohistochemical localization of PGF2 alpha receptor in the mouse testis.

As a step towards understanding the role of prostaglandin F2 alpha (PGF2 alpha) in male reproductive tract physiology, a rabbit polyclonal antiserum reactive with purified PGF2 alpha receptor (PGF2 alpha-R) was produced. Here we describe the use of this anti-PGF2 alpha-R antiserum in immunohistochemical staining of mouse testis to ascertain which cell types, in vivo, possess immunoreactive PGF2 alpha-R. As an initial control Western blot analysis was performed to show that the anti-PGF2 alpha-R antiserum recognizes only one antigen in the testis, and that this molecule is similar in molecular mass (by PAGE) to the previously described, purified PGF2 alpha-R molecule. Immunohistochemical staining demonstrates that adult mouse testis contains a single subpopulation of cells with PGF2 alpha-R and that subpopulation is the interstitial or Leydig cell subpopulation. Cell and tissue types negative for immunoreactive PGF2 alpha-R include: the capsule (tunica albuginea) and subcapsular stroma, all histologic layers of the vasculature (both venules and arterioles), peritubular stroma, peritubular boundary tissue, spermatogonia, primary and secondary spermatocytes, spermatids, Sertoli cells, and spermatozoa. While the above described localization of PGF2 alpha-R is also seen in rat, there are fewer rat Leydig cells and this subpopulation appears to atrophy and stain less intensely with increasing age of the animal. Preabsorption of the PGF2 alpha-R antiserum with a corpora lutea homogenate acetone powder eliminated immunohistochemical staining of the Leydig cell subpopulation further suggesting that the antigenic determinant detected here is related to that in the ovary (PGF2 alpha-R).

Aging↗
PubMed2002

Expression and distribution of inducible nitric oxide synthase in mouse testis.

Nitric oxide (NO) is a simple and relatively unstable radical under physiological conditions. It is synthesized by three isoforms of NO synthase, that is neuronal, endothelial and inducible (iNOS) isoforms. In the present study, we investigated the distribution of iNOS with immunohistochemical methods in the mouse testis. The iNOS-immunoreactivity was detected on the basal region of the seminiferous tubules, where the cytoplasm of Sertoli cells was selectively immunolabeled. This immunoreactivity was observed by both immunofluorescent and immunoenzyme methods. Weak immunoreactivity was detected on the perinuclear cytoplasm of Sertoli cells throughout the seminiferous stages, whereas in stages I-VIII, it was remarkable on the processes of Sertoli cells surrounding the spermatogonia and early spermatocytes, and elongating into the lumina of seminiferous tubules. By reverse transcriptase-polymerase chain reaction, mRNA for iNOS was found to be expressed in the mouse testis. These results reveal that iNOS is consistently distributed at the front of the testicular environment.

Animals↗
PubMed1982

Microvasculature of the mouse testis and excurrent duct system.

The microvascular architecture of the mouse testis and excurrent duct system was examined by a casting method with the aid of scanning electron microscopy and by sectioning techniques for light and transmission electron microscopy. Two fundamental types of peritubular capillary arrangements were noted in relation to the tubules. The first type most typically appeared in the testis. In this type, peritubular capillaries interconnected intertubular blood vessels to form a rope-ladder-like structure. The length of the individual peritubular capillaries was short, and a number of the capillaries encircled each tubule. Adjacent tubules shared a single layer of capillaries. The vascular organization of the ductuli efferentes and of the middle segment of the epididymis was also of this type, but the number of peritubular capillaries was much fewer compared with that of the testis. In the second type, the peritubular capillaries formed a network encircling each tubule in its subepithelial layer. The capillary organization in the initial and terminal segments of the epididymis and the ductus deferens belonged to the second type. In the distal part of the ductus deferens, a prominent venous plexus was formed in the most peripheral subepithelial layer.

Animals↗
PubMed1975

Subcellular compartmentation of free and esterified cholesterol in the interstitial cells of the mouse testis.

The subcellular distribution of free and esterified cholesterol in mouse testis and the changes occurring in cholesterol content of whole testes and cell fractions after inhibition of gonadotropins with methallibure (ICI 33, 828) and restimulation with human chorionic gonadotropin (HCG) are reported in the present paper. In subcellular fractions, the bulk of free cholesterol is associated with organelles sedimented in the microsomal fraction while esterified cholesterol is mainly stored in isolated lipid droplets. The latter compartment increases in methallibure-treated mice 2.3 fold and is remarkably depleted after administration of HCG. There is a close parallelism between the changes in esterified cholesterol content and the variation in the numbers of lipid droplets found in electron micrographs of interstitial cells of mice receiving similar treatments. By contrast no significant changes were noticed in either free cholesterol concentrations of the microsomal fractions or in the fine structure of organelles associated with this fraction. The dynamic nature of steroidogenesis in the microsomal fraction requires the existence of a free cholesterol pool with a high turnover rate for use as an intermediate in androgen synthesis. On the other hand, the large content of free cholesterol in microsomes and its stability under different conditions suggest the presence of a cholesterol compartment with a slow turnover, as a constituent of the membranes.

Animals↗
PubMed1987

Light and electron microscopic observations of giant cells in the mouse testis after efferent duct ligation.

Multinucleated giant cells in the mouse testis after ligation of the efferent duct were examined by light and electron microscopy. The findings suggest that the giant cells are formed as a result of the fusion of spermatids due to alterations in the intercellular bridges. Further, the orientation of acrosomes, nuclei, and organelles for formation of the tail in the giant cells is related to the polarity of the spermatids.

Animals↗