Germ Cells Of Rat Testis Research Articles

Acrosome‐specific gene AEP1: Identification, characterization and roles in spermatogenesis

Spermatogenesis is a tightly regulated process leading to the development of spermatozoa. To elucidate the molecular spermatogenic mechanisms, we identified an acrosome-specific gene AEP1 in spermatids, which is located in rat chromosome 17p14 with a transcript size of 3,091 bp encoding a signal peptide, zinc finger-like motif, coiled-coil region, several predicted glycosylation and phosphorylation sites. Northern blot and RT-PCR analyses revealed the restricted expression of AEP1 to the testis only. In postnatal rat testes, AEP1 mRNA became detectable from postnatal 25 dpp (round spermatids) and onwards. By using in situ hybridization (ISH) and flow cytometry-fluorescent ISH, only the haploid spermatids yielded the positive AEP1 signal. Immunohistochemistry showed that AEP1 was expressed in the acrosomal cap of late-staged germ cells in rat testis, and co-localized with the acrosomal marker, peanut agglutinin. The spatial expression of AEP1 immunoreactivity in testis was conserved among diverse mammalian species (rat, pig, monkey, human). To further study its roles in spermatogenesis, we showed AEP1 and beta-actin was associated together in complex by co-immunoprecipitation in adult germ cells and by immunofluorescence assay in isolated spermatozoon. In human testes diagnosed with hypospermatogenesis, lower expression of AEP1 was observed, whereas there was no detectable signal in undescended testes. In short, AEP1 is an evolutionary-conserved acrosome-specific gene and likely functions in acrosome-cap formation.

Enhanced Expression of Tyrosine Kinase Receptor A in Germ Cells of Rat Testis with Acute Experimental Testicular Torsion

Objective and Methods: To examine the involvement of neurotrophic factor receptors in the testis with acute experimental testicular torsion, the expression of tyrosine kinase receptors (trk) A and B, and p75 nerve growth factor receptor (NGFR) were studied in the rat testis with ischemia/reperfusion (I/R) injury, using Western blotting and immunohistochemistry. Apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Results: There was a significant increase in TUNEL-positive reaction in spermatogonia in the seminiferous tubules in rat testes after testicular torsion. Western blot analysis showed that trk A expression reached a significant peak at 12 h after reperfusion (p < 0.01), as compared to sham-operated controls, whereas trk B was not increased in the testis after I/R. Constitutive expression of p75 NGFR was at or below the level detectable by Western blot analysis, and it remained unchanged in the testis after I/R. Immunohistochemistry demonstrated that after I/R trk A expression was increased in spermatocytes and spermatids in the seminiferous tubules, in contrast to the basal location of the TUNEL-positive reaction. Immunoreactivity of trk B was seen mainly in the interstitial cells in the sham-operated testis, and its localization was not changed after I/R. Conclusion: It is postulated that trk A and B, but not p75 NGFR, are involved differently in the survival of testicular cells during acute experimental testicular torsion. In particular, increased trk A seems to be related to germ cell survival following I/R.

Identification and characterization of a novel splice variant of β3 subunit of GABA A receptor in rat testis and spermatozoa

γ-Aminobutyric acid type A (GABA A) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of β3 subunit of GABA A receptor, designated as β3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the β3 subunit of neuronal GABA A receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The β3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the β3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of β3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the β3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the β3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of β3 subunit of GABA A receptor, designated as β3t, is found in germ cells of rat testis and sperm. The inability of the β3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.

A novel gene, RSD-3/HSD-3.1, encodes a meiotic-related protein expressed in rat and human testis.

The expression of stage-specific genes during spermatogenesis was determined by isolating two segments of rat seminiferous tubule at different stages of the germinal epithelium cycle delineated by transillumination-delineated microdissection, combined with differential display polymerase chain reaction to identify the differential transcripts formed. A total of 22 cDNAs were identified and accepted by GenBank as new expressed sequence tags. One of the expressed sequence tags was radiolabeled and used as a probe to screen a rat testis cDNA library. A novel full-length cDNA composed of 2228 bp, designated as RSD-3 (rat sperm DNA no.3, GenBank accession no. AF094609) was isolated and characterized. The reading frame encodes a polypeptide consisting of 526 amino acid residues, containing a number of DNA binding motifs and phosphorylation sites for PKC, CK-II, and p34cdc2. Northern blot of mRNA prepared from various tissues of adult rats showed that RSD-3 is expressed only in the testis. The initial expression of the RSD-3 gene was detected in the testis on the 30th postnatal day and attained adult level on the 60th postnatal day. Immunolocalization of RSD-3 in germ cells of rat testis showed that its expression is restricted to primary spermatocytes, undergoing meiosis division I. A human testis homologue of RSD-3 cDNA, designated as HSD-3.1 (GenBank accession no. AF144487) was isolated by screening the Human Testis Rapid-Screen arrayed cDNA library panels by RT-PCR. The exon-intron boundaries of HSD-3.1 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The cDNA consisted of 12 exons that span approximately 52.8 kb of the genome sequence and was mapped to chromosome 14q31.3.

