Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development.

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

2MD (DP001), a Single Agent in the Management of Hemodialysis Patients: A Randomized Trial.

BACKGROUND: Vitamin D analogs and calcimimetics are used to manage secondary hyperparathyroidism (SHPT) in dialysis patients. DP001 is an oral vitamin D analog that suppresses parathyroid hormone (PTH) in uremic rats, osteopenic women, and hemodialysis patients. The safety and effectiveness of DP001 suppressing PTH in dialysis patients previously managed with active vitamin D with or without a calcimimetic are presented. METHODS: A multicenter, randomized, double-blind study compared DP001 to placebo in hemodialysis patients with serum-intact PTH (iPTH) ≥300 pg/ml. The primary efficacy endpoint was the proportion of patients achieving 2 consecutive ≥30% decreases in iPTH levels during the 12 weeks of treatment. Calcium, phosphorus, calcium × phosphorus product and safety were also evaluated. The responses to DP001 were compared in patients previously treated with both active vitamin D and a calcimimetic to those previously on active vitamin D alone. RESULTS: Sixty-two patients were randomized (n = 34 DP001; n = 28 placebo). At week 12, 78% of all DP001-treated patients and 7% of all placebo-treated patients achieved the primary endpoint (p < 0.0001); iPTH fell 45% in the DP001 group and increased 37% in the placebo group. No patient exceeded the safety threshold of 2 consecutively corrected serum calcium levels ≥11.0 mg/dl. Patients previously on cinacalcet plus active vitamin D also responded to DP001 (n = 10) resulting in a 55% decrease in iPTH, while those on placebo (n = 9) increased by 70%. CONCLUSION: DP001 safely and effectively suppressed iPTH in hemodialysis patients with SHPT that were previously managed with active vitamin D alone or with a calcimimetic (www.clinicaltrials.gov, NCT01922843).

Use of 2MD, a Novel Oral Calcitriol Analog, in Hemodialysis Patients with Secondary Hyperparathyroidism.

BACKGROUND: Use of existing therapies for secondary hyperparathyroidism (SHPT), such as calcitriol or paricalcitol, is frequently limited by the development of hypercalcemia. 2-Methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD; DP001) is a novel and a more potent vitamin D receptor activator (VDRA) that more selectively localizes in the parathyroid gland, and has a wider therapeutic margin in the uremic rat model than calcitriol and paricalcitol. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Hemodialysis patients were enrolled and dosed with 110, 220, 330, 440, or 550 ng of 2MD orally thrice weekly for 4 weeks. Responders were defined as patients having a ≥30% reduction in parathyroid hormone (PTH) from baseline, and were assessed at weeks 2 and 4. RESULTS: Of 31 patients recruited, 24 completed the 4-week treatment. There was little or no reduction in PTH in the 110 and 220 ng dose cohorts. Higher dose cohorts had greater PTH suppression with more than half the patients in the 440 and 550 ng dose cohorts considered responders (≥30% PTH reduction from baseline). None had oversuppression of PTH or hypercalcemia (corrected serum calcium >10.6 mg/dl). Plasma drug concentration increased with increasing dose, and all responders achieved a 2MD concentration of ≥1.5 pg/ml. All dose levels of 2MD were well tolerated without safety concerns. CONCLUSIONS: In hemodialysis patients with SHPT, 2MD, at thrice weekly oral doses of 440 and 550 ng, is well tolerated and effectively suppresses PTH without hypercalcemia. Future studies are needed to study the long-term implications of treating ESRD patients with this novel VDRA.

A complex RARE is required for the majority of Nedd9 embryonic expression.

