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Biology
Agricultural And Food Sciences

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Evolution and Expression Characteristics of Receptor-Like Cytoplasmic Protein Kinases in Maize, Rice and Arabidopsis

Receptor-like cytoplasmic protein kinases (RLCKs) are involved in various activities in plant growth and development. We have totally identified 162, 160, and 402 RLCK genes in maize, rice, and Arabidopsis genomes, respectively. Phylogenetic analyses divided 724 RLCK genes into 15 subfamilies and similar structural patterns of kinase activity sites and functional sites were observed within the subfamilies. Furthermore, the structural patterns of intron/exon in the same subfamilies were similar, implicating their close evolutionary relationship. Chromosome distribution indicated that segmental duplication of RLCK genes might be a major mechanism contributing to the expansion of the RLCK superfamilies in maize, rice, and Arabidopsis, respectively. The analysis of the synteny relationship and gene structure indicated that the evolution of most RLCKs in maize were prior to rice and Arabidopsis. Most of the ratio of Ka/Ks is inferior to one, suggesting that RLCK genes have experienced the negative selection in maize, rice and Arabidopsis. Duplication time revealed that the maize was the earliest emergence among these three species. The expression profiles showed that there are some specifically expressed RLCK genes in maize root, leaf, ear, and tassel. These specific expression genes may participate in the developmental regulation of these maize tissues. Our results will be useful in providing new insights into evolution of RLCKs and revealing the regulatory network of maize, rice, and Arabidopsis development.

Introduction
Receptor-like protein kinases (RLKs), widely existing in plants, are kinds of enzymes which recognize the signals by extracellular domains, and transfer the signals to intracellular domains through the transmembrane domains [1]. According to domains, RLKs can be classified into receptor-like protein kinases (RLP), transmembrane receptor kinases (TMRK), and receptor-like cytoplasmic protein kinases (RLCKs). RLCKs lack transmembrane domains and extracellular domains, only containing intracellular domains. Previous studies revealed that there were approximately 25% and 17% of RLKs members belonging to RLCKs in Arabidopsis and rice, respectively [2][3][4]. RLKs can regulate plant development, growth and fertilization, self-incompatibility [5,6].
The first RLK gene in maize was cloned by Walker and Zhang [7]. After that, a large number of RLKs, including RLCKs, have been found successively in plants. Yang et al. [8] reported that A novel CRCK1 in Arabidopsis was up-regulated expression under the abiotic stress treatment, suggested that CRCK1 may be involved in various stress-resistance signal transduction pathways. The novel RLCKs NtPK1 and NtPK2 could regulate the signaling pathways of pollen germination and pollen tube growth [9]. The triggered stomata immunity by pathogen-associated molecular patterns can be mediated through BIK1 [10]. OsRLCK102 regulates development by Xa21-mediated immunity in BR signaling pathway [11]. GsRLCK acted as an important function in response to abscisic acid (ABA), drought and salt stresses [12]. Hence, RLCKs may play important roles in stress-resistance, diseases-resistance, and developmental regulation in plants.
Here intron numbers, the distribution of genes on chromosomes, and the evolutionary characteristics of RLCK genes, combining with the genes synteny relations among maize, rice, and Arabidopsis, were illustrated. The RLCK superfamilies are expanded due to segmental duplications among maize, Arabidopsis, and rice chromosomes. Meanwhile, the ratio of Ka/Ks and the duplication time among these three species showed that RLCK genes in monocotyledons have undergone a longer evolutionary time than dicotyledons. Meanwhile, RLCK genes expression profiles and sequence analysis highlighted their potential functional diversity.

Identification of RLCKs in Maize, Rice and Arabidopsis
The RLKs, including the RLCK subfamily, were searched in the databases of maize, rice, and Arabidopsis databases and the relevant articles. A total of 2449 sequences were retrieved and regarded as "potential" RLKs. The sub-cellular localization of all obtained RLKs was predicted. After removing the incomplete sequences manually, we totally retrieved 724 "candidate" RLCKs, including 162, 160, and 402 in maize, rice, and Arabidopsis, respectively (Tables S1 and S2).

Conservative Evolution of RLCKs in Maize, Rice and Arabidopsis
A total of 724 RLCK kinase domain sequences in maize, rice, and Arabidopsis were aligned and 15 groups were generated ( Figure 1a). Each group was named after the RLCK gene classification according to the "PlantsP Kinase Classification" database (http://plantsp.genomics.purdue.edu/). Most RLCK genes were classified into RLCK V~RLCK IX groups. RLCK OS only contained maize and rice RLCK genes, indicating the differentiation between monocotyledons and dicotyledons during the evolution. Based on conservative motifs of kinase domains, the genetic distance of some genes in RLCK VII was more closely with RLCK OS1 and RLCK OS4, indicating that kinase domains among RLCK VII, RLCK OS1, and RLCK OS4 were similar and conservative, but other domains may be different (Figure 1a). It was suggested that RLCK genes with a conservative structure may perform a similar function in plants. It is also implied that RLCK genes had a common ancestor during the evolution. The predicted kinase activity sites were shown in Figure 1b. The kinase domains between the adjacent groups were conservative and the kinase activity sites were contained in the conserved sequences. We found that most RLCK kinase activity sites were aspartic acid (D) (Figure 1b). The aspartic acid (D) was also discovered between the fourth and fifth amino acid in all the RLCK motifs ( Figure 1c). These results showed the RLCK functional sites may be the same amino acid between the different groups. In Figure 1c, the different groups were shown to be significantly conservative, such as RLCK II and RLCK XII, RLCK VII, and RLCK OS. RLCK VII and RLCK OS motifs were well conserved over the first 15 and the last six amino acid residues. Above all, the RLCK amino acid sequences were well conserved in the period of evolution in maize, rice, and Arabidopsis.
The conservation of gene structure, related with the number of introns, plays a crucial role in the occurrence of evolution [13]. Thus, we analyzed the intron numbers and the distribution of RLCK genes of maize, rice and Arabidopsis. In Figure 2, the third circle is the statistics of intron numbers ranging from 0 to 15. There were 50, 67, and 123 RLCK genes with more than five introns; there were 25, 19, and 46 RLCK genes with five introns; 86, 49, and 233 RLCK genes with less than five introns in maize, rice, and Arabidopsis, respectively.

The Evolution Selection of RLCKs in Maize, Rice and Arabidopsis
To understand the evolutionary characteristics, the synonymous mutations (Ks) and non-synonymous mutations (Ka) were calculated. It is generally believed that the value of Ks was not affected by natural selection, but the Ka was affected by natural selection. The Ka/Ks value can also explain positive selection (Ka/Ks > 1), neutral selection (Ka/Ks = 1) and negative selection (Ka/Ks < 1) during the evolution [14,15]. The ratio distribution of each pair was shown in Figure 3a-c. The percent of Ka/Ks < 1 were 86%, 67% and 70% in these three species, respectively. The negative selection of maize RLCK genes (Ka/Ks < 1) was higher than Arabidopsis and rice. These results illustrated that RLCK genes in these three species were undergoing the negative selection and the monocotyledons maybe performed more negative selection than dicotyledons during the evolution. The maize Ka/Ks frequency mainly distributed between 0.4 and 0.8, rice between 0 and 0.4, and Arabidopsis between 0.6 and 1 (Figure 3d-f). Thess results declared that the negative selection in maize was stronger than Arabidopsis but weaker than rice. The duplication time (T) of RLCK genes is from 7.1 to 153.7 Mya ago in maize, from 5.5 to 66.5 Mya in Arabidopsis, and from 0.15 to 151.3 Mya in rice, suggesting that the maize RLCK genes have been undergone a longer evolutionary time than Arabidopsis and rice. ranging from 0 to 15. There were 50, 67, and 123 RLCK genes with more than five introns; there were 25, 19, and 46 RLCK genes with five introns; 86, 49, and 233 RLCK genes with less than five introns in maize, rice, and Arabidopsis, respectively.

