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PubMed2005

Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis.

In postmeiotic male germ cells, TSN, formerly known as testis brain-RNA binding protein, is found in the cytoplasm and functions as a posttranscriptional regulator of a group of genes transcribed by the transcription factor CREM-tau. In contrast, in pachytene spermatocytes, TSN is found predominantly in nuclei. Tsn-null males show a reduced sperm count and high levels of apoptosis in meiotic cells, suggesting a critical function for TSN during meiosis. To identify meiotic target RNAs that associate in vivo with TSN, we reversibly cross-linked TSN to RNA in testis extracts from 17-day-old and adult mice and immunoprecipitated the complexes with an affinity-purified TSN antibody. Extracts from Tsn-null mice were used as controls. Cloning and sequencing the immunoprecipitated RNAs, we identified four new TSN target mRNAs, encoding diazepam-binding inhibitor-like 5, arylsulfatase A, a tetratricopeptide repeat structure-containing protein, and ring finger protein 139. In contrast to the population of postmeiotic translationally delayed mRNAs that bind TSN, these four mRNAs are initially expressed in pachytene spermatocytes. In addition, anti-TSN also precipitated a nonprotein-coding RNA (ncRNA), which is abundant in nuclei of pachytene spermatocytes and has a putative polyadenylation signal, but no open reading frame. A second similar ncRNA is adjacent to a GGA repeat, a motif frequently associated with recombination hot spots. RNA gel-shift assays confirm that the four new target mRNAs and the ncRNA specifically bind to TSN in testis extracts. These studies have, for the first time, identified both mRNAs and a ncRNA as TSN targets expressed during meiosis.

Animals↗
PubMed2006

Leukemia inhibitory factor enhances formation of germ cell colonies in neonatal mouse testis culture.

Spermatogonial stem cells continuously divide in the testis to support spermatogenesis throughout the life of adult male animals. Although very few spermatogonial stem cells are present in vivo, we recently succeeded in expanding these cells in vitro. Germ cells from postnatal testes were able to proliferate in the presence of several types of cytokines, and they formed uniquely shaped colonies of spermatogonia (germline stem or GS cells). These cells reinitiated normal spermatogenesis when transplanted into seminiferous tubules. However, much remains unknown about the contributions of cytokines to successful stem cell culture. In the present study, we examined the role of leukemia inhibitory factor (LIF) in GS cell culture. We found that the addition of LIF to newborn testis cell culture enhances the formation of germ cell colonies. Ciliary neurotrophic factor, but not oncostatin M, had the same effect, although they both bind to the IL-6ST (gp130) receptor. On the other hand, GS cells could be established from pup or adult testes in the absence of LIF. No phenotypic or functional difference was found between GS cells established from different stages, and normal offspring were born from pup-derived GS cells that had been maintained in the absence of LIF, indicating that LIF per se is not involved in the self-renewal of GS cells. These results demonstrate that LIF is useful in the initiation of GS cell culture and suggest that LIF or a related cytokine is involved in the maturation of gonocytes into spermatogonia.

Animals↗
PubMed1991

Effects of in utero ultrasound exposure on the development of the fetal mouse testis.

The effect on the developing fetal testis of in utero exposure to 1-MHz, continuous-wave ultrasound in the spatial peak, temporal average intensity range 0.5-10 W/cm2 for durations of 400-30 sec on Day 9, 12, or 15 of gestation was determined. Results show that two subtle, yet potentially deleterious, effects occurred: a reduction in the Sertoli cell population and an apparent delay in the cessation of gonocyte mitosis. An increase was also seen in the number of fetal resorptions and stillborn pups per number of implantation sites in the exposed specimens as compared to the sham and cage controls. Because the reduction in testis weight was proportional to decreased body weight and because there was no difference in Sertoli cell numbers due to day of treatment, the testicular effects may reflect a generalized delay in growth. Whether this effect of ultrasound on fetal testis will be translated into an equal reduction in germ cell numbers in the adult testis remains to be determined.

