Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.     

Metallothionein Induction in Neonatal Rat Primary Astrocyte Cultures Protects Against Methylmercury Cytotoxicity

Abstract: Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [α-³²P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, ∼550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 × 10⁻⁶ M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic d -[³H]aspartate uptake. Preexposure of astrocytes to CdCl₂, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on d -[³H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl₂ treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl₂ pretreatment) attenuate MeHg-induced toxicity. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.     

Stage- and cell-specific expression of cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat seminiferous epithelium.

Expression of mRNAs in the rat testis encoding cyclic AMP (cAMP)-dependent protein kinases (PKAs) was studied. A microdissection method was used to isolate 10 pools of seminiferous tubules representing various stages of the cycle of the seminiferous epithelium in combination with Northern blots and in situ hybridization. The results showed a differential expression of the four isoforms of the regulatory subunits (PKA-R) at various stages of the cycle. RI alpha mRNA was detected at approximately the same levels at all stages while expression of RI beta mRNA was low at stages XIII-III, started to increase at stages IV-V, and reached a maximum at stages VIII-XI. The level of RII alpha mRNA was low at stages II-VI, increased markedly at stage VIIa,b, and reached maximal levels at stages VIIc,d and VIII, followed by a reduced expression at later stages, RII beta mRNA levels increased significantly at stage VI with maximal levels at stages VII and VIII. In situ hybridization of sections from the adult rat testis revealed RI alpha mRNA in the layers of pachytene spermatocytes and round spermatids of all stages. RI beta mRNA was detected over late pachytene spermatocytes and round spermatids of stages VII-XIII. RII alpha mRNA was seen in the layers of round spermatids of stages VII-VIII and elongating spermatids of later stages while RII beta mRNA was detected only in the round spermatid region of stages VII-VIII and in some tubules of stages I-VI. These data show that mRNAs encoding PKA-R are expressed in a stage-specific manner in differentiating male germ cells with different patterns of expression for each subunit; this suggests specific roles for these protein kinases at different times of spermatogenesis.     

Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.     

Expression of nuclear transport importins beta 1 and beta 3 is regulated during rodent spermatogenesis

Spermatogenic differentiation requires progressive gene expression changes, and proteins required for this must be transported into the nucleus. Many of these contain a nuclear localization signal and are likely to be transported by importin protein family members, each of which recognizes and transports distinct cargo proteins. We hypothesized that importins, as modulators of protein nuclear access, would display distinct expression profiles during spermatogenesis, indicating their potential to regulate key steps in cellular differentiation. This was tested throughout testicular development in rodents. Real-time PCR analysis of postnatal mouse testes revealed changing expression levels of Knpb1 (encoding importin beta 1) and Ranbp5 (encoding beta 3) mRNAs, with Knpb1 highest at 26 days postpartum and Ranbp5 highest in Day 26 and adult testis. Their distinctive cellular expression patterns visualized using in situ hybridization and immunohistochemistry were identical in mouse and rat testes where examined. Within the seminiferous epithelium, Knpb1 mRNA and importin beta1 protein were detected within mitotic Sertoli and germ cells during fetal and early postnatal development, becoming restricted to spermatogonia and spermatocytes in adulthood. Importin beta 3 protein in fetal germ cells displayed a striking difference in intracellular localization between male and female gonads. In adult testes, Ranbp5 mRNA was detected in round spermatids and importin beta 3 protein in elongating spermatids. This is the first comprehensive in situ demonstration of developmentally regulated synthesis of nuclear transport components. The contrasting expression patterns of importins beta 1 and 3 identify them as candidates for regulating nuclear access of factors required for developmental switches.     

Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats.

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.     

A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse.

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno-protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S- and 3H-labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno-protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno-protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno-protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis.     

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.