The Evolution Selection of RLCKs in Maize, Rice and Arabidopsis
To understand the evolutionary characteristics, the synonymous mutations (Ks) and nonsynonymous mutations (Ka) were calculated. It is generally believed that the value of Ks was not affected by natural selection, but the Ka was affected by natural selection. The Ka/Ks value can also explain positive selection (Ka/Ks > 1), neutral selection (Ka/Ks = 1) and negative selection (Ka/Ks < 1) during the evolution [14,15]. The ratio distribution of each pair was shown in Figure 3a-c. The percent of Ka/Ks < 1 were 86%, 67% and 70% in these three species, respectively. The negative selection of maize RLCK genes (Ka/Ks < 1) was higher than Arabidopsis and rice. These results illustrated that RLCK genes in these three species were undergoing the negative selection and the monocotyledons maybe performed more negative selection than dicotyledons during the evolution. The maize Ka/Ks frequency mainly distributed between 0.4 and 0.8, rice between 0 and 0.4, and Arabidopsis between 0.6 and 1 (Figure 3d-f). Thess results declared that the negative selection in maize was stronger than Arabidopsis but weaker than rice. The duplication time (T) of RLCK genes is from 7.1 to 153.7 Mya ago in maize, from 5.5 to 66.5 Mya in Arabidopsis, and from 0.15 to 151.3 Mya in rice, suggesting that the maize RLCK genes have been undergone a longer evolutionary time than Arabidopsis and rice.

Segmental Duplication of RLCKs in Maize, Rice and Arabidopsis
The gene duplication events were significantly occurred in the plants more than other

Segmental Duplication of RLCKs in Maize, Rice and Arabidopsis
The gene duplication events were significantly occurred in the plants more than other eukaryotes [13]. The gene duplication mainly includes whole genome duplication (WED), segmental duplication, tandem duplication, and transposon-mediated duplication [16,17]. There were nine, eight, and eleven segmental duplication events founded in maize, rice, and Arabidopsis, respectively ( Figure 4). These results suggested that the segmental duplication played an important role during the RLCK genes evolution. Gene cluster refers to the genes within a 2 MB region in a chromosome [18]. In Figure 4, there were 25 gene clusters in maize, less than 30 and 63 in Arabidopsis and rice, respectively.

The Distribution and Synteny of RLCK Genes in Maize, Rice and Arabidopsis
To understand the chromosomal distributions of RLCK genes, MapInspect software was employed to analyze the RLCK gene starting positions on chromosomes (Figure 4). Maize RLCK genes were unevenly distributed on 10 chromosomes (Figure 4a). On chromosome 3, 7, and 10, there were

The Distribution and Synteny of RLCK Genes in Maize, Rice and Arabidopsis
To understand the chromosomal distributions of RLCK genes, MapInspect software was employed to analyze the RLCK gene starting positions on chromosomes (Figure 4). Maize RLCK genes were unevenly distributed on 10 chromosomes (Figure 4a). On chromosome 3, 7, and 10, there were the minimum number of genes (10 genes) and the maximum number was on chromosome 1 which more evenly distributed 29 genes. The 162 maize RLCK genes have showed a high density in the terminal of chromosome 1, 2, and 9 and on the end of chromosome 4, 6, 8, and 9. In Figure 4b,c, the rice and Arabidopsis RLCK genes were more evenly distributed than maize.
The innermost map showed the synteny relations of RLCK genes among maize, rice, and Arabidopsis ( Figure 2). In total, 106 RLCK genes in maize showed synteny relationships with Arabidopsis and rice, indicating that most RLCK genes in maize were conservative during the evolution comparing with Arabidopsis and rice, and, further, these three species shared common ancestors. Figure 5 showed the maize RLCK genes expression profile based on 131 maize RLCK genes. Table S3 listed RLCK genes expression data from the Maize eFP Browser [19]. The other 31 maize RLCK genes, lacking expression data, were not shown in the expression profile. Leaf, root, ear, and tassel in different development stages displayed in the bottom of the hierarchial clustering heatmap. Based on expression data, maize RLCK genes were classified into four groups. There were 45 RLCK genes in group I and also in group II; 27 in group III, and 13 in group IV. RLCK genes in group I had the low expression levels in the leaf, ear, and tassel. The log2 values were from −3 to 2. The genes in group II had high expression levels in root with log2 values from 2 to 6. The genes in group III showed a widely low expression levels in leaf, root, ear, and tassel. The genes in group IV showed a low expression levels in ear, tassel, and root, but relatively high levels in leaf. The expression profile showed that RLCK genes were widely expressed in maize, but there were some RLCK genes were specific expression in one tissue. For example, GRMZM2G15290, GRMZM2G473411, GRMZM2G090732, and GRMZM2G139223 had specific expression in root and GRMZM2G406601 and GRMZM2G055154 had specific expression in leaf. GRMZM2G091338 only had a relatively high expression in stele. GRMZM2G169080 and GRMZM2G177445 were only expressed in the base zones of leaf. GRMZM2G365319 and GRMZM2G073359 were expressed in the leaf zones of Base and −1 from ligule and root. GRMZM2G114899, GRMZM2G378547, GRMZM2G070961, and GRMZM2G301647 were only expressed in ear and tassel. The mentioned above indicated that these genes may have a specific function in the development processes of maize leaf, root, ear, and tassel.

Discussion
Overall, here we thought out analysis of genomic phylogeny, gene structure, chromosomal location, expansion mechanism of the maize, rice, and Arabidopsis RLCK superfamily. Previous research showed that the segmental duplication and the tandem duplication led to the expansion of RLKs in Arabidopsis and rice [13]. Our study results indicated that the segmental duplication might lead to the evolutionary differences of maize RLCK genes from Arabidopsis and rice. Gene duplication may be the main reason for the functional diversity on the gene level [20]. The maize RLCK genes distribution on chromosomes were near the ends of telomeres, indicating that a number

Discussion
Overall, here we thought out analysis of genomic phylogeny, gene structure, chromosomal location, expansion mechanism of the maize, rice, and Arabidopsis RLCK superfamily. Previous research showed that the segmental duplication and the tandem duplication led to the expansion of RLKs in Arabidopsis and rice [13]. Our study results indicated that the segmental duplication might lead to the evolutionary differences of maize RLCK genes from Arabidopsis and rice. Gene duplication may be the main reason for the functional diversity on the gene level [20]. The maize RLCK genes distribution on chromosomes were near the ends of telomeres, indicating that a number of redundant or conservative sequences maybe existed in the positions which were far away from the centromere. The genes which were far away from the centromere may mutate more easier and thus produce the novel genes in the evolution [21]. The novel function may be generated by new genes to support survival and adapt for the changed environment.
The conservative kinase activity sites usually provide a stable environment for RLCKs to perform the function in cytoplasm. In our study, most of RLCK kinase activity sites were aspartic acid (D). The conservative kinase activity sites usually provide a stable environment for RLCKs to perform the functions in cytoplasm. In the phylogenetic tree, RLCK OS only contained the maize and rice RLCK genes, suggesting that RLCK OS may be a specific receptor like protein kinase of monocotyledon. It may provide strong evidence for the differentiation of monocotyledonous and dicotyledonous. The syntenic relationship between two species could provide an important basis for gene evolution [22]. Our research results further proved that the RLCKs among maize, rice, and Arabidopsis had a common ancestor, even orthologous in the evolutionary events [23]. The analysis of gene synteny and structure showed that some RLCK genes have appeared before the differentiation of monocotyledons and dicotyledons. Intron number might impact on the evolution in monocotyledons and dicotyledons, further influencing the diversity and complexity of RLCK genes structure during the long progress of evolution in maize [13].
It is important to understand Ka/Ks value for the evolution analysis. Generally, the genes which have undergone negative selection could add to the gene dosage [14]. The maize negative selection happened more strongly than other two species, suggesting that maize RLCK genes maybe have more chances to increase the gene dosage and generate the novel functions during evolution compared with Arabidopsis and rice. The earliest duplication time of RLCK genes in maize is 153.7 Mya which is prior to other two species, indicating that the maize had originated earlier than Arabidopsis and rice. Our results provide new insights into the evolution of RLCKs in maize, rice, and Arabidopsis, and will be useful in studies monocotyledonous and dicotyledonous development.

Identification and Protein Domain Prediction of RLCK Genes
In the present study, we chose RLKs from maize, rice, and Arabidopsis databases and relevant articles as base data sources. The related RLCK sequences were obtained from Phytozome (Available online: https://phytozome.jgi.doe.gov/pz/portal.html). The results were merged, and redundant sequences were deleted. For genes with more than one predicted isoforms, we analyzed only the longest peptide sequence of each gene. Online software PSORTb version 3.0.2 (Available online: http://www.psort.org/psortb/) was employed to predict sub-cellular localization using amino acid sequences of all obtained RLKs, and RLKs located in cytoplasm were selected to analyze kinase domains according to the SMART databases (http://smart.embl-heidelberg.de/smart/set_mode.cgi? NORMAL=1).