Animals↗
PubMed1994

Developmental expression of DNA methyltransferase messenger ribonucleic acid, protein, and enzyme activity in the mouse testis.

Sex- and sequence-specific patterns of methylation of mammalian DNA are established during gametogenesis and are believed to be important for genomic imprinting and developmental gene regulation. DNA methylation in mammalian cells is performed predominantly by the enzyme DNA (cytosine-5)-methyltransferase (DNA MTase). For a better understanding of how DNA methylation events are regulated during spermatogenesis, a developmental study comparing the expression of DNA MTase mRNA, protein, and enzyme activity was performed. Northern and Western blotting and enzyme activity assays were carried out on testes and purified populations of cells from the testes of mice aged 6-70 days. The 5.2-kb DNA MTase transcript was most abundant in testes of mice aged 6-10 days (2-3-fold the 70-day values); it had decreased in abundance by 40% by Day 20 and reached steady adult levels by 63 days. On Western blot analysis, developmental changes in the relative abundance of DNA MTase protein paralleled the changes seen in mRNA concentrations. DNA MTase enzyme activity in the testis was highest at 6 days of age (10-fold the 70-day values); it had decreased by more than 65% by 20 days and reached steady adult levels at 35 days. Analysis of purified germ cells from the adult testis revealed high levels of expression of both DNA MTase mRNA and protein in haploid round spermatids. In conclusion, DNA MTase is clearly developmentally regulated during spermatogenesis at the level of mRNA, protein, and enzyme activity. These results argue for an important role for this DNA-methylating enzyme during spermatogenesis.

Aging↗
PubMed2000

Mouse testis brain ribonucleic acid-binding protein/translin colocalizes with microtubules and is immunoprecipitated with messenger ribonucleic acids encoding myelin basic protein, alpha calmodulin kinase II, and protamines 1 and 2.

Testis brain RNA-binding protein (TB-RBP) is a sequence-dependent RNA-binding protein that binds to conserved Y and H sequence elements present in many brain and testis mRNAs. Using recombinant TB-RBP and a highly enriched tubulin fraction, we demonstrate here that recombinant TB-RBP binds to microtubules assembled in vitro. The interaction between recombinant TB-RBP and microtubules was inhibited by high salt and by the microtubule disassembling agents colcemid and calcium, but not by the microfilament-disassembling agent cytochalasin D. Confocal microscopy confirmed colocalization of TB-RBP and tubulin in the cytoplasm of male germ cells. An affinity-purified antibody prepared against recombinant TB-RBP specifically precipitated mRNAs encoding myelin basic protein and alpha calmodulin-dependent kinase II-two transported mRNAs, and protamines 1 and 2-two translationally regulated testicular mRNAs. These data indicate an intracellular association between TB-RBP and specific target mRNAs and suggest an involvement of TB-RBP in microtubule-dependent mRNA transport in the cytoplasm of cells.

Amino Acid Sequence↗
PubMed2000

Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules.

Testes from adult and prepubertal mice lacking the Desert hedgehog (DHH:) gene were examined in order to describe further the role of Dhh in spermatogenesis because, in a previous report, DHH:-null male mice were shown to be sterile. Dhh is a signaling molecule expressed by Sertoli cells. Its receptor, patched (Ptc), has been previously localized to Leydig cells and is herein described as being localized also to peritubular cells. Two phenotypes of the mice were observed: masculinized (7.5% of DHH:-null males) and feminized (92.5%), both of which displayed abnormal peritubular tissue and severely restricted spermatogenesis. Testes from adult feminized animals lacked adult-type Leydig cells and displayed numerous undifferentiated fibroblastic cells in the interstitium that produced abundant collagen. The basal lamina, normally present between the myoid cells and Sertoli cells, was focally absent. We speculate that the abnormal basal lamina contributed to other characteristics, such as extracordal gonocytes, apolar Sertoli cells, and anastomotic seminiferous tubules. The two DHH:-null phenotypes described have common peritubular cell defects that may be indicative of the essential role of peritubular cells in development of tubular morphology, the differentiation of Leydig cells, and the ultimate support of spermatogenesis.

