Ethnic variation in the association between sleep and body mass among US adolescents.

OBJECTIVE: We investigate whether differences in sleep duration help explain ethnic disparities in body mass index (BMI) among US adolescents. We also evaluate the functional form of the association between sleep duration and BMI, and investigate whether this association varies by sex and ethnicity. PARTICIPANTS AND METHODS: We analyzed restricted-use data from the first two waves of the National Longitudinal Study of Adolescent Health (n=30 133) to evaluate linear and quadratic associations between sleep duration and BMI. Through a series of models that incorporated interaction terms between sex, ethnicity and sleep duration, we also assessed whether (1) sleep duration mediates associations between ethnicity and BMI, and (2) associations between sleep duration and BMI differ for girls and boys from different ethnic groups. RESULTS: A linear association between sleep duration and BMI best fits the data in this large sample of US adolescents. We find no evidence that sleep duration contributes substantially to ethnic disparities in BMI. However, we detect significant differences in the association between sleep duration and BMI by sex and ethnicity. Sleep duration is negatively associated with BMI among White, Hispanic and Asian boys, positively associated with BMI among Black girls and is not related to BMI among Black boys or girls from White, Hispanic or Asian ethnic groups. CONCLUSIONS: Despite significant associations between sleep duration and BMI for certain groups of adolescents, we find no evidence that ethnic differences in sleep duration exacerbate ethnic disparities in BMI. Future research should explore mechanisms that underlie ethnic differences in the association between sleep and BMI.

Current SEM techniques for de- and re-construction of centromeres to determine 3D CENH3 distribution in barley mitotic chromosomes.

Combined light microscopic (LM) and field emission scanning electron microscopic (FESEM) techniques with FluoroNanogold labelling allowed quantification and high resolution analysis of 3D distribution of the centromere-specific histone H3 variant CENH3 in barley mitotic chromosomes. Chromosomes were investigated with fluorescence LM, conventional FESEM, low-voltage FESEM and combined FIB/FESEM techniques for unprecedented comprehensive analysis to determine chromatin distribution patterns in the centromere. Using data from FIB/FESEM sectioning of centromeric regions of chromosomes, it was possible to render 3D reconstruction of the CENH3 distribution with highest resolution achieved to date. Complementary data derived from each approach show that CENH3 localizes not only to the primary constriction, but also in the pericentric regions and is distributed exclusively in the interior, rather than on the surface, of the centromere. This is relevant for understanding kinetochore assembly and digresses from current models of centromere structure. We emphasize here this broad microscopic approach, focusing on technical aspects of combined FESEM techniques, for which advantages and limitations are discussed, providing a relevant example--in the field of centromeric research--for application to investigations of other subcellular biological structures.

Holocentric chromosomes of Luzula elegans are characterized by a longitudinal centromere groove, chromosome bending, and a terminal nucleolus organizer region.

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability.

Chromosome centromeres: structural and analytical investigations with high resolution scanning electron microscopy in combination with focused ion beam milling.

Whole mount mitotic metaphase chromosomes of different plants and animals were investigated with high resolution field emission scanning electron microscopy (FESEM) to study the ultrastructural organization of centromeres, including metacentric, acrocentric, telocentric, and holocentric chromosome variants. It could be shown that, in general, primary constrictions have distinctive ultrastructural features characterized by parallel matrix fibrils and fewer smaller chromomeres. Exposure of these structures depends on cell cycle synchronization prior to chromosome isolation, chromosome size, and chromosome isolation technique. Chromosomes without primary constrictions, small chromosomes, and holocentric chromosomes do not exhibit distinct ultrastructural elements that could be directly correlated to centromere function. Putative spindle structures, although rarely observed, spread over the primary constriction to the bordering pericentric regions. Analytical FESEM techniques, including specific DNA staining with Pt blue, staining of protein as a substance class with silver-colloid, and artificial loosening of fixed chromosomes with proteinase K, were applied, showing that centromere variants and ultrastructural elements in the centromere differ in DNA and protein distribution. Immunogold localization allowed high-resolution comparison between chromosomes with different centromere orientations of the distribution of centromere-related histone variants, phosphorylated histone H3 (ser10), and CENH3. A novel application of FESEM combined with focused ion beam milling (FIB) provided new insights into the spatial distribution of these histone variants in barley chromosomes.