The Phylogram and Selection of RLCKs
All RLCK amino sequences in maize, rice, and Arabidopsis were aligned by ClustalX v1.83 [24] with default parameters. MEGA 5.0 [25] with NJ method was used to generate the phylogenetic tree. The RLCK gene intron information of maize, rice, and Arabidopsis were from the Ensembl Genomes (Available online: http://ensemblgenomes.org/).

Distribution and Synteny of RLCK Genes
The starting positions on chromosome and synteny data of RLCK genes among maize, rice, and Arabidopsis were from Ensembl (Available online: http://www.ensembl.org/index.html). The chromosomal maps of RLCK genes in maize, rice, and Arabidopsis were visualized using MapInspect (http://www.plantbreeding.wur.nl/uk/software_mapinspect.html).

Differential Expression of Maize RLCK Genes
For analyzing the gene expression of RLCK genes in maize, the gene expression data were retrieved from Maize eFP Browser (http://bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi). The RLCK gene expression values were normalized by log2 values. Then the Heml software (http://hemi. biocuckoo.org/down.php) was employed to generate the maize RLCK gene expression profiles in hierarchial clustering.


Stage-Specific Expression Profiles of Anti-Müllerian Hormone and its Type II Receptor in Germ Cells during Spermatogenic Cycle of Rats

Although anti-Müllerian hormone (AMH) is present in seminiferous tubules, its physiological roles in testis remain unclear. We defined expression patterns of AMH and its type II receptor (AMHR2) in germ cells during spermatogenic cycles. To this end, we performed in situ hybridization of Amh and Amhr2, and immunohistochemical staining using antibodies against AMH and AMHR2 in testes from rats aged 10, 15, 21, 35, and 49 days (d). In addition, quantitative RT-PCR analyses were performed to determine relative expression levels of Amh, Amhr2, synaptonemal complex protein 1 (Scp1) and round spermatid basic protein 1 (Rsbn1), and Smads 1, 5, and 8 in germ cells isolated from 49d testes. In 49d testes, synthesis of AMH and AMHR2 corresponded with spermatocyte expression of Amh and Amhr2. Spermatocytes of stages VII-XIV synthesized AMH, and AMH staining was decreased in round spermatids and was not detectable in elongated spermatids. Spermatocytes of stages VII-XIV synthesized AMHR2, and round and elongated spermatids continued expressing AMHR2. The strongest staining of AMH was observed in spermatocytes at stage VIII, and that of AMHR2 was observed in round spermatids at the same stage. When isolated germ cells were cultured with anti-AMH antibody, expression levels of Amh and Amhr2 were significantly increased, those of Smads 1, 5 and Scp1 increased simultaneously, and that of Rsbn1 decreased. We speculate that the AMH-SMADs axis is associated with morphological change from round to elongated spermatids at stage VIII.

Introduction
Anti-Müllerian hormone (AMH) is also known as Müllerian inhibiting substance (MIS), and is a member of the TGF-β superfamily [1]. AMH plays an essential role in the regression of Müllerian ducts and descent of the testes during sexual differentiation of male fetuses [2,3]. Fetal Sertoli cells secrete AMH until birth, even after Müllerian ducts regression [4,5]. Moreover, male transgenic mice overexpressing Amh manifest feminized genitalia [6], in accordance with decreased Leydig cell numbers and testosterone secretion [7]. Conversely, Amh knockout mice manifested Leydig cell hyperplasia and infertility [8,9]. It is therefore likely that AMH plays an important role in fetal Leydig cell function [10,11].
Postnatally, expression of Amh and Amhr2 has been observed in rat testes and ovaries [12,13]. During postnatal maturation of the testis, Sertoli cells change morphologically and functionally from immature type to mature type cells, although slightly immature Sertoli cells persist even in mature testes [14].
However, there were few reports that examine AMH-producing cells of postnatal mature testes in detail. Recently, we were the first to show expression of AMH and AMH type II receptor (AMHR2) in spermatocytes from rats aged 21 days (21d) using immunohistochemical (IHC) staining and in situ hybridization (ISH).
During the development of spermatogenesis in maturing rat testis, germ cells of neonatal testes are only spermatogonia, and spermatocytes appear during weaning prior to formation of round and elongated spermatids [16]. Spermatogonia and spermatocytes are present in 21d rat testis, whereas round and elongated spermatids are not. Moreover, in postnatal maturing rat testis, Sertoli cells and spermatocytes express and synthesize AMH and AMHR2 [15]. AMH signals are initiated by binding to an AMHR2, and can activate other signaling pathways such as those of β-catenin or NFkB [17]. In previous reports of AMH-AMHR2 signal transduction, AMH was shown to bind AMHR2 and activate the SMAD 1, 5, and 8 signaling pathway through one or several type 1 receptors [18]. SMAD proteins are intracellular mediators of TGF signaling and in postnatal mouse testes; SMAD1 was detected in spermatocytes and round spermatids [19], whereas SMAD5 was detected mainly in spermatogonia [20]. AMH activates the SMAD1, 5, and 8 signaling pathway through AMHR2 and inhibits SMAD6 [20]. We also reported that 21d testes and isolated germ cells expressed Smad1, 5, and 8 [15].
These data indicated that AMH is involved in spermatogenesis. However, although AMH is present in seminiferous tubules, its physiological roles in mature testis are not entirely clear. Thus, in this Journal of Steroids & Hormonal Science study we characterized expression patterns of AMH and AMHR2 in germ cells from mature rat testis during the spermatogenic cycle.

Animals
All experiments were approved by the Animal Care and Use Committee of the University of Yamanashi, Japan.
Normal Sprague-Dawley (SD) rats aged 5, 10, 16, 30, and 44 days (d) were purchased from Japan SLC and were housed for 5 days in an air-conditioned animal room at an ambient temperature of 22°C and a relative humidity of 55% ± 10% under a 12 h light/12 h dark cycle. Male rats were bred with maternal rats until postnatal day 20 (20d). The day of birth was designated as 0d and testes were collected from 10, 15, 21, 35 and 49d rats for use in assays.

In situ hybridization (ISH)
To determine the localization of Amh and Amhr2 in 49d rat testis, ISH was performed using antisense probes comprising 55-mer oligonucleotides, which were labeled at the 5′-end using digoxigenin (DIG). Two non-overlapping probes were designed based on the Amh and Amhr2 sequences and were commercially synthesized (BEX, Tokyo, Japan). Amh and Amhr2 antisense probe sequences were previously reported [15]. Paraffin-embedded rat testis sections of 8 μm in thickness, were prepared and mounted on glass slides, which were pre-coated with 3-aminopropyltriethoxysilane (Shin-Etsu Chemical Co., Tokyo, Japan). All hybridization steps, including washing and visualization, were performed using a commercial kit (IsHyb In situ Hybridization Kit; Biochain Institute, Newark, CA) according to the manufacturer's instructions. Probe signals were detected with an alkaline phosphatase-conjugated anti-DIG antibody and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) solution. ISH was performed by mixing two individual mRNA probes (AS1 and AS2), and specificity of hybridization was confirmed using sense oligonucleotide probes located at the same position as the antisense probes, which were used as a negative controls.

AMH and AMHR2 antibodies
AMH and AMHR2 polyclonal antibodies [15] were raised by Operon Biotechnologies K.K. custom synthesis service (Tokyo, Japan). Briefly, the AMH peptides, C+KFQEPPPGGASRWE (143-156) and CAWPQSDRNPRYGNH (485-499) were synthesized, and mixed, and rabbits were immunized four times with the mixture and with the carrier protein KLH every week. In identical procedures, the antigen peptides, CDPVPRAHPSPSSTL (92-106) and CSATDPRGLRELLED (470-484) were used to raise the AMHR2 antibody in rabbits. Polyclonal antisera were purified using affinity column chromatography with synthesized antigens.