Neural precursor cell expressed, developmentally down-regulated 9 (Nedd9, Casl, Hef1, p105cas, Ef1) is a scaffolding protein that assembles complexes involved in regulating cell adhesion, migration, division, and survival. Nedd9 is found very early in the developing embryonic nervous system. A highly conserved complex retinoic acid response element (RARE) is located 485 base pairs (bp) upstream of exon 2B in the promoter of the Nedd9 gene. Mice transgenic for a 5.2 kilobase (kb) region of the 2B Nedd9 promoter containing the RARE upstream of a lacZ reporter gene [Nedd9(RARE)-lacZ] show a large subset of the normal endogenous Nedd9 expression including that in the caudal hindbrain neuroepithelium, spinal cord, dorsal root ganglia (drg) and migrating neural crest (ncc). However, the transgenic mice do not recapitulate the native Nedd9 expression pattern in presumptive rhombomeres (pr) 3 and 5 of the early hindbrain, the base of the neuroepithelium in the midbrain, nor the forebrain telencephalon. Thus, the 5.2 kb region containing the intact RARE drives a large subset of Nedd9 expression, with additional sequences outside of this region needed to define the full complement of expression. When the 5.2 kb construct is modified (eight point mutations) to eliminate responsiveness of the RARE to all-trans retinoic acid (atRA) [Nedd9(mutRARE)-lacZ], virtually all ß-galactosidase (ß-gal, lacZ) expression is lost. Exposure of Nedd9(RARE)-lacZ transgenic embryos to excess atRA at embryonic day 8.0 (E8.0) leads to rostral ectopic transgene expression within 6 h whereas the Nedd9(mutRARE)-lacZ mutant does not show this effect. Thus the RARE upstream of the Nedd9 2B promoter is necessary for much of the endogenous gene expression during early development as well as ectopic expression in response to atRA.

Novel, selective vitamin D analog suppresses parathyroid hormone in uremic animals and postmenopausal women.

BACKGROUND: The use of 1α-hydroxylated vitamin D therapy to control secondary hyperparathyroidism in renal failure patients has been a success story, culminating with the demonstration of increased life expectancy in patients treated with these compounds. However, hypercalcemic episodes have been a recurrent problem with these therapies and have resulted in the added use of calcium mimetics. Clearly there is good reason to search for improved vitamin D therapy. In our inventory of vitamin D compounds, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) surfaced as a potential candidate. This was based on its preferential localization in the parathyroid gland and a clear suppression of serum parathyroid hormone (PTH) levels without a change in serum calcium in a clinical trial in postmenopausal women. METHODS: 2MD has now been tested in the rat 5/6-nephrectomy model of renal failure, and in postmenopausal women to determine if it can suppress serum PTH at doses that do not elevate serum calcium and serum phosphorus concentrations. RESULTS: Daily oral treatment of uremic rats on 2.5 ng/bw/day of 2MD dramatically suppressed PTH without a change in serum calcium or serum phosphorus. Further, PTH was suppressed in postmenopausal women after only 3 daily oral doses of 2MD that continued for 4 weeks with no change in serum calcium or serum phosphorus. CONCLUSION: These results coupled with a pharmacokinetic half-life of ~24 h suggest that 2MD given either daily or at the time of dialysis may be a superior therapy for secondary hyperparathyroidism in chronic renal failure patients.

14-3-3ε and NAV2 interact to regulate neurite outgrowth and axon elongation.

Neuron navigator 2 (NAV2) is required for all-trans retinoic acid (atRA) to induce neurite outgrowth in human neuroblastoma cells. Further, ectopic overexpression of full-length human NAV2 rescues an axonal elongation defect in the Caenorhabditis elegans unc-53 (NAV2 ortholog) mutant. Using a region of NAV2 that independently associates with the cytoskeleton as bait in a yeast-two-hybrid screen, 14-3-3ε was identified as a novel NAV2 interacting partner. Amino acids 761-960 of NAV2 are sufficient to confer a positive interaction with 14-3-3ε as evidenced by a two-hybrid screen and co-immunoprecipitation assay. Knockdown of 14-3-3ε leads to a decrease in atRA-mediated neurite outgrowth, similar to the elongation defects observed when NAV2 is depleted or mutated. Likewise, posterior lateral microtubule (PLM) defects in C. elegans fed unc-53 RNAi are similar to those fed ftt-2 (14-3-3 homolog) RNAi. The discovery of an interaction between NAV2 and 14-3-3ε could provide insight into the mechanism by which NAV2 participates in promoting cell migration and neuronal elongation.

The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells.

The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21(Cip1), a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G(1)/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation.

26- and 27-Methyl groups of 2-substituted, 19-nor-1α,25-dihydroxylated vitamin D compounds are essential for calcium mobilization in vivo.