Consensus statement on the diagnosis and treatment of children with idiopathic short stature: a summary of the Growth Hormone Research Society, the Lawson Wilkins Pediatric Endocrine Society, and the European Society for Paediatric Endocrinology Workshop.

OBJECTIVE: Our objective was to summarize important advances in the management of children with idiopathic short stature (ISS). PARTICIPANTS: Participants were 32 invited leaders in the field. EVIDENCE: Evidence was obtained by extensive literature review and from clinical experience. CONSENSUS: Participants reviewed discussion summaries, voted, and reached a majority decision on each document section. CONCLUSIONS: ISS is defined auxologically by a height below -2 sd score (SDS) without findings of disease as evident by a complete evaluation by a pediatric endocrinologist including stimulated GH levels. Magnetic resonance imaging is not necessary in patients with ISS. ISS may be a risk factor for psychosocial problems, but true psychopathology is rare. In the United States and seven other countries, the regulatory authorities approved GH treatment (at doses up to 53 microg/kg.d) for children shorter than -2.25 SDS, whereas in other countries, lower cutoffs are proposed. Aromatase inhibition increases predicted adult height in males with ISS, but adult-height data are not available. Psychological counseling is worthwhile to consider instead of or as an adjunct to hormone treatment. The predicted height may be inaccurate and is not an absolute criterion for GH treatment decisions. The shorter the child, the more consideration should be given to GH. Successful first-year response to GH treatment includes an increase in height SDS of more than 0.3-0.5. The mean increase in adult height in children with ISS attributable to GH therapy (average duration of 4-7 yr) is 3.5-7.5 cm. Responses are highly variable. IGF-I levels may be helpful in assessing compliance and GH sensitivity; levels that are consistently elevated (>2.5 SDS) should prompt consideration of GH dose reduction. GH therapy for children with ISS has a similar safety profile to other GH indications.

Idiopathic short stature: management and growth hormone treatment.

In the management of ISS auxological, biochemical, psychosocial and ethical elements have to be considered. In boys with constitutional delay of growth and puberty androgens are effective in increasing height and sexual characteristics, but adult height is unchanged. GH therapy is efficacious in increasing height velocity and adult height, but the inter-individual variation is considerable. The effect on psychosocial status is uncertain. Factors affecting final height gain include GH dose, height deficit in comparison to midparental height, age and first year height velocity. In case of a low predicted adult height at the onset of puberty, addition of a GnRH analogue can be considered. Although GH therapy appears safe, long-term monitoring is recommended.

Sniffing the diagnosis: Olfactory testing in neurodegenerative parkinsonism.

OBJECTIVE: To determine the diagnostic utility of olfactory testing in patients with neurodegenerative parkinsonism. METHODS: The Sniffin' Sticks test battery for assessment of odor identification, discrimination, and threshold was applied to patients with Parkinson's disease (PD), multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) as well as healthy controls (HC). Two different cohorts were analyzed: A PD/healthy control that included PD patients and HC as well as a PD/diseased control cohort for which patients PD, MSA and PSP were recruited. The former cohort was exploited to calculate cut-off values that discriminate PD patients from HC with a sensitivity (sensitivity-weighted cut-off) or specificity (specificity-weighted cut-off) exceeding 95%, respectively. The PD/diseased controls cohort was used to determine the diagnostic accuracy using these cut-off values in discriminating patients with neurodegenerative parkinsonism. RESULTS: PD patients (n = 67) performed significantly worse in olfactory testing than HC (n = 41) and patients with MSA (n = 23) or PSP (n = 23). There was no significant difference in olfactory function between MSA and PSP patients. Diagnostic performance of the identification subscore was similar to the sum score of the Sniffin' Sticks test (AUC identification test 0.94, AUC sum score 0.96), while threshold and discrimination subscores were inferior. In patients with parkinsonism, the specificity-weighted cut-off predicted a diagnosis of PD with a sensitivity and specificity of 76.6 and 87.0%, respectively. The discriminative value of this cut-off in separating PD from MSA was 76.7% (sensitivity) and 95.7% (specificity). The corresponding, prevalence-adjusted positive predictive value of olfactory testing exceeded 95%. CONCLUSIONS: Our data suggest that assessment of olfactory function, particularly odor identification, can be useful to discriminate PD from atypical parkinsonian disorders, particularly MSA patients.