Immunohistochemical (IHC) staining
Testes from 10d-49d rats were fixed in buffered neutral formalin, were embedded in paraffin, and were sectioned (5-μm, thickness) for IHC staining, following the procedure prescribed by Morpho Technology (Sapporo, Japan). IHC procedures were performed as previously reported [15]. Briefly, fixed tissues were deparaffinized, rehydrated, and washed with tris-buffered solution (TBS). Antigen retrieval was then performed using a high temperature antigen unmasking technique, in which sections were incubated with EDTA buffer solution (pH 9.0) at 95°C for 20 min. Sections were then rinsed with TBS, and endogenous peroxidase activity was inactivated with 0.1% H 2 O 2 at room temperature for 10 min. Tissue sections were then immunostained using an automated staining system (Autostainer Plus, Dako). Sections were incubated with anti-AMH antibody (1:100 dilution) or anti-AMHR2 antibody (1:100 dilution) at room temperature for 60 min and were then washed with TBS. Sections were then incubated with Histofine® Simple Stain MAX-PO(R) (Nichirei, Tokyo) at room temperature for 30 min, and were visualized with DAB solution at room temperature for 5 min. Sections were counterstained with hematoxylin for 1.5 min and negative controls were processed simultaneously by omitting the primary antibodies.

Germ-cell isolation
Germ cells were isolated from 49d testes to examine the effects of the AMH antibody on AMH expression. Testicular cells were isolated as described previously by Galardo et al. [21] with minor modifications [15]. Germ cells were isolated from 4 testes of 49d rats in each experiment. Testes were decapsulated and treated with 0.1% collagenase type 2 (Worthington Biochemical Corp., Lakewood, NJ) and 10 U/ml DNase I (D7291, Sigma-Aldrich, St Louis, MO) in 20 ml of Hanks' balanced salt solution (HBSS) for 10 min at 35°C in a Bio Shaker (BR-30L, Taitec, Tokyo) at 100 oscillations per min. Collagenase solution was diluted with 20 ml of HBSS and seminiferous tubules were allowed to sediment for 2 min. Supernatants were then discarded and the tubular pellets were digested with 20 ml of 0.1% trypsin (Gibco) in HBSS for 20 min at 35°C in a Bio Shaker at 100 oscillations per min. Trypsin-digested solutions were supplemented with 10 ml of 10% fetal calf serum (FCS) in HBSS and were filtered with a Steriflip 100 μm nylon net filter (Millipore) and then centrifuged at 400 × g for 5 min. Cell pellets were re-suspended in a 1:1 mixture of DMEM-Ham's F-12 medium supplemented with 10% FCS, 15-mM NaHCO 3 , 100 IU/ml penicillin, 2.5 mg/ml amphotericin B, and 20-mM HEPES (pH 7.4; DMEM-F12). Isolated cells from seminiferous tubules were seeded in 10 culture plates (10 cm 2 /plate) and were cultured at 35°C in a 5% CO 2 incubator for 6 h. During this period, Sertoli cells were attached to the surface of the plate. Attached cells (Sertoli cells) were washed twice with HBSS, and cultured again with DMEM-F12 for 12 h. These cells were then washed twice with HBSS, scraped, centrifuged, and frozen for total RNA isolation. Floating germ cells were collected by carefully removing the medium and centrifuging at 400 × g for 5 min. Germ cell pellets were resuspended in HBSS and were loaded on a discontinuous four-layer (20%, 25%, 32%, and 37%) Percoll density gradient (Percoll PLUS, GE Healthcare Bio-Sciences) for gradient centrifugation at 800 × g for 30 min at 4°C. The fraction between the 25%-32% Percoll interface was collected. To remove Percoll, 4 volumes of HBSS were added to the recovery solution, and were centrifuged at 400 × g for 5 min at 4°C. Germ cell pellets were re-suspended in DMEM-F12, and were then cultured in 10 cm 2 plates at 35°C in a CO 2 incubator for 12 h. Few Sertoli cells remained in the germ cell population, and these were attached to the dish surface. Floating germ cells were obtained by carefully removing the medium and were precipitated from the medium by centrifugation at 400 × g for 5 min. Germ cell pellets were re-suspended in 60 ml of DMEM-F12 (without FCS), and cell suspensions were then divided into 6 plates (10 ml/plate). Subsequently, 50 μl of anti-AMH antibody was added to 3 of 6 plates (final dilution 200 X). Re-suspended germ cells in these 6 plates were cultured at 35°C in a CO 2 incubator for 2 h, and were then centrifuged. Germ cell pellets collected from 6 plates were frozen for total RNA isolation and subsequent quantitative real-time polymerase chain reaction (qRT-PCR). Experiments were performed in triplicate.

Quantitative real-time polymerase chain reaction
Total RNA was extracted from germ cells and Sertoli cells that were isolated from 49d rat testes, using RNeasy Mini Kit (QIAGEN, Tokyo). Subsequently, cDNA was synthesized from 1 μg of total RNA template using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). For qRT-PCR, triplicated samples containing 9 μl of cDNA with 10 μl of Taqman Universal PCR Master Mix (Applied Biosystems) and 1 μl of 20 X Assays-on-demand Gene Expression Product (Applied Biosystems) were pre-incubated at 50°C for 2 min and then at 95°C for 10 min. Amplification was performed by 40 cycles of reaction at 95°C for 15 s and 60°C for 1 min. Fluorescence intensity was quantitatively analyzed using an ABI Prism 7500 Sequence Detection System (Applied Biosystems). Genes that were analyzed using PCR included Amh, Amhr2, Scp1, Rsbn1, Ngfr, Rhox5, and Smads 1, 5, and 8 (Table 1). Testicular expression of individual genes in whole testis from 49 d rats was set at 1.0. Relative expression levels of genes in isolated germ cells were normalized to that of β-actin.

Gene
Assay ID

Statistical analysis
Relative expression levels of Amh, Amhr2, Scp1, Rsbn1, and Smads 1, 5, and 8 in isolated germ cells were log-transformed, and the effects of anti-AMH antibody were analyzed separately using linear regression models that were adjusted for the order of experiments. P-values of less than 0.05 were considered indicative of statistical significance.  (Figures 1b and   1e). Signals of Amh and Amhr2 in round and elongated spermatids were negative at all stages, but were unclear in spermatogonia, and were faint in Sertoli cells.  Figure 2 demonstrates age-related changes in AMH and AMHR2 IHC staining. Only spermatogonia and Sertoli cells were present in the seminiferous tubules of 10d testis, whereas few spermatocytes appeared in those of 15d testes. In 10d testes, AMH and AMHR2 stainings were strong in Sertoli cells, but were not present in spermatogonia (Figures 2a and 2e). When spermatocytes appeared in the seminiferous tubules of 15d testes, AMH and AMHR2 staining's observed in Sertoli cells and were weakly present in spermatocytes, but were absent in spermatogonia (Figures 2b and 2f). In 21d testis, many spermatogonia had differentiated into spermatocytes, but no spermatids were observed. Strong AMH and AMHR2 staining's observed in spermatocytes, but were absent in Sertoli cells and spermatogonia (Figures 2c and 2g). Spermatocytes had differentiated into spermatids in 35d seminiferous tubules, and AMH staining was strong in spermatocytes, weak in Sertoli cells, and not present in elongated spermatids and spermatogonia (Figure 2d). In contrast, AMHR2 staining was strong in spermatocytes and elongated spermatids, but was unclear in Sertoli cells (Figure 2h). Figures 3 and 4 shows the spermatogenic stage-specific IHC staining of AMH and AMHR2 in 49d seminiferous tubules. In spermatocytes, AMH staining was negative or weak at stage I-IV (Figure 3a), but was strong at stages VII-XIV. The strongest AMH staining was observed at stage VIII (Figure 3c and Figure 4a). In contrast, AMH staining was clearly observed in round spermatids at stages I-IV (Figure 3a), but was decreased at stage VII, and was diminished in elongated spermatids at stages VIII-XIV (Figures 3c, 3d  and 3e. Figure 4a). AMHR2 staining was negative or weak in spermatocytes at stages I-IV (Figure 3f), but was strong at stages VII-XIV. Unlike the staining of AMH in spermatids, AMHR2 staining was present in round and elongated spermatids at all stages, and was especially strong in cell membranes of round spermatids at stage VII-VIII (Figures 4b and 4c). Moreover, AMHR2 staining was observed in residual bodies (Figure 4c). AMH and AMHR2 staining's were absent in spermatogonia at all stages, and were clear in Sertoli cells at stages I-IV.    As shown in Figure 6, germ cells from 49d testes expressed Amh, Amhr2, Scp1 and Rsbn1, and Smads 1, 5, and 8. After incubation of isolated germ cells with anti-AMH antibody, expression levels of Amh, Amhr2, Scp1, Smad1 and Smad5 were significantly increased, whereas that of Rsbn1 was significantly decreased.