Twelve new analogs of 19-nor-1α,25-dihydroxyvitamin D36-17, were prepared by a multi-step procedure from known alcohols 18 and 19. We have examined the influence of removing two methyl groups located at C-25, as well as the 25-hydroxy group, on the biological in vitro and in vivo biological activity. Surprisingly, removal of the 26- and 27-methyl groups from either the 2α-methyl or 2-methylene-19-nor-1α,25-dihydroxyvitamin D3 reduced vitamin D receptor binding, HL-60 differentiation, and 25-hydroxylase transcription in vitro only slightly to moderately (compounds 6-13). However, these compounds were devoid of in vivo bone mobilization activity and had markedly reduced activity on intestinal calcium transport. The analogs 14-17 with a 2ß-methyl substitution had little or no activity in vitro and in vivo as expected from previous work.

Nav2 hypomorphic mutant mice are ataxic and exhibit abnormalities in cerebellar development.

Development of the cerebellum involves a coordinated program of neuronal process outgrowth and migration resulting in a foliated structure that plays a key role in motor function. Neuron navigator 2 (Nav2) is a cytoskeletal-interacting protein that functions in neurite outgrowth and axonal elongation. Herein we show that hypomorphic mutant mice lacking the full-length Nav2 transcript exhibit ataxia and defects in cerebellar development. At embryonic day (E)17.5, the mutant cerebellum is reduced in size and exhibits defects in vermal foliation. Reduction in cell proliferation at early times (E12.5 and E14.5) may contribute to this size reduction. The full-length Nav2 transcript is expressed in the premigratory zone of the external granule layer (EGL). Granule cells in the germinal zone of the EGL appear to proliferate normally, however, due to the reduction in cerebellar circumference there are fewer total BrdU-labeled granule cells in the mutants, and these fail to migrate normally toward the interior of the cerebellum. In Nav2 hypomorphs, fewer granule cells migrate out of cerebellar EGL explants and neurite outgrowth from both explants and isolated external granule cell cultures is reduced. This suggests that the formation of parallel axon fibers and neuronal migration is disrupted in Nav2 mutants. This work supports an essential role for full-length Nav2 in cerebellar development, including axonal elongation and migration of the EGL neurons.

Astrocytes as a regulated source of retinoic acid for the brain.

Retinaldehyde dehydrogenases (RALDH) catalyze the synthesis of the regulatory factor retinoic acid (RA). Cultured astrocytes express several of the RALDH enzyme family, and it has been assumed that this can be extrapolated to astrocytes in vivo. However, this study finds that few astrocytes in the rodent brain express detectable RALDH enzymes, and only when these cells are grown in culture are these enzymes upregulated. Factors controlling the expression of the RALDHs in cultured astrocytes were explored to determine possible reasons for differences between in vitro versus in vivo expression. Retinoids were found to feedback to suppress several of the RALDHs, and physiological levels of retinoids may be one route by which astrocytic RALDHs are maintained at low levels. In the case of RALDH2, in vivo reduction of vitamin A levels in rats resulted in an increase in astrocyte RALDH2 expression in the hippocampus. Other factors though are likely to control RALDH expression. A shift in astrocytic RALDH subcellular localization is a potential mechanism for regulating RA signaling. Under conditions of vitamin A deficiency, RALDH2 protein moved from the cytoplasm to the nucleus where it may synthesize RA at the site of the nuclear RA receptors. Similarly, in conditions of oxidative stress RALDH1 and RALDH2 moved from the cytoplasm to a predominantly nuclear position. Thus, the RALDHs have been revealed to be dynamic in their expression in astrocytes where they may maintain retinoid homeostasis in the brain.

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.

4-Hydroxybenzyl modification of the highly teratogenic retinoid, 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB), yields a compound that induces apoptosis in breast cancer cells and shows reduced teratogenicity.