The ERK-dependent signalling is stage-specifically modulated by FSH, during primary Sertoli cell maturation.

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent differentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA- and also Src-dependent manner. This activation is required for FSH-induced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic effect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their differentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite effects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells.

3D analysis of chromosome architecture: advantages and limitations with SEM.

Three-dimensional mitotic plant chromosome architecture can be investigated with the highest resolution with scanning electron microscopy compared to other microscopic techniques at present. Specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis. Applied to investigate the structural effects of different preparative procedures, these techniques were the groundwork for the "dynamic matrix model" for chromosome condensation, which postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.

Management of cryptorchidism.

The forces guiding testicular descent have not been completely elucidated. Both testosterone and anti-Müllerian hormone might play a role. Available evidence suggests that malfunction of the testes of some sort usually precedes maldescent. The proper management of cryptorchidism has long been a controversial issue. In unilateral cryptorchidism, hormonal function and fertility are generally normal. To maximize fertility in patients with bilateral cryptorchidism, surgical treatment should be completed ideally by the first birthday. GnRH is unlikely to be of much help in initiating testicular descent. Cryptorchidism is associated with a three- to tenfold increase in testicular cancer. Twenty percent of tumors in unilateral cryptorchidism are in the normally descended testes. The condition of all boys and men with a history of cryptorchidism should be followed by physicians their entire lives, and these boys and men must become proficient in self-examination.

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.

Urinary excretion of IGF-I and growth hormone in children with IDDM.

OBJECTIVE: To compare the urinary output of insulinlike growth factor I (IGF-I) and growth hormone (GH) in prepubertal and pubertal children with insulin-dependent diabetes mellitus (IDDM) versus nondiabetic subjects and to analyze the relationship between the urinary excretion of these peptides and degree of metabolic control. RESEARCH DESIGN AND METHODS: Group 1 included 30 IDDM patients who had had diabetes for 4.9 +/- 0.7 yr and had normal renal function (mean age 11.6 +/- 0.9 yr); group 2 consisted of 31 control subjects (mean age 9.2 +/- 0.6 yr). Sensitive radioimmunoassays were used to measure IGF-I and GH in urine aliquots from 12-h timed overnight collections that had been dialyzed, concentrated 50-fold, and lyophilized. RESULTS: Significantly lower IGF-I and GH outputs per kilogram body weight per 12 h were observed in IDDM subjects compared with control subjects. When data were expressed per kilogram of body weight, no difference was observed between the urinary output of IGF-I and GH between prepubertal and pubertal subjects within group 1 or group 2. The prepubertal children had significantly lower HbA1 than the pubertal population; however, no correlation was found between urinary output of IGF-I or GH and HbA1. A positive correlation was observed between urinary IGF-I and GH (r = 0.85, P less than .001). CONCLUSIONS: Patients with long-standing IDDM excrete significantly lower urinary levels of IGF-I and GH compared with normal subjects. Serial measurements of these peptides from onset of IDDM are needed to define whether the changes observed are present at diagnosis or are secondary to duration of disease.

Interleukin-10 inhibits burst-forming unit-erythroid growth by suppression of endogenous granulocyte-macrophage colony-stimulating factor production from T cells.