Discussion
Josso et al. were the first to report evidence that Sertoli cells in fetal testes produce AMH [22]. Based on studies of fetal testes, it was thought that AMH was produced only by Sertoli cells in males. During postnatal spermatogenesis, Sertoli cells and spermatogonia are present in 10d rat testes, and whereas spermatocytes appear after 15d testis, round spermatids do not appear in testes until 21d. Recently, we showed that spermatocytes of rat testes synthesize AMH and AMHR2 at 21d [15]. Similarly, several recently reports show AMH expression in oocytes and primordial follicles [23,24], although other studies show that AMH is absent in oocytes of all follicular categories [25,26]. Hence, although the present experiments indicate that germ cells of matured gonads express AMH, the physiological role of AMH in gonads remains unclear.
In this study, we first clarified that spermatocytes expressed and synthesized AMH and AMHR2 during spermatogenic cycle in 49 d rat testes. The stage-specific expression profiles of AMH and AMHR2 in spermatocytes, spermatids and Sertoli cells are showed in Table 2.
Spermatocytes expressed Amh and Amhr2, and synthesized AMH and AMHR2. IHC staining of AMH in spermatocytes was negative or weak during stage I-IV of the spermatogenic cycle, but became strong during stages VII-XIV. Moreover, the intensity of AMH staining in spermatocytes during the spermatogenic cycle corresponded with that of Amh expression in spermatocytes. In contrast, Amh expression was not detected in spermatids, despite positive staining of AMH protein, suggesting that during differentiation of spermatocytes to round spermatids, AMH is transferred from spermtocytes to round spermatids. Accordingly, staining of the transferred AMH in round spermatids decreased with the progression of the spermatogenic cycle, was diminished at stage VIII, and was not observed in elongated spermatids. AMH staining was also weak in Sertoli cells at stage I to IV. Taken together, these data indicate that AMH is predominantly produced by spermatocytes rather than Sertoli cells in matured testes.  In this study, we first clarified that spermatocytes expressed and synthesized AMH and AMHR2 during spermatogenic cycle in 49 d rat testes. The stage-specific expression profiles of AMH and AMHR2 in spermatocytes, spermatids and Sertoli cells are showed in Table 2.
Spermatocytes expressed Amh and Amhr2, and synthesized AMH and AMHR2. IHC staining of AMH in spermatocytes was negative or weak during stage I-IV of the spermatogenic cycle, but became strong during stages VII-XIV. Moreover, the intensity of AMH staining in spermatocytes during the spermatogenic cycle corresponded with that of Amh expression in spermatocytes. In contrast, Amh expression was not detected in spermatids, despite positive staining of AMH protein, suggesting that during differentiation of spermatocytes to round spermatids, AMH is transferred from spermtocytes to round spermatids. Accordingly, staining of the transferred AMH in round spermatids decreased with the progression of the spermatogenic cycle, was diminished at stage VIII, and was not observed in elongated spermatids. AMH staining was also weak in Sertoli cells at stage I to IV. Taken together, these data indicate that AMH is predominantly produced by spermatocytes rather than Sertoli cells in matured testes.
Moreover, expression patterns of Amhr2 and AMHR2 in spermatocytes closely resembled those of Amh and AMH, with high expression during stages VII-XIV. Amhr2 expression was not detected in spermtids, despite positive staining of AMHR2, suggesting that AMHR2 was transferred from spermatocytes to round spermatids. However, unlike the expression pattern of AMH, AMHR2 was observed in round spermatids and elongated spermatids. AMHR2 staining was clearly observed at cell membranes of round spermatids, especially during stages VII-VIII. Moreover, AMHR2 was present in residual bodies at stage VIII. Because spermatocytes synthesize both AMH and AMHR2, these data indicate that 1) AMH acts on spermatocytes in an autocrine fashion during stages VII-XIV, 2) AMH is mainly produced by spermatocytes and acts on round and elongated spermatids through AMHR2 in a paracrine fashion from stages VIII to XIV, especially at stage VIII, and 3) AMH from Sertoli cells acts on elongated spermatids during the early stages I-IV. These observations suggest that AMH contributes to morphological changes from round to elongated spermatid forms at stages VIII-IX, and to morphological changes in elongated spermatids.
In our experiments with isolated testicular cells, expression of Sertoli cell specific genes (Ngfr and Rhox5) was not observed in isolated germ cells. The result indicates that cultured germ cells did not have the contamination of Sertoli cells. In cultured germ cells isolated from 49d testes, expression levels of Amh, Amhr2, Smad1, and Smad5 were significantly increased by the addition of AMH antibody. In a previous study, AMH through AMHR2 activated the SMAD1/5/8 signaling pathway and inhibited SMAD6 [20]. SMAD1 was also detected in spermatocytes and round spermatids [19], and SMAD5 was detected mainly in spermatogonia [20]. In the present study, although spermatogonia did not express Amhr2 and AMHR2, expression levels of Smads1 and 5 were increased in isolated germ cells, indicating that SMADs1 and 5 are present in spermatocytes and spermatids. Moreover, treatment with anti-AMH antibody led to increased Scp1 expression and decreased Rsbn expression in isolated germ cells. Scp1 is pachytene spermatocyte specific gene and Rsbn1 is round spermatids specific gene. These results may suggest that AMH mainly secreted by spermatocytes controls differentiation from spermatocytes to round spermatids and morphological changes from round spermatids to elongated form.
In AMHR2 expressing ovarian granulose cell tumor cells, exogenous AMH activated SMADs 1 and 5, and augmented caspase-3 activity, a marker of apoptosis induction [27]. Caspase-3 is present in spermatocytes and residual bodies and may be associated with germ cell apoptosis [28]. During terminal morphological spermatogenic changes, spermatid cytoplasm's are eliminated and finally collected in residual bodies that display several apoptotic features. Male mice with a targeted deletion of the septin4 gene (Sept4) are sterile due to abnormal spermatozoa with attached residual cytoplasm. In accordance, caspase inhibitor XIAP was induced following mutation of Sept4 [29]. These reports suggest that AMH activates caspase-3 through AMHR2-SMAD signaling in germ cells, and that caspase-3 contributes to cytoplasmic apoptosis in elongated spermatids.
In conclusion, the present data show that the spermatocytes stagespecifically express and synthesize AMH and AMHR2 during the spermatogenic cycle, and that AMH and AMHR2 are transferred to spermatids. AMH was expressed in spermatocytes at stages VII-XIV, and AMHR2 was most strongly expressed in round spermatids at stage VIII, and then in elongated spermatids and residual bodies. Finally, Smad 1 and 5 expressions were increased in response to AMH in cultured isolated germ cells, indicating that the AMH-SMAD pathway may be associated with morphological changes from round to elongated spermatids at stage VIII. However, the inferred cytoplasmic apoptosis of spermatids remains to be fully established. Future studies are required to define further details of the physiological role of AMH during spermatogenic cycles.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Corticotropin releasing hormone can selectively stimulate glucose uptake in corticotropinoma via glucose transporter 1

BACKGROUND
Pre-operative detection of corticotropin (ACTH) secreting microadenomas causing Cushing's disease (CD) improves surgical outcomes. Current best magnetic resonance imaging fails to detect up to 40% of these microadenomas. 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) is specific, but not sensitive in detecting corticotropinomas. Theoretically, secretagogue stimulation with corticotropin releasing hormone (CRH) could improve detection of adenomas with 18F-FDG PET. Previous attempts with simultaneous CRH stimulation have failed to demonstrate increased 18F-FDG uptake in corticotropinomas. We hypothesized that CRH stimulation leads to a delayed elevation in glucose uptake in corticotropinomas.

METHODS
Clinical data was analyzed for efficacy of CRH in improving 18FDG-PET detection of corticotropinomas in CD. Glucose transporter 1 (GLUT1) immunoreactivity was performed on surgical specimens. Ex-vivo, viable cells from these tumors were tested for secretagogue effects (colorimetric glucose uptake), and for fate of intracellular glucose (glycolysis stress analysis). Validation of ex-vivo findings was performed with AtT-20 cells.

