Cellular localization of mRNAs for retinoic acid receptor-alpha, cellular retinol-binding protein, and cellular retinoic acid-binding protein in rat testis: evidence for germ cell-specific mRNAs

In the present study we have examined the cellular localization and developmental changes of mRNAs for retinoid-binding proteins in rat testis. We demonstrate that mRNA (0.7 kb) for cellular retinol-binding protein (CRBP) is expressed only in Sertoli cells and peritubular cells. The mRNA for CRBP could not be detected in other testicular cells. In contrast, mRNA for cellular retinoic acid-binding protein (CRABP) was detected primarily in germ cells and to a small extent in tumor Leydig cells. The mRNA for CRABP in germ cells revealed distinct size heterogeneity and three distinct mRNA species were observed (1.0, 1.8, and 1.9 kb), in contrast to previous data for somatic cells where only the 1.0-kb mRNA has been reported. Messenger RNAs for retinoic acid receptor-alpha (RAR alpha) were detected in both somatic and haploid germ cells. The highest level of RAR alpha was seen in Sertoli cells, round spermatids, and tumor Leydig cells. Lower, but distinct, levels were observed in peritubular cells. Furthermore, we observed germ cell-specific species of RAR alpha mRNA (4 kb and approximately 7 kb). The smallest mRNA for RAR alpha (2.7 kb) in somatic cells was absent in germ cells. The levels of mRNAs for the various retinoid-binding proteins in whole testis obtained from rats of various ages confirmed this cellular localization. The mRNAs for CRBP, the small molecular size (2.7 kb) mRNA for RAR alpha (localized to somatic cells), and the 1-kb mRNA for CRABP showed an age-dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular expression of retinal dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA

We examined expression of retinal dehydrogenase (RALDH) types 1 and 2 in liver and lung, and the effect of vitamin A status on testis expression by in situ hybridization. Liver expressed RALDH1 and RALDH2 only in stellate cells and hepatocytes, respectively. Lung expressed RALDH1 and RALDH2 throughout the epithelia of the airways, from the principal bronchi to the respiratory bronchiole. Vitamin A-sufficient rats expressed RALDH1 in spermatocytes, with less intense expression in spermatogonia and spermatids, and expressed RALDH2 in interstitial cells, spermatogonia, and spermatocytes. Neither Sertoli nor peritubular cells showed detectable RALDH1 or RALDH2 mRNA. Vitamin A deficiency produced a sevenfold increase in RALDH1 and a 70-fold decrease in RALDH2 mRNA in testis. In each case, the net change reflected extensive loss of germ cells, increased intensity of expression in residual germ cells, and expression in Sertoli and peritubular cells. Low-dose RA relatively early during vitamin A depletion supported spermatogenesis and affected expression of both RALDHs, but did not reinstate "vitamin A normal" expression patterns. These results show that: RALDH1 and RALDH2 have distinct mRNA expression patterns in multiple cell types in three vitamin A target tissues; RALDH expression occurs in cell types that express cellular retinol-binding protein and retinol dehydrogenase isozymes (except stellate cells, for which retinol dehydrogenase expression remains unknown); vitamin A deficiency and RA supplementation affects the loci and intensity of RALDH mRNAs in testis; and low-dose RA does not substitute completely for retinol. Overall, these data provide insight into the unique functions of RALDH1 and RALDH2 in retinoid metabolism.     

Cellular localization and age-dependent changes in mRNA for cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat testis

Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes. The regulatory subunit (R) designated RII51 was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated C alpha, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the C alpha, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis. In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.     

Vitamin A metabolism in cultured somatic cells from rat testis

Sertoli and peritubular myoid cells, the somatic cells of the seminiferous tubule, support growth and differentiation of developing germ cells. This action strictly depends on the availability of in situ synthesized retinoic acid and we have previously documented the ability of Sertoli, but not peritubular cell extracts, to support the oxidation of retinol to retinoic acid. Using primary cultures of somatic cells treated with a physiological concentration of free retinol, we show here that the same is essentially true also for whole cultured cells. Sertoli cells are capable of producing not only retinoic acid, but are also the major site of retinyl ester (mainly, retinyl palmitate) formation. Compared with retinyl palmitate accumulation, retinoic acid synthesis was both faster and positively influenced by prior exposure to retinol. This increase in retinoic acid synthesis was further augmented by treatment with the retinoic acid catabolic inhibitor liarozole, thus indicating that enhanced synthesis, rather than reduced catabolism, is responsible for such an effect. Myoid cells had a higher capacity to incorporate exogenously supplied retinol, yet retinoic acid synthesis, and even more so retinyl palmitate formation, were considerably lower than in Sertoli cells. Retinoic acid synthesis in myoid cells was not only depressed, but also very little influenced by prior retinol exposure and totally insensitive to liarozole. These data further support the view that myoid cells are involved in retinol uptake from the blood and its transfer to other cells, rather than in metabolic interconversion or long-term storage of vitamin A, two processes that mainly take place in Sertoli cells.     

Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin a and retinoic acid

Lecithin:retinol acyltransferase (LRAT), present in microsomes, catalyzes the transfer of the sn-1 fatty acid of phosphatidylcholine to retinol bound to a cellular retinol-binding protein. In the present study we have cloned mouse and rat liver LRAT cDNA and tested the hypothesis that LRAT mRNA, like LRAT activity, is regulated physiologically in a liver-specific manner. The nucleotide sequences of mouse and rat liver LRAT cDNA each encode a 231-amino acid protein with 94% similarity between these species, and approximately 80% similarity to a cDNA for LRAT from human retinal pigment epithelium. Expression of rat LRAT cDNA in HEK293T cells resulted in functional retinol esterification and storage. RNA from several rat tissues hybridized with liver LRAT cDNA. However, LRAT mRNA was virtually absent from the liver of vitamin A-deficient animals, while being unaffected in intestine and testis. LRAT mRNA was rapidly induced by retinoic acid (RA) in liver of vitamin A-deficient mice and rats (P < 0.01). LRAT mRNA and enzymatic activity were well correlated in the same livers of rats treated with exogenous RA (r = 0.895, P < 0.0001), and in a dietary study that encompassed a broad range of vitamin A exposure (r = 0.799, P < 0.0001). Liver total retinol of <100 nmol/g was associated with low LRAT expression (<33% of control).We propose that RA, derived exogenously or from metabolism, serves as an important signal of vitamin A status. The constitutive expression of liver LRAT during retinoid sufficiency would serve to divert retinol into storage pools, while the curtailment of LRAT expression in retinoid deficiency would maintain retinol for secretion and delivery to peripheral tissues.     

Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus

Cellular retinoic acid-binding protein (CRABP), a potential mediator of retinoic acid action, enables retinoic acid to bind in a specific manner to nuclei and chromatin isolated from testes of control and vitamin A-deficient rats. The binding of retinoic acid was followed after complexing [3H]retinoic acid with CRABP purified from rat testes. The binding was specific, saturable, and temperature dependent. If CRABP charged with nonlabeled retinoic acid was included in the incubation, binding of radioactivity was diminished, whereas inclusion of free retinoic acid, or the complex of retinol with cellular retinol binding protein (CRBP) or serum retinol binding protein had no effect. Approximately 4.0 X 10(4) specific binding sites for retinoic acid were detected per nucleus from deficient animals. The number of binding sites observed was influenced by vitamin A status. Refeeding vitamin A-deficient rats (4 h) with retinoic acid lowered the amount of detectable binding sites in the nucleus. CRABP itself did not remain bound to these sites, indicating a transfer of retinoic acid from its complex with CRABP to the nuclear sites. Further, CRBP, the putative mediator of retinol action, was found to enable retinol to be bound to testicular nuclei, in an interaction similar to the binding of retinol to liver nuclei described previously.     

In vivo metabolism of topically applied retinol and all-trans retinoic acid by the rabbit cornea

Corneas of normal and vitamin A-deficient rabbits were treated topically with [11, 12-3H] retinol or [11, 12-3H] all-trans retinoic acid. Methanol extracts of these corneas were analyzed by high pressure liquid chromatography. Radiolabeled compounds were extracted from the corneas which co-migrated chromatographically with known retinoid standards. In agreement with studies on other tissues and organs, retinol was metabolized to retinoic acid and more polar compounds by corneas of normal and vitamin A-deficient rabbits. All-trans retinoic acid was isomerized to 13-cis retinoic acid in normal rabbit corneas; however, this trans-cis isomerization did not occur in vitamin A-deficient, xerophthalmic corneas.     

Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture

A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules.     

Retinoic acid inhibits rat XY gonad development by blocking mesonephric cell migration and decreasing the number of gonocytes

Vitamin A (also called retinol) and its derivatives, retinoic acids (RAs), are required for postnatal testicular function. Abnormal spermatogenesis is observed in rodents on vitamin A-deficient diets and in retinoic acid receptor alpha (RARalpha) knockout mice. In contrast, RA has an inhibitory effect on the XY gonad development in embryos. To characterize this inhibitory effect of RA, we investigated the cellular events that are required for the XY gonad development, including cell migration from the adjacent mesonephros into the gonad, fetal Sertoli cell differentiation, and survival of gonocytes. In organ cultures of Embryonic Day 13 (E13) XY gonads from rats, all-trans-retinoic acid (tRA) inhibited mesonephric cell migration into the gonad. Moreover, treatment with tRA decreased the expression of Müllerian-inhibiting substance in Sertoli cells and dramatically reduced the number of gonocytes. Increased apoptosis was detected in the XY gonads cultured with tRA, suggesting that the loss of gonocytes could be due to increased apoptosis. In addition, Am580, a synthetic compound that exhibits RARalpha-specific agonistic properties, mimicked the inhibitory effects of tRA on the XY gonad development including mesonephric cell migration and gonocyte survival. Conversely, a RARalpha-selective antagonist, Ro 41-5253, suppressed the inhibitory ability of tRA on the XY gonad development. These results suggest that retinoic acid acting through RARalpha negatively affects fetal Sertoli cell differentiation and gonocyte survival and blocks the migration of mesonephric cells, thereby leading to inhibition of the XY gonad development.     