Numerous cytokines released from accessory cells have been shown to exert either stimulatory or inhibitory growth signals on burst-forming unit-erythroid (BFU-E) growth. Because of its cytokine synthesis-inhibiting effects on T cells and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirectly affecting erythropoiesis. We investigated the effects of IL-10 on BFU-E growth from normal human peripheral blood mononuclear cells (PBMC) using a clonogenic progenitor cell assay. The addition of recombinant human IL-10 to cultures containing recombinant human erythropoietin suppressed BFU-E growth in a dose-dependent manner (by 55.2%, range 47.3-63.3%, p < 0.01, at 10 ng/mL). In contrast, no inhibitory effect of IL-10 was seen when cultivating highly enriched CD34+ cells. BFU-E growth from PBMC also was markedly suppressed in the presence of a neutralizing anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (by 48.7%, range 32.9-61.2% inhibition,p < 0.01), but not by neutralizing antibodies against granulocyte colony-stimulating factor and interleukin-3. This suggests a stimulatory role of endogenously released GM-CSF on BFU-E formation. Also, the addition of exogenous GM-CSF completely restored IL-10-induced suppression of BFU-E growth. To determine the cellular source of GM-CSF production, we analyzed GM-CSF levels in suspension cultures containing PBMC that were either depleted of monocytes or T cells. Monocyte-depleted PBMC showed spontaneous production of increasing amounts of GM-CSF on days 3, 5, and 7, respectively, which could be suppressed by IL-10, whereas GM-CSF levels did not increase in cultures containing T-cell-depleted PBMC. Our data indicate that IL-10 inhibits the growth of erythroid progenitor cells in vitro, most likely by suppression of endogenous GM-CSF production from T cells.

Diverse deletions in the growth hormone receptor gene cause growth hormone insensitivity syndrome.

Growth hormone insensitivity syndrome (GHIS; also known as Laron syndrome), is characterized by severe postnatal growth failure and normal growth hormone. The syndrome is frequently caused by point mutations in the growth hormone receptor gene (GHR). Here we report five families with GHIS and partial deletions of the GHR gene. The deletion breakpoints were sequenced and PCR-based diagnostic tests were developed. In a Cambodian family, a novel deletion removed part of exon 5 and 1.2 kb of the preceding intron. The deletion occurred by recombination within four identical nucleotides. In the mutant transcript, skipping of the truncated exon 5 leads to a frameshift and premature termination codon (PTC). A previously reported discontinuous deletion of GHR exons 3, 5, and 6 was identified in three Oriental Jewish families. An unaffected individual was heterozygous for the exon 5 and 6 deletion, but homozygously deleted for exon 3 suggesting that the exon 3 deletion is a polymorphism. The pathogenic deletion of exons 5 and 6 spans about 7.5 kb. Sequence analysis of the breakpoints revealed an imperfect junction between introns 4 and 6, with a four basepair insertion. A novel deletion of 13 nucleotides within exon 9 was identified in a Caucasian girl with GHIS who carries the I153T missense mutation on her other allele. The exon 9 deletion leads to a frameshift and PTC. The predicted protein retains the transmembrane domain and a short cytoplasmic tail. Four family members in three generations were carriers of this deletion, but only two of them were below normal for height, suggesting that this mutation by itself does not act as a dominant negative, as was reported for two other GHR mutations which lead to truncation of the intracellular domain.

Expression and functionality of luteinizing hormone/chorionic gonadotropin receptor in the rat prostate.