RESULTS
CRH increased glucose uptake in human-derived corticotroph tumor cells and AtT-20, but not in normal murine or human corticotrophs (p < 0.0001). Continuous and intermittent (1 h) CRH exposure increased glucose uptake in AtT-20 with maximal effect at 4 h (p = 0.001). Similarly, CRH and 8-Br-cAMP led to robust GLUT1 upregulation and increased membrane translocation at 2 h, while fasentin suppressed baseline (p < 0.0001) and CRH-mediated glucose uptake. Expectedly, intra-operatively collected corticotropinomas demonstrated GLUT1 overexpression. Lastly, human derived corticotroph tumor cells demonstrated increased glycolysis and low glucose oxidation.

CONCLUSION
Increased and delayed CRH-mediated glucose uptake differentially occurs in adenomatous corticotrophs. Delayed secretagogue-stimulated 18F-FDG PET could improve microadenoma detection.

Corticotropin releasing hormone (CRH) has secretagogue effects on adrenocorticotropic hormone or corticotropin (ACTH) secreting pituitary adenomas (Takano et al., 1996), but not on suppressed adjacent normal gland. This effect could theoretically improve the imaging detection of adenomas with 18 F-FDG PET imaging following secretagogue stimulation. However, our group (unpublished data) and others (Patt et al., 2014), have failed to demonstrate increased 18 F-FDG uptake in corticotropinomas by simultaneous CRH administration. To date, it remains unclear whether corticotropinomas are resistant to CRH-mediated glucose uptake, or if the effects are delayed.
We hypothesized that CRH stimulation leads to a delayed elevation in glucose uptake in corticotropinomas. In this study, we investigated the kinetics of CRH-modulated glucose uptake. A timecourse in-vitro study revealed that maximum glucose uptake occurs approximately 4 h post CRH administration. Moreover, we demonstrate for the first time that CRH stimulation results in a differential glucose uptake in adenomatous, but not in normal corticotrophs. Mechanistically, this was associated with a robust increase in glucose transporter 1 (GLUT1) expression. Taken together, these novel findings support the potential use of delayed 18 F-FDG PET imaging following CRH stimulation to improve microadenoma detection in CD.

Tissue sample collection
A total of 10 patients with CD were enrolled in a clinical trial (NIH 12-N-0067, NCT01459237) conducted at the National Institute of Neurological Diseases and Stroke (NINDS) to evaluate the utility of CRH-stimulated 18 F-FDG PET. Study was approved by the Combined Neuroscience Institutional Review Board (IRB). Pituitary adenoma tissues were obtained from these patients at the time of transsphenoidal adenomectomy at the National Institutes of Health Clinical Center (NIHCC) under a clinical trial (NIH 03-N-0164, NCT00060541) for subsequent immunohistochemical and metabolic analyses. Written informed consent was obtained from each patient for research study participation. The study was conducted in accordance to the standards and guidelines established by the IRB.

CRH-stimulated 18 F-FDG PET study
PET imaging was performed using a high-resolution research tomography scanner (Siemens AG). Each subject underwent two randomly ordered 18 F-FDG high resolution PET (hrPET) studies with and without CRH stimulation separated by at least 24 h. For CRH stimulated studies, ovine CRH (oCRH or Acthrel ® ) 1 mcg/kg (up to a maximum dose of 100 mcg) was administered immediately preceding 18 F-FDG administration (10 mCi). Pituitary metabolic activity was quantified using the standardized uptake values (SUV). Maximum SUV (SUV-Max) and averaged SUV (SUV-Avg) were calculated as previously described (Chittiboina et al., 2014).

Cell culture
AtT-20/D16:16 (a generous gift from Dr. Steven L. Sabol at the National Heart Lung and Blood Institute) murine corticotroph tumor cell lines were cultured in DMEM (Thermo Fisher Scientific, USA), 10% FCS (Thermo Fisher Scientific) following comparison with commercially available AtT-20 cells ( Supplementary Fig. 1). Cells were incubated for 2 h prior to all experiments in serum-free DMEM supplemented with 0.25% BSA (hereafter DMEM-BSA) and 90 mg/dL glucose, to simulate physiologic glucose levels. Pooled corticotroph cells were harvested (<30 min post-euthanasia) from female, BALB/c mice (aged 6e8 weeks; Taconic Biosciences, USA), digested and homogenized in 1 mg/mL collagenase (Sigma-Aldrich, USA) for 30 min, and cultured as above. All animals were euthanized in accordance with the standards and guidelines of the Institutional Animal Care and Use Committee of the National Institutes of Health (NIH). Western blot analysis was performed with anti glucocortidoid receptor antibody (Santa Cruz Biotechnology), anti Serine 211 phophorylated glucocorticoid receptor antibody (antibodies-online.com), and anti CRH receptor 1 antibody (Thermo Fisher Scientific).

Fluorescence activating sorting of murine corticotrophs
After dissociation of murine hypophyseal cells, corticotrophs were isolated by flow cytometry. Briefly, pooled cells were incubated with anti-CRH receptor 1 antibody (CRH-R1) (Thermo Fisher Scientific) for 1 h, followed by Alexa Fluor-555 conjugated antibody (Invitrogen, USA) and finally stained with DAPI (Thermo Fisher Scientific) for 30 min. Cell sorting analysis was carried out using MoFlo Astrios Cell Sorter and Summit Acquisition and Analysis software (Beckman Coulter, USA).

ACTH release studies
Following several washes in PBS, cells were incubated in serumfree DMEM-BSA and stimulated with 50 nM CRH (Sigma-Aldrich, USA), 100 nM arginine vasopressin (Sigma-Aldrich), 5 mM 8bromoadenosine 3 0 ,5 0 -cyclic monophosphate (8-Br-cAMP; Sigma-Aldrich), and/or 10 nM dexamethasone (DEX; Sigma-Aldrich) for the time points indicated. Media was then diluted and assessed via enzyme linked immunosorbant assay (MD Bioproducts, USA) according to manufacturer instructions. Glucose uptake experiments were conducted with Screen Quest Colorimetric Glucose Uptake Assay Kit (AAT Bioquest, USA) per manufacturer instructions. In brief, cells were cultured in DMEM-BSA on 96-well plates, washed with Krebs-Ringer-Phosphate-HEPES (KRPH) buffer and incubated in 90 ml/well of Glucose Uptake Buffer (AAT Bioquest) for 1 h. Subsequently, cells were exposed to 10 ml/well of Deoxy-D-glucose (2DOG) for 40min; cells in the CRH stimulation arm were exposed to 50 nM of CRH for 1 h. Following treatment, cells were washed with KRPH buffer and lysed. Lastly, 50 ml of the Uptake Assay Mixure (AAT Bioquest) was added to each sample and absorbance ratio was read with 570 nm wavelength.

RNA extraction and quantitative real-time PCR
Total RNA was extracted from cells using RNeasy Mini Kit (Qiagen, Germany) and reverse transcribed to complementary DNA (cDNA) with Superscript III qRT-PCR Supermix (Life Technologies, USA). Quantitative RT-PCR was performed using target-specific primers (Sigma-Aldrich; Supplementary Table 1) and SYBR Select Master Mix (Life Technologies) on an Illumina Eco Real-Time PCR System (Illumina, USA). Each reaction was performed in triplicate (technical and biological), and relative gene expression values were calculated using the DDCt method with ACTB as an internal control.

Immunofluorescence and immunohistochemistry
After CRH stimulation for various time periods, cells were fixed using 4% paraformaldehyde (Thermo Fisher Scientific) and blocked using 2% donkey serum in 2% PBSA. Tris-buffered saline with Tween was used as wash buffer. Cells were then incubated with primary antibody against GLUT1 (Abcam, UK) for 1 h and secondary donkey anti-rabbit FITC antibody (Jackson ImmunoResearch, USA) along with DAPI (Thermo Fisher Scientific) was applied to the cells for 30 min. Rabbit IgG was used as negative control. Plasma membrane translocation of GLUT1 was calculated as previously described (Cai et al., 2013). Briefly, ImageJ software (ImageJ 1.50i, NIH, Bethesda) was used to measure the relative fluorescence intensity of GLUT1 in the plasma membrane adjacent region and the cytosolic region. The ratio of these measurements was calculated in at least 8 cells per condition tested. Formalin fixed human derived surgical adenoma specimens were stained with GLUT1 antibody (Abcam, UK) using Leica BondMax (Leica Biosystems, Buffalo Grove, IL) and reviewed by blinded neuropathologist (ARC).