Novel Low Molecular Weight Microtubule-associated Protein-2 Isoforms Contain a Functional Nuclear Localization Sequence

Known high and low molecular weight (LMW) MAP2 protein isoforms result from alternative splicing of the MAP2 gene. Contrary to previous reports that MAP2 is neural-specific, we recently identified MAP2 mRNA and protein in somatic and germ cells of rat testis, and showed the predominant testicular isoform is LMW. Although cytoplasmic in neural tissue, MAP2 appeared predominantly nuclear in germ cells using immunohistochemistry. We sought to determine whether this unexpected localization was due to the inclusion of exon 10 within novel LMW MAP2 isoforms. Normally excluded from the LMW MAP2c, exon 10 harbors a putative CcN motif, comprising a nuclear localization sequence (NLS) flanked by regulatory phosphorylation sites for protein kinase CK2 and cdc2 kinase. Characterization of MAP2 mRNA in adult and immature brain and testis, by reverse transcriptase-polymerase chain reaction/Southern analysis and Northern blot, identified novel LMW forms containing exons 10 and 11, previously detected only in high molecular weight MAP2a and 2b. The MAP2 NLS targeted a large heterologous protein to the nucleus, as demonstrated using bacterially expressed MAP2-CcN-beta-galactosidase fusion protein and an in vitro nuclear import assay. Antibodies raised against the fusion protein produced a testicular immunohistochemical staining pattern correlating with MAP2 protein distribution in the nucleus of most germ cells, and precipitated both approximately 70-kDa and >220-kDa proteins recognized by the commercial MAP2-specific HM2 monoclonal antibody, supporting our hypothesis of a novel LMW MAP2 isoform. These results demonstrate the presence of a functional NLS in MAP2 and indicate that novel LMW MAP2 isoforms may be targeted to the nucleus in both neural and non-neuronal tissues.

Behaviour of the germ cell specific lamin through mammalian spermatogenesis as probed with monoclonal antibodies.

We had earlier identified a 60 kDa nuclear lamin protein (lamin(g)) unique to the germ cells of rat testis which was subsequently shown to be antigenically conserved in germ cells of grasshopper, rooster, frog and plants. We have now obtained eight monoclonal antibodies in mouse against this lamin(g) antigen. While all the eight Mabs reacted with lamin(g) antigen in an immunoblot analysis, only three Mabs (A11C7, A11D4, C1F7) showed strong reactivity in the immunofluorescence analysis of the germ cells. The Mabs A11C7 and A11D4 showed a slight cross-reactivity with rat liver lamin B. Indirect immunofluorescence analysis of pre-meiotic, meiotic and post-meiotic germ cells with Mabs have shown that while the lamin(g) is localized in the lamina structures of spermatogonia and round spermatids, it is localized to the phase dense regions of pachytene spermatocytes which is in conformity with our previous observations using rabbit polyclonal antibodies. The localization of the antigen in the germ cells was also confirmed by immunohistochemical staining of the thin sections of seminiferous tubules. By immunostaining the surface spread pachytene spermatocytes, the antigen was further localized to the telomeric ends of the paired homologous chromosomes. Using anti-somatic lamin B antibodies, we have also demonstrated the absence of somatic lamins in meiotic and post-meiotic germ cells. The lamina structure of pre-meiotic spermatogonial nucleus contains both somatic lamin B and lamin(g) as evidenced by immunofluorescence studies with two differently fluorochrome labelled anti-lamin B and anti-lamin(g) antibodies. The selective retention of lamin(g) in the pachytene spermatocytes is probably essential for anchoring the telomeric ends of the paired chromosomes to the inner nuclear membrane.

A transferrinlike (hemiferrin) mRNA is expressed in the germ cells of rat testis.