In addition to androgens that are essential for maintenance of prostate growth and function, nonandrogenic hormones must also be considered to explain some regulatory events occurring in the prostate. The detection of LH/CG receptor (LH/CG-R) gene expression in some nongonadal tissues, has led us to consider LH as a potential regulatory factor of prostatic development and function. In this study, we have demonstrated by RT-nested polymerase chain reaction and Northern blotting that the rat prostate contains the same LH/CG-R transcript as the gonads. Western immunoblotting, ligand blotting, and binding analysis have shown that rat prostate also contains a 93-kDa receptor protein that is able to bind [125I]hCG specifically and with a high affinity (6.0 x 10(9) M-1). Our results also indicated that the concentration of binding sites was lower in the prostate than in the gonads. LH/CG-R sites of expression have been localized in the prostate by immunohistochemistry: specific staining was observed in all the epithelial cells of the gland, but the ventral lobes are much more immunoreactive than the lateral and dorsal lobes. Finally, the ability of this prostatic LH/CG-R to induce a physiological response was evaluated in an explant culture system. A time course experiment was carried out, and we observed a significant dose-dependent stimulation of cAMP production after 3 h of treatment: 3.0 +/- 0.4, 4.2 +/- 0.4, and 5.0 +/- 0.5 pmol/ml of cAMP for 0, 100, and 500 ng/ml of LH, respectively. In conclusion, LH is able to act directly on the prostatic gland through specific receptors that are structurally and functionally very similar to those expressed in the gonads and are mainly localized in the ventral lobe of the organ. These data suggest that LH plays a significant physiological role in the prostate.

Synthesis of 1,25-dihydroxyvitamin D in the nephrectomized pregnant rat.

Pregnant rats were maintained on diets either adequate or deficient in vitamin D. On the 20th day of gestation, animals were either nephrectomized bilaterally or sham operated. Immediately therafter, four groups of nephrectomized or sham-operated pregnant rats received iv [26,27-3H]25-hydroxyvitamin D3 ([26,27-3H]25OHD3), while two groups received [1,2-3H,4-14C]D3. The animals were sacrificed 10-24 h later. The distribution of the radiolabeled metabolites of vitamin D3 was determined in extracts of maternal plasma, maternal intestinal tract, placentae, and fetuses after Sephadex LH-20 column chromatography. Both vitamin D3 and 25OHD3 crossed the placenta and entered the fetus. In anephric animals receiving [26,27-3H]-25OHD3, 24,25-dihydroxyvitamin D and a polar peak eluting in the position of 1,25-dihydroxyvitamin D [1,25(OH)2D] and 25,26-dihydroxyvitamin D were identified in extracts of maternal plasma and intestinal tracts and of placentae and fetuses. The identities of 24,25-dihydroxyvitamin D and 1,25 (OH)2D were confirmed by high pressure liquid chromatography. In rats receiving [1,2-3H,4-14C]D3, approximately 50% of the polar metabolite consisted of 1,25(OH)2D. We conclude that the anephric pregnant rat is able to synthesize 1,25(OH)2D, that the fetal portion of the feto-placental unit is the most likely site of production of this hormone, and that this metabolite of vitamin D is able to cross the placenta from the fetus to the mother.

Growth hormone directly affects the function of the different lobes of the rat prostate.