Statistical analysis
Statistical analyses were determined by one-factor ANOVA using GraphPad Prism version 6.0 software program (GraphPad, USA) for in-vitro and in-vivo results. Grouped analyses were evaluated using two-way ANOVA. Alpha level threshold of 0.05 was used to assess statistical significance.
Murine pituitary cells also expressed GR, phosphorylated GR similar to AtT20 cells. However, CRHR1 expression was lower in unsorted normal murine pituitary cells, with non-significant change upon dexamethasone exposure ( Supplementary Fig. 3). As expected, levels of measured ACTH were substantially lower in isolated normal murine corticotrophs (following fluorescence activating sorting), when compared to AtT-20 cells (Fig. 1A). When stimulated with CRH, a significant increase in ACTH secretion was observed in AtT-20 cells after 4 h (mean difference 84.7%, [95% CI 64.5e104.9], ANOVA p < 0.0001) (Fig. 1A). AtT-20 cells exposed to CRH displayed a significant increase in 2DOG uptake, while the same effect was not observed in normal murine corticotrophs (Fig. 1B). We then tested the effect of continuous CRH stimulation (at 1 and 4 h) on human-derived pituitary corticotroph tumor cells. At 1 and 4 h following initiation of CRH stimulation, a consistent increase in 2DOG uptake (mean difference 216 ± 38% and 222 ± 59%, respectively) was observed (Fig. 1C). We then confirmed the selectivity of secretagogue effect on glucose uptake in normal pituitary and corticotropinoma tissues ex-vivo from one patient. Expectedly, CRH stimulation led to a significant increase in ACTH secretion from human derived corticotroph tumor cells but not normal corticotrophs cells in primary culture (mean difference 225.6% [95% CI 21.1e430.2], ANOVA p ¼ 0.04) (Fig. 1D). Furthermore, 2DOG uptake was differentially increased in human derived corticotroph tumor cells but not in normal corticotrophs when exposed to CRH (mean difference 76.3%, [95% CI 50.5e102.2], ANOVA p < 0.0001) (Fig. 1E). These results suggest that adenomatous cells but not normal corticotrophs have increased glucose uptake that may be modulated with secretagogues stimulation.
Intracellular effects of CRH stimulation are largely mediated by the ubiquitous second messenger cyclic adenosine monophosphate (cAMP) (Gigu ere et al., 1982), therefore, we hypothesized that glucose uptake would increase in response to cAMP. Continuous exposure to 8-Br-cAMP revealed a slightly different pattern of delayed increase in 2DOG uptake as significant glucose uptake was seen at the 1, 2, 4 and 8-h mark ( (Fig. 2D). Taken together, these results suggest that secretagogues stimulation may lead to a delayed (2e4 h) increase in glucose uptake in adenomatous corticotrophs. Fig. 1. CRH stimulation results in selective augmented glucose uptake in adenomatous but not normal corticotrophs. Normal murine corticotrophs cells were separated from harvested pituitary glands using CRH-R1 antibody and flow cytometry. As expected, murine derived AtT-20/D16:16 cells demonstrated a robust CRH-stimulated ACTH secretion response not seen in normal murine corticotrophs (A). At 4 h, a significant increase in normalized 2DOG uptake was noted in AtT-20 cells exposed to CRH but not in normal murine corticotrophs (B). At 1 and 4 h, CRH stimulation resulted in a consistent increase in 2DOG uptake in human derived corticotroph tumor cells (C). Horizontal line at 100 represents 2DOG uptake by adenomatous cells at baseline. The individual data points represent 2DOG uptake normalized to adenoma without stimulation (y axis ¼ 100) at 1 and 4 h. When human derived corticotroph tumor cells were exposed to CRH for 4 h, a substantial increase in both ACTH release and glucose uptake was seen (D and E). Normal corticotrophs did not respond to CRH stimulation for both ACTH secretion or 2DOG uptake demonstrating the selective CRH-mediated effect on adenomatous cells only. *p 0.05, ****p 0.0001 compared with corresponding control values. Horizontal bars represent mean ± standard deviation. Abbreviations: ACTH -adrenocorticotropic hormone, CRH -corticotropinreleasing hormone, hCt e normal corticotrophs, hCtT e human derived corticotroph cells, mCt e normal murine corticotrophs. Fig. 2. Continuous and short-term exposure to CRH or 8-Br-cAMP leads to increased glucose uptake in AtT-20/D16:16 cells. Panels A and B represent normalized 2DOG uptake by AtT-20 cells. When exposed to CRH continuously (A), no increase in 2DOG was seen at early time points (1 and 2 h). 2DOG uptake was significantly elevated at 4 h with further delay up to 24 h. With a short-term 1 h (Tr-CRH) exposure to CRH (B), increased 2DOG was seen at 2 h followed by later time points. Exposure to 8-Br-cAMP resulted in delayed increases in 2DOG uptake with both continuous (Cont-cAMP) and transient (Tr-cAMP) exposure (C and D). The increase in CRH-mediated 2DOG uptake in AtT-20 cells was reduced in the presence of DEX (E). Despite a 3 h exposure to DEX, CRH stimulation still resulted in a significant increase in 2DOG uptake when compared to the control group (E). In AtT20 cells, AVP failed to induce significant increase in 2DOG uptake, with or without DEX (E). The data presented is representative of experiments carried out with biological duplicates and technical quadruplicates. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001 compared with corresponding control values. The horizontal line at 100 represents 2DOG uptake under control conditions. Horizontal bars represent mean ± standard deviation. Abbreviations: 2DOG -2deoxyglucose, 8-Br-cAMP -8Br-cyclic adenosine monophosphate, CRH -corticotropin-releasing hormone, DEX e dexamethasone, Cont-CRH e continuous corticotropin-releasing hormone, Cont cAMP e continuous cyclic adenosine monophosphate Tr-CRH e transient corticotropin-releasing hormone, Tr-cAMP e transient cyclic adenosine monophosphate.
Next, we wanted to understand the contribution of cAMP mediated effects of CRH receptor activation on glucose uptake. When AtT-20 cells were stimulated with continuous 8-Br-cAMP, GLUT1 exhibited significant transcriptional upregulation at 1 h, 4 h, 8 h and 24 h ( Supplementary Fig. 2). Expression of the remaining glucose transporters decreased when exposed to 8-Br-cAMP ( Supplementary Fig. 2). These findings also suggest transcriptional regulation of GLUT1 with secretagogue stimulation via cAMP production.

CRH augments GLUT1 expression and translocation to the membrane
Next, we confirmed the effect of secretagogue stimulation on GLUT1 expression and translocation in AtT-20/D16:16 cells. We exposed cells to both CRH and 8-Br-cAMP continuously for 2 h, 8 h and 24 h. Immunofluorescence revealed upregulation of GLUT1 within cells in response to stimulation (Fig. 4). This effect was seen as early as 2 h when compared to control (Fig. 4A-a, b and c). Relative fluorescence ratio of GLUT1 signal between plasma membrane adjacent region versus cytosolic region was increased from 0.9 to 1.16 within 2 h (p < 0.05). At 24 h of continuous CRH exposure, the ratio increased from 0.68 to 1.21 (p < 0.0001) (Fig. 4B). GLUT1 plasma membrane translocation was not increased in AtT20 cells exposed to 8-Br-cAMP (Fig. 4A-c, f and I, 4B), suggesting cAMP independent effect of CRH receptor activation.

Corticotropinomas have increased GLUT1 expression compared to suppressed normal gland
Clinically, concurrent CRH stimulation and 18 F-FDG PET failed to improve standardized uptake value of 18 F-FDG or adenoma detection in 10 patients (Table 1). A histopathological analysis was performed blindly on intra-operatively obtained human tissue by an experienced neuropathologist (A.R.) with both H&E stained, and GLUT1 immunostained sections available (Fig. 4C). Corticotroph adenomas in 9 out of 10 patients demonstrated immunoreactive cyptosolic GLUT1 expression (Table 1). In 3 patients where adjacent normal gland was available, the adjacent normal gland was negative for immunoreactive GLUT1 (Table 1). Increased immunoreactive cytosolic GLUT1 expression in corticotropinoma was not correlated with increased glucose uptake on FDG-PET scanning ( Table 1), suggesting that basal cytosolic GLUT1 expression was not related to increased glucose uptake per se.