Retinoids are a class of compounds with structural similarity to vitamin A. These compounds inhibit the proliferation of many cancer cell lines but have had limited medical application as they are often toxic at therapeutic levels. Efforts to synthesize retinoids with a greater therapeutic index have met with limited success. 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB) is one of the most biologically active all-trans-retinoic acid (atRA) analogues and is highly teratogenic. In this study, we show that modification of the TTNPB carboxyl group with an N-(4-hydroxyphenyl)amido (4HPTTNPB) or a 4-hydroxybenzyl (4HBTTNPB) group changes the activity of the compound in cell culture and in vivo. Unlike TTNPB, both compounds induce apoptosis in cancer cells and bind poorly to the retinoic acid receptors (RARs). Like the similarly modified all-trans-retinoic acid (atRA) analogues N-(4-hydroxyphenyl)retinamide (4-HPR/fenretinide) and 4-hydroxybenzylretinone (4-HBR), 4HBTTNPB is a potent activator of components of the ER stress pathway. The amide-linked analogue, 4HPTTNPB, is less toxic to developing embryos than the parent TTNPB, and most significantly, the 4-hydroxybenzyl-modified compound (4HBTTNPB) that cannot be hydrolyzed in vivo to the parent TTNPB compound is nearly devoid of teratogenic liability.

Calmin expression in embryos and the adult brain, and its regulation by all-trans retinoic acid.

The vitamin A metabolite, all-trans retinoic acid (atRA), is a regulator of nervous system development. Using a subtracted cDNA library constructed from neuroblastoma cells, the atRA-responsive gene calmin (Clmn) was identified (Merrill et al. [2004] Biol Chem 385:605-614). The Clmn transcript is detected very early in rat embryonic development and is sensitive to retinoid status. In vitamin A-deficient embryos, Clmn mRNA is dramatically down-regulated in the neuroepithelium adjacent to the somites, and this expression can be rescued with the addition of atRA. In embryonic day 18.5 embryos, CLMN is detected in regions where newly differentiated neurons are found, including the neural retina and the cortical plate; and in the adult brain, CLMN is most highly expressed in the neuron cell bodies of the hippocampus, cerebellum, and olfactory bulb. Thus, Clmn is sensitive to retinoid status during early gestational stages, and its expression is relegated to postmitotic neuronal cells in the adult rat brain.

The temporal requirement for vitamin A in the developing eye: mechanism of action in optic fissure closure and new roles for the vitamin in regulating cell proliferation and adhesion in the embryonic retina.

Mammalian eye development requires vitamin A (retinol, ROL). The role of vitamin A at specific times during eye development was studied in rat fetuses made vitamin A deficient (VAD) after embryonic day (E) 10.5 (late VAD). The optic fissure does not close in late VAD embryos, and severe folding and collapse of the retina is observed at E18.5. Pitx2, a gene required for normal optic fissure closure, is dramatically downregulated in the periocular mesenchyme in late VAD embryos, and dissolution of the basal lamina does not occur at the optic fissure margin. The addition of ROL to late VAD embryos by E12.5 restores Pitx2 expression, supports dissolution of the basal lamina, and prevents coloboma, whereas supplementation at E13.5 does not. Surprisingly, ROL given as late as E13.5 completely prevents folding of the retina despite the presence of an open fetal fissure, showing that coloboma and retinal folding represent distinct VAD-dependent defects. Retinal folding due to VAD is preceded by an overall reduction in the percentage of cyclin D1 positive cells in the developing retina, (initially resulting in retinal thinning), as well as a dramatic reduction in the cell adhesion-related molecules, N-cadherin and beta-catenin. Reduction of retinal cell number combined with a loss of the normal cell-cell adhesion proteins may contribute to the collapse and folding of the retina that occurs in late VAD fetuses.

Expression pattern of Nav2 in the murine CNS with development.