Comparison of the effects of vitamin A and its analogs upon rabbit ear cartilage in organ culture and upon growth of the vitamin A-deficient rat

A study was conducted to explore the relationship between the effects of vitamin A upon cartilage and the biological role of vitamin A in maintaining growth and life. Retinol, retinoic acid, alpha-retinoic acid, and ROB-7699 (a cyclopentyl analog of retinoic acid) were highly effective in promoting the lysis of the extracellular matrix of cartilage grown in organ culture in vitro. Retinoic acid and its two analogs were quantitatively more active than was retinol in bringing about lysis of matrix and release of proteoglycan into the culture medium. A bioassay was then conducted to determine the ability of each compound to promote growth of vitamin A-deficient rats. In contrast to their effects upon cartilage, retinoic acid and its two analogs were considerably less active quantitatively than retinol in promoting growth of vitamin A-deficient rats. Moreover, the three acids tested showed graded biological activity in the growth bioassay, with alpha-retinoic acid showing reduced bioactivity (approx. one-fourth that of retinoic acid) and ROB-7699 being virtually inactive. The lysis of cartilage produced by these compounds was presumably caused by release of lysosomal enzymes as a result of the membrane-labilizing effects of the compounds. Thus, these membrane effects of the vitamin A-related compounds are poorly correlated with their biological growth-promoting activity. The alpha-ionone analogs of retinol and retinoic acid were able to maintain good health and growth of vitamin A-deficient rats, although their quantitative activity was low. Rats fed alpha-retinyl acetate showed high liver stores of alpha-retinyl esters and low levels of serum retinol-binding protein (similar to the levels seen in retinoic acid-fed rats). The biological activity of the alpha-ionone analogs was apparently not due to contamination with or conversion to the normal beta-ionone compounds.     

Retinol and retinoic acid increase MMP-2 activity by different pathways in cultured Sertoli cells

Diseases such as atherosclerosis, arthritis and cancer have been related with imbalance in ROS production and failures in regulation of the MMPs. Authors suggested a relationship between MPP activity and ROS. Our research group has demonstrated that retinol 7µM induced changes in Sertoli cell metabolism linking retinol treatment and oxidative stress. We verified MMP activity in Sertoli cells treated with vitamin A using gelatin zymography. We found that retinol (7µM) and retinoic acid (1nM) induced MMP-2 activity in Sertoli cells. Antioxidants reversed retinol-induced but not retinoic acid-induced MMP-2 activity. Moreover, retinol but not retinoic acid increased ROS production quantified by DCFH-DA oxidation. We found that retinol and retinoic acid induced ERK1/2 phosphorylation, but only retinol-increased MMP-2 activity was inhibited by UO126, an ERK1/2 phosphorylation inhibitor. Our findings suggested that retinol-induced MMP-2 activity, but not retinoic acid-induced MMP-2 activity, was related to ERK1/2 phosphorylation and ROS production.     

Retinol and retinoic acid increase MMP-2 activity by different pathways in cultured Sertoli cells

Diseases such as atherosclerosis, arthritis and cancer have been related with imbalance in ROS production and failures in regulation of the MMPs. Authors suggested a relationship between MPP activity and ROS. Our research group has demonstrated that retinol 7µM induced changes in Sertoli cell metabolism linking retinol treatment and oxidative stress. We verified MMP activity in Sertoli cells treated with vitamin A using gelatin zymography. We found that retinol (7µM) and retinoic acid (1nM) induced MMP-2 activity in Sertoli cells. Antioxidants reversed retinol-induced but not retinoic acid-induced MMP-2 activity. Moreover, retinol but not retinoic acid increased ROS production quantified by DCFH-DA oxidation. We found that retinol and retinoic acid induced ERK1/2 phosphorylation, but only retinol-increased MMP-2 activity was inhibited by UO126, an ERK1/2 phosphorylation inhibitor. Our findings suggested that retinol-induced MMP-2 activity, but not retinoic acid-induced MMP-2 activity, was related to ERK1/2 phosphorylation and ROS production.