Androgen-Insensitivity Syndrome↗
PubMed2001

Expression of Fas and Fas ligand in normal and ischemia-reperfusion testes: involvement of the Fas system in the induction of germ cell apoptosis in the damaged mouse testis.

Apoptosis of germ cells is very common in normal and injured mammalian testes. The aim of this study was to examine the possible involvement of the Fas and Fas ligand (FasL) system in the induction of germ cell apoptosis in normal and ischemia-reperfusion testes of adult mice. Apoptosis was assessed by the TUNEL method and by DNA gel electrophoresis. Fas and FasL mRNAs were detected by Northern blotting and reverse transcription polymerase chain reaction techniques, and proteins were analyzed by Western blotting and immunohistochemistry. Apoptosis of germ cells was identified in the normal testis especially around stages XI and XII, whereas the expression of Fas and FasL was largely confined to Leydig cells and Sertoli cells, respectively. However, in the testes reperfused after 1 h of ischemia, a high number of TUNEL-positive cells were identified in parallel with increased Fas-positive germ cells, whereas FasL expression in Sertoli cells was almost constant irrespective of the duration of reperfusion. Moreover, i.p. injection of anti-Fas antibody, which blocks the interaction between Fas and FasL, inhibited apoptosis, as indicated by the reduced number of TUNEL-positive cells, except for apoptosis at stages XI and XII. Our results indicate that the Fas/FasL system mediates apoptosis of spermatogenic cells in the injured testis but not spontaneous apoptosis in the normal testis.

Animals↗
PubMed2001

Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis.

Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis.

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.

Animals↗
PubMedDate not supplied

Relative positions of the spermatogenic stages in the mouse testis: morphological evidences for their non-randomized adjacencies.

The topographical distribution pattern of the stages of the murine seminiferous epithelium cycle was investigated. PAS-hematoxylin stained testicular sections from adult mice representative of the apical, equatorial and caudal region of both testes, were used. The relative frequencies (RF) of the stages of the seminiferous epithelium cycle was estimated on the basis of more than 10,000 cross-sectioned seminiferous tubules identified according to the criteria of Leblond and Clermont (1952). It was found that in the testicular sections the stages of adjacent seminiferous tubules are not distributed randomly. The comparison of the RF of the stages (calculated over all the testicular sections) with the RF of the stages that are adjacent to a given seminiferous tubule stage suggests a clustered occurrence of numerically identical stages. These comparisons very often show statistically significant differences. The finding of such associations among adjacent segments of seminiferous tubules (stages) suggest the existence of an ordered distribution of the seminiferous tubules inside the testis possibly controlled by substances with local control capacity of spermatogenesis. On the basis of the findings here presented, it is suggested an interpretation of the phenomenon of modulations of the waves of the seminiferous epithelium.

Animals↗
PubMed1988

Melatonin does not affect in vitro secretion of testosterone in white-footed mouse testis.

There is evidence that melatonin may exert its antigonadal effect by acting in the gonad to inhibit steroidogenesis. To explore this possibility a hormonally sensitive in vitro system of dispersed Peromyscus leucopus testis was used to assess the effects of melatonin on human chorionic gonadotropin (hCG)- and dibutyryl-cAMP (dbcAMP)-stimulated secretion of testosterone (T). At near-maximally stimulating doses of hCG and dbcAMP, melatonin in concentrations of 2 x 10(-12) M to 2 x 10(-7) M did not affect the rate of secretion of T in cells from active or regressed testes. These results do not support the idea that melatonin helps to regulate seasonal reproduction by acting in the testes to inhibit steroidogenesis.

Animals↗