Neuron navigator 2 (NAV2, RAINB1, POMFIL2, HELAD1, unc53H2) is essential for nervous system development. In the present study the spatial distribution of Nav2 transcript in mouse CNS during embryonic, postnatal and adult life is examined. Because multiple NAV2 proteins are predicted based on alternate promoter usage and RNA splicing, in situ hybridization was performed using probes designed to the 5' and 3' ends of the Nav2 transcript, and PCR products using primer sets spanning the length of the mRNA were also examined by real time PCR (qPCR). These studies support full-length Nav2 transcript as the predominant form in the wild-type mouse CNS. The developing cortex, hippocampus, thalamus, olfactory bulb, and granule cells (GC) within the cerebellum show the highest expression, with a similar staining pattern using either the 5'Nav2 or 3'Nav2 probe. Nav2 is expressed in GC precursors migrating over the cerebellar primordium as well as in the postmitotic premigratory cells of the external granule cell layer (EGL). It is expressed in the cornu ammonis (CA) and dentate gyrus (DG) throughout hippocampal development. In situ hybridization was combined with immunohistochemistry for Ki67, CTIP2 and Nissl staining to follow Nav2 transcript location during cortical development, where it is observed in neuroepithelial cells exiting the germinal compartments, as well as later in the cortical plate (CP) and developing cortical layers. The highest levels of Nav2 in all brain regions studied are observed in late gestation and early postnatal life which coincides with times when neurons are migrating and differentiating. A hypomorphic mouse that lacks the full-length transcript but expresses shorter transcript shows little staining in the CNS with either probe set except at the base of the cerebellum, where a shorter Nav2 transcript is detected. Using dual fluorescent probe in situ hybridization studies, these cells are identified as oligodendrocytes and are detected using both Olig1 and the 3'Nav2 probe. The identification of full-length Nav2 as the primary transcript in numerous brain regions suggests NAV2 could play a role in CNS development beyond that of its well-established role in the cerebellum.

A New 1,25 Dihydroxy Vitamin D Analog with Strong Bone Anabolic Activity in OVX Rats with Little or no Bone Resorptive Activity.

A new 1α,25-dihydroxy vitamin D3 analog (2-methylene-22(E)-(24R)-22-dehydro-1α,24,25-trihydroxy-19-norvitamin D3 or WT-51) has been tested as a possible therapeutic for osteoporosis. It is 1/10th as active as 1,25(OH)2 D3 in binding affinity for the vitamin D receptor but is at least 200 times more active than 1,25(OH)2 D3 and equal to that of 2MD (2-methylene-19-nor-(20S)-1α,25(OH)2 D3 , an analog previously tested in postmenopausal women), in supporting bone formation by isolated osteoblasts in culture. However, in contrast to 2MD, it is virtually inactive on bone resorption in vivo. WT-51 markedly increased bone mass (lumbar and femur) in ovariectomized (OVX) female rats. Further, bone strength tested by the three-point bending system is significantly increased by WT-51. Thus, WT-51 is an attractive candidate for the treatment of postmenopausal osteoporosis. © 2019 American Society for Bone and Mineral Research.

A nutritional model of late embryonic vitamin A deficiency produces defects in organogenesis at a high penetrance and reveals new roles for the vitamin in skeletal development.

Vitamin A plays an essential role in vertebrate embryogenesis. In the present study, pregnant vitamin A-deficient (VAD) rats were maintained during early pregnancy on the short half-life vitamin A metabolite, all-trans retinoic acid (atRA), in an amount sufficient to support normal development to E10.5, with a higher level of atRA (250 microg atRA/g diet) provided from embryonic day (E) 8.5-10.5 to prevent mid-gestational resorption. When limiting amounts of atRA (1.5 or 12 microg/g diet) were provided after E10.5, a highly reproducible and penetrant state of late fetal vitamin A deficiency (late VAD) was induced in the organs of developing fetuses. In addition, late VAD fetuses displayed both anteriorization of cervical regions and novel posteriorization events at the thoracic and sacral levels of the skeleton, and showed sternal and pelvic malformations not previously observed in early VAD or genetic models. The expression of several Hox genes (Hoxd3 and Hoxb4) was altered in late VAD embryos, with a reduction in Hoxd3 noted as early as 1 day after instituting deficiency. All late VAD-induced malformations were prevented by the addition of retinol starting at E10.5, whereas provision of a high level of atRA throughout pregnancy improved but could not completely rescue the development of all organ systems. This work defines a nutritional model in which vitamin A deficiency can be induced during fetal development, and reveals new functions for the vitamin in the development of the axial and appendicular skeleton.

Vitamin A deficiency blocks the initiation of meiosis of germ cells in the developing rat ovary in vivo.