In the testis, germ cells which are separated from the serum by the blood-testis barrier rely primarily on the Sertoli cell to obtain nutrients. For example, transferrin synthesized by the Sertoli cell is important in delivering iron from the serum to the developing germ cells. Because of its role in the testis, Sertoli cell transferrin protein and mRNA have been extensively studied. By using RNA blot analysis of rat testicular tissue, we detected a transcript of 2.6 kb which is attributed to transferrin. In addition, we detected a novel mRNA of 0.9 kb which had sequence similarity to the 3' end of transferrin. This 0.9-kb mRNA was present in germ cells, but not Sertoli cells, liver, or brain. The primary source of this mRNA in the testis was round spermatids. Sequence analysis of a cDNA clone showed that this mRNA encoded a protein with sequence similarity to the carboxy terminus of transferrin. Polysome analysis indicated that this transcript was translated and may therefore have importance in the iron metabolism of germ cells. The evolutionary implications between the transferrinlike mRNA germ cells and the gene duplication event which resulted in the diferric binding of transferrin are discussed.

A Transferrinlike (Hemiferrin) mRNA Is Expressed in the Germ Cells of Rat Testis

In the testis, germ cells which are separated from the serum by the blood-testis barrier rely primarily on the Sertoli cell to obtain nutrients. For example, transferrin synthesized by the Sertoli cell is important in delivering iron from the serum to the developing germ cells. Because of its role in the testis, Sertoli cell transferrin protein and mRNA have been extensively studied. By using RNA blot analysis of rat testicular tissue, we detected a transcript of 2.6 kb which is attributed to transferrin. In addition, we detected a novel mRNA of 0.9 kb which had sequence similarity to the 3' end of transferrin. This 0.9-kb mRNA was present in germ cells, but not Sertoli cells, liver, or brain. The primary source of this mRNA in the testis was round spermatids. Sequence analysis of a cDNA clone showed that this mRNA encoded a protein with sequence similarity to the carboxy terminus of transferrin. Polysome analysis indicated that this transcript was translated and may therefore have importance in the iron metabolism of germ cells. The evolutionary implications between the transferrinlike mRNA germ cells and the gene duplication event which resulted in the diferric binding of transferrin are discussed.

Immunocytochemical demonstratin of c‐fos protein in sertoli cells and germ cells in rat testis

Immunocytochemical demonstratin of c‐fos protein in sertoli cells and germ cells in rat testis

Relative cellular distribution of particulate androgen binding between sertoli and germ cells in rat testis

An androgen binding activity with characteristics similar to ABP is present in a particulate fraction (105,000 g pellet) obtained by differential centrifugation of seminiferous tubules, impure Sertoli cells and impure germ cells homogenates. Purification of germ cells through albumin gradients, results in almost a complete loss of androgen binding activity in the purified germ cell suspensions. Furthermore, Sertoli cell enriched testes from 22-, 32- and 40-day old rats showed increases in particulate androgen binding, when compared to matched controls, parallel to increments in the activity of a Sertoli cell marker enzyme (β-glucuronidase). These results suggest that particulate androgen binding activity is only present in Sertoli cells and this protein may play a role in the compartmentalization of androgens in the testis.

Effect of medroxyprogesterone acetate of 3 alpha-hydroxysteroid oxidoreductase of testicular germ cells and prostate of the rat.

On incubation with testosterone (T), nonflagellate germ cells of rat testis produced dihydrotestosterone (DHT) and 5a-androstane-3cs, I 7�I-diol (3o-diol). When the concentration of T was adjusted to 0.1 �zM (its level in rete testis fluid), 4-androstene-3, 17-dione (A) and DHT accumulated during the first 30 mm; during the subsequent 90 mm, A declined and 3&-diol accumulated, DHT remaining at almost constant level. Medroxyprogesterone acetate (MPA, 5 pM) abolished 3s-diol production, but allowed DHT to accumulate at linear rates. Separation of nonflagellate germ cells by velocity gradient sedimentation yielded spermatocytes and spermatids as major fractions. On incubation with 0.1 pM T, the spermatocytes produced A and DHT; the spermatids, although metabolizing T at lower rates, produced relatively large amounts of 3cs-diol. In the presence of T in physiological concentration, MPA did not inhibit Scs-reductase or 1 713-hydroxysteroid oxidoreductase of germ cells. It abolished 3o-diol formation in the spermatids, allowing DHT to accumulate in equivalent amounts. The action of MPA as a specific inhibitor of 3o-hydroxysteroid oxidoreductase without effect on Scs-reductase was also observed in rat prostate extracts when