In this study, we investigated the involvement of GH in rat prostate function. First, we demonstrated that specific transcripts corresponding to the GH receptor (4.5 kilobases) and to the GH-binding protein (1.2 kilobases) were expressed in the normal rat prostate, but also in all prostatic carcinoma cell lines tested (LNCaP, PC-3, MAT-Lu, MAT-LyLu, and Pif-1). Moreover, these transcripts were much more abundant in the human and rat carcinoma cells than in the normal tissue. One-year-old dwarf rats were supplemented for 7 days with saline (group DR1) or highly purified rat GH (group DR2). Northern blotting and quantitation of prostatic messenger RNAs (mRNAs) revealed that GH increases the steady state levels of transcripts coding for androgen receptor (2.4-fold), type I and II 5 alpha-reductases (2.6- and 2.2-fold), and several androgen-dependent proteins [prostatein C3 subunit (3.6-fold), probasin (11.0-fold), and R. W. B. (Royal Winnipeg Ballet) (12.5-fold)]. This suggests that GH might either potentiate the action of androgens on the prostate or act directly on this gland by a mechanism that does not depend on testicular androgens. To address this question, we supplemented hypophysectomized and castrated adult rats for 7 days with saline (group HC1), rat GH (group HC2), testosterone propionate (group HC3), or GH plus testosterone (group HC4), starting 3 days after castration. In this animal model, the abundance of C3 mRNA increased in all hormone-treated rats; the stimulation factors were 3.5 (group HC2), 25.5 (group HC3), and 9.5 (group HC4) compared to group HC1. Analysis of prostatein synthesis by Western blotting confirmed these results at the protein level. The same trend was observed for probasin and RWB mRNA levels. Probasin mRNA increased 4.5-fold in group HC2 and 12-fold in group HC3, but did not increase in group HC4 (both hormones combined); enhancement of RWB mRNA was, respectively, 5.0-, 28.0-, and 15.0-fold in groups HC2, HC3, and HC4. GH did not affect the abundance of androgen receptor mRNA. As described previously, the level of this mRNA dropped significantly in group HC3. GH alone did not significantly alter the level of either 5 alpha-reductase mRNA, whereas testosterone, alone or with GH, produced a 2-fold increase in type II 5 alpha-reductase mRNA (groups HC3 and HC4). Type I isoenzyme mRNA reached 1.6 times the control level (group HC1) in groups HC3 and HC4.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat G protein-coupled receptor kinase GRK4: identification, functional expression, and differential tissue distribution of two splice variants.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G protein-coupled receptors, leading to the homologous mode of desensitization. We report here on the cloning of complementary DNAs that encode two rat GRK4 variants. Rat GRK4A (575 amino acids) displays 76% identity with the long human GRK4 splice variant. Rat GRK4B (545 amino acids) delineates a new variant that is identical to GRK4A except for a 31-amino acid deletion in the N-terminal domain, corresponding to exon VI in the human GRK4 gene. GRKs4A and B are likely produced by alternative splicing from a single gene, the partial characterization of which revealed a structural organization similar to that of the human GRK4 gene. GRK4A messenger RNA (mRNA) is abundant only in testis. A combination of in situ hybridization and quantitative RT-PCR studies demonstrated that GRK4A mRNA level increases during testicular development and predominates in leptotene to late pachytene primary spermatocytes and round spermatids. GRK4B mRNA is poorly expressed in testis and most rat tissues but is heterogeneously distributed in the kidney, with 20-fold enrichment in the outer medulla. GRKs4A and B are both functional protein kinases, as demonstrated in a rhodopsin phosphorylation assay. The differential tissue distribution of GRKA4 and GRK4B suggests that individual GRK4 variants may serve distinct physiological functions.

A novel messenger ribonucleic acid homologous to human MAGE-D is strongly expressed in rat Sertoli cells and weakly in Leydig cells and is regulated by follitropin, lutropin, and prolactin.

We have cloned a novel complementary DNA whose expression was decreased in rat Sertoli cell cultures after treatment with FSH. This complementary DNA encodes a protein of 570 amino acids and shares 92% homology with the human MAGE-D protein. In contrast to other MAGE genes (A, B, or C), we have shown that MAGE-D expression was ubiquitous in healthy rat tissues. In the seminiferous tubules, the MAGE-D was expressed in Sertoli cells but not in germ cells as demonstrated by RT-PCR and in situ hybridization, whereas for the other MAGE genes, expression has been shown to be restricted to germ cells. Interestingly, MAGE-D was also detected for the first time in the female gonad by Northern blotting. In MLTC-1 cells (mouse Leydig tumor cell line-1), LH and PRL stimulated MAGE-D expression. Using hypophysectomized rats, it was confirmed that FSH decreased MAGE-D expression, whereas LH and PRL increased MAGE-D messenger RNA level in the whole testis most probably through a direct action on Leydig cells. As MAGE-D is present in both the seminiferous compartment and interstitium and hormonally regulated in each, it is possible that it has specific functions in each compartment during the development and the maintenance of the testis.