Vitamin A (retinol) is required for male and female reproduction as well as to support many developmental processes. In the male, meiotic entry of germ cells occurs after birth and throughout adulthood, whereas in the female, the entry into meiosis I occurs during embryonic development. Evidence from cultured embryonic ovaries suggests that the vitamin A metabolite, all-trans retinoic acid (atRA), initiates this process. However, in vivo evidence to support a normal role for atRA in meiotic entry is lacking. The present study demonstrates that although germ cell number is normal in ovaries from both vitamin A-sufficient (VAS) embryos and those that are deficient in atRA, the majority of germ cells in the most severely atRA-deficient group fail to enter meiosis and remain in an undifferentiated state. In contrast, in a group that is only moderately deficient in atRA, a small number of ovarian germ cells enter meiosis (30%) compared with 75% of cells in the VAS control group. The expression of the atRA-responsive gene, Stra8, is reduced by approximately 90% and 50% in the severely and moderately atRA-deficient ovaries, respectively, compared with the VAS controls. These results provide the first in vivo evidence that vitamin A regulates the entry of germ cells into meiosis in the developing ovary.

13-Methyl-substituted des-C,D analogs of (20S)-1alpha,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): synthesis and biological evaluation.

Analogs of (20S)-1alpha,25-dihydroxy-2-methylene-19-norvitamin D(3) (2, 2MD), substituted at C-13 but lacking both C and D rings, were prepared in convergent syntheses, starting with the chiral ester 14 and the phosphine oxide 29. Two of the synthesized vitamins (11 and 32) were analogs in which the 13-methyl group constituted a substituent of an unsaturated fragment, that is, C(13)-C(17) double bond, whereas in the two other cases (12 and 13), the methyl group belonged to a ternary carbon stereogenic center. The aim of these studies was to further explore extensive modifications in the 'upper' part of the vitamin D skeleton in the hope of finding biologically active analogs of potential therapeutic value. The commercial (R)-(-)-methyl-3-hydroxy-2-methylpropionate (14) was converted in six steps to alcohol 18, the vitamin D side chain fragment. Its subsequent three-step transformation led to aldehyde 20 which was subjected to the Still-Gennari HWE reaction with anion derived from ester 21. The obtained alpha,beta-unsaturated esters 22 and 23 served as convenient starting compounds to the syntheses of the corresponding chiral acyclic aldehydes, beta,gamma-unsaturated (28) and saturated (39 and 40), required for the final Wittig-Horner coupling with the anion of the phosphine oxide 29. After hydroxyl deprotection, the synthesized vitamin D analogs 11-13 and 32 were purified and biologically tested. Only the (13R,20S)-analog 12 retained substantial, although 30 times lower than 1alpha,25-(OH)(2)D(3), binding ability to the full-length rat recombinant vitamin D receptor (VDR). This analog was also very effective in differentiation of HL-60 cells, and it exerted significant transcriptional activity (2 times and 15 times less potent, respectively, as compared to the native hormone). The in vivo tests showed that all synthesized vitamin D analogs were devoid of calcemic activity.

Removal of the 20-methyl group from 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD) selectively eliminates bone calcium mobilization activity.

The 18-nor (7), 21-nor (8) and 18,21-dinor (9) analogs of (20S)-1alpha,25-dihydroxy-2-methylene-19-norvitamin D(3) (6, 2MD) were prepared by convergent syntheses. The known phosphine oxide 10 was coupled by the Wittig-Horner process with the corresponding C,D-fragments (13-15), obtained by a multi-step procedure from commercial vitamin D(2). The goal of our studies was to examine the influence of removal of the methyl groups located at carbons 13 and 20 on the biological potency of 2MD in the hope of finding analogs with improved therapeutic profiles. Replacement of the 20-methyl with hydrogen in 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD) did not affect binding to the rat vitamin D receptor and had little effect on transcription activity and on HL-60 differentiation. However, the mobilization of calcium from bone was largely eliminated while intestinal calcium transport remained strong. Curiously, removal of both the C-13-methyl and 20-methyl restored slightly the bone calcium mobilizing activity. Thus, the 21-nor analog of 2MD may provide a potent analog with a greater margin of safety than 2MD.