Human-derived corticotroph tumor cells are highly dependent on glycolysis
Lastly, we investigated the fate of glucose within humanderived corticotroph tumor cells. Normally, the pituitary gland depends on free fatty acids as an energy source (Vannucci and Hawkins, 1983;Viña et al., 1984). Expectedly (Oldfield and Merrill, 2015), we found that corticotropinoma cells demonstrated higher glycolytic activity compared to normal gland (50.47% vs. 38.99%, Fig. 5A). However, the absolute level of glucose oxidation in corticotropinoma cells was quite low (Fig. 5B). In order to establish the dependency of corticotroph tumors cells on mitochondrial oxidative phosphorylation and/or anaerobic glycolysis, ETO, BPTES, and UK5099 were employed to block oxidation of fatty acids, glutamine, and glucose, respectively. Similarly, FCCP, an electron transport chain uncoupling agent was subsequently utilized to block oxidative phosphorylation pathways. Human-derived corticotroph tumor cells utilized up to 70e80% of their glycolytic capacity but less than 15% of their glucose oxidation capacity (Fig. 5) suggesting that these cells were highly dependent on anaerobic glycolysis.

Discussion
18 F-FDG PET reveals increased glucose uptake in both nonfunctioning and hormone secreting pituitary adenomas (De Souza et al., 1990;Bergstr€ om et al., 1987), whilst minimal 18 F-FDG uptake is usually seen in the normal gland (Ju et al., 2017;Hyun et al., 2011;Jeong et al., 2010). Sellar 18 F-FDG uptake is highly specific (Ju et al., 2017;Jeong et al., 2010;Koo et al., 2006), but a relatively insensitive (40%) finding for pituitary adenomas (Chittiboina et al., 2014;Alzahrani et al., 2009). Low sensitivity of 18 F-FDG-PET suggests the need for modulation of glucose uptake in adenomas to improve detection. In hormone secreting cells, secretagogues can further stimulate glucose uptake (Filetti et al., 1987;Harris et al., 2012), offering an ability to modulate glucose uptake in these cells. Consistent with other reports (Patt et al., 2014), our study failed to demonstrate an immediate change in standardized uptake value (SUV) with concomitant administration of CRH and 18 F-FDG in 10 human subjects (Table 1). These findings led us to investigate whether corticotropinomas are resistant to CRH-mediated glucose uptake, or if the effects are delayed. We hypothesized that CRH stimulation leads to delayed increased glucose uptake in corticotropinomas. Glucose kinetic studies demonstrated an increase in Fig. 3. CRH stimulation results in transcriptional upregulation of GLUT1. Expression of glucose transporters was calculated by DDCt method following RT-qPCR. GLUT1 is overexpressed throughout the period of continuous exposure to CRH in AtT20 D16:16 cells starting at 2 h (A). Other glucose transporters are variably expressed during CRH exposure. The response to CRH was modest at early time points for GLUT2 (B) and GLUT 3 (C) whilst GLUT3 seemed to have a delayed response at 24 h (C). No significant increase was noted for GLUT4 (D). GLUT8 reflected a similar pattern of expression when compared to GLUT1 with a less robust increase (E). The duration of exposure is denoted within each panel with an arrow underneath 'CRH'. CRH-mediated glucose uptake in AtT-20 cells was blunted by GLUT1 inhibitor Fasentin following a 3-h exposure (F). However, CRH exposure reversed the decreased glucose uptake caused by a lower dose of Fasentin (1 mM). A higher dose of fasentin (40 mM) dose prevented CRH mediated normalization of glucose uptake.
This reduction below baseline likely due to the large effect of high dose Fasentin on of glucose uptake at baseline (F). The data shown is representative of experiments carried out in both biological and technical triplicates. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001 compared with corresponding control values. Horizontal bars represent mean ± standard errors of mean (SEM). Abbreviations: CRH -corticotropin-releasing hormone, GLUT e glucose transporter, PCR -polymerase chain reaction. glucose uptake in ACTH-secreting AtT-20/D16:16 cells but not in normal murine corticotrophs cells in response to CRH stimulation. Further, selective modulation of glucose uptake with CRH was confirmed in human-derived corticotropinoma when compared to adjacent normal pituitary tissue. These findings are consistent with evidence suggesting that 18 F-FDG uptake is specific to residual/ recurrent pituitary adenoma following adenomectomy (Zhao et al., 2014).
A time-course study revealed that both continuous and transient administration of CRH leads to delayed increase glucose uptake in AtT-20 cells. CRH-mediated glucose uptake was maximal at 4 h for continuous CRH exposure, and 2e4 h for transient (1 h) CRH exposure. As intracellular effects of CRH stimulation are largely mediated by cAMP (Gigu ere et al., 1982), we deemed important to assess whether it was the main downstream mediator of CRH effects on glucose uptake. Continuous 8-Br-cAMP administration led to increased uptake at early time points from 1 to 4 h whereas transient exposure maximal effect was seen at 8 h. The difference in Fig. 4. GLUT1 is overexpressed in pituitary adenomas and its translocation increases with CRH administration. Fluorescence immunocytochemistry revealed increase in total GLUT1 content within AtT20/D16:16 cells with CRH and 8-Br-cAMP stimulation (A: b to i). This effect was seen as early as 2 h (A: b and c). Continuous CRH stimulation resulted in a robust and sustained GLUT1 membrane translocation (A: e and h). Representative immunofluorescence images are shown. GLUT1 membrane tranlocation was quantified as relative fluorescence ratio between averaged plasma membrane region versus cytosolic region using eight representative cells for each condition (B). The middle bars demonstrate a significant increase in GLUT1 localization to plama membrane region with 2-h (p < 0.05) and 24-h (p < 0.0001) exposure to CRH. Pituitary adenomas (asterix) harvested from patients who underwent transsphenoidal adenomectomy had higher GLUT1 expression than the surrounding pituitary gland (black arrowhead) (C). Adjacent histopathological sections of surgical specimens were examined with hematoxylin and eosin staining (top) and with GLUT1 immunohistochemical staining (bottom). Corticotropinoma demonstrated increased GLUT1 expression not associated with normal anterior pituitary gland (bottom, black arrowhead). Scale bars for fluorescence immunocytochemistry ¼ 10 mm. Scale bars for immunohistochemistry ¼ 100 mm. Abbreviations: DAPI -4 0 ,6-diamidino-2-phenylindole, GLUT1 e glucose transporter 1. time-dependent stimulation effect between CRH and 8-Br-cAMP might be due to the broad actions of CRH on AtT-20 cells not solely relying on the well-characterized cAMP transduction pathway. Interestingly, previous literature showed that little net oxidation of glucose by either the Krebs' cycle or the pentose phosphate pathway occurs in the pituitary gland in-vivo and that nonesterified fatty acids are one of the main energy substrates (Vannucci and Hawkins, 1983;Viña et al., 1984). Our results suggest that pituitary adenomas undergo metabolic reprogramming to rely on glycolysis as the primary energy source, similar to the Warburg effect in malignant tumors (Vander Heiden et al., 2009). CD is characterized by a state of hypercortisolemia that could inhibit glucose uptake in adenomatous (or AtT-20) cells (Booth et al., 1998). Our results suggest that even in the presence of DEX, CRH stimulation may lead to significant delayed increase in glucose uptake in corticotropinomas. A number of glucose transporters are involved in glucose uptake by cells of the central nervous system (Maher et al., 1994). We identified GLUT1 overexpression in 10 human corticotropinomas and identified GLUT1 as the predominant glucose transporter modulated by CRH stimulation. Both CRH and 8-Br-cAMP led to a robust increase in GLUT1 transcription maintained up to 24 h. This is consistent with previous studies in which gonadotrophin releasing hormone (GnRH) increased GLUT1 expression and stimulated glucose uptake in the gonadotrophs (Harris et al., 2012). Intriguingly, CRH stimulation resulted in a more robust and sustained membrane translocation of GLUT1 when compared to 8-Br-cAMP. We found a discordance between human corticotropinoma cytosolic GLUT1 expression and 18 F-FDG uptake (Table 1) presumably due to delayed glucose uptake effects. We thus suspect that in clinical use, exposure to oCRH may lead to delayed increased membrane translocation of GLUT1 within corticotropinomas, and consequently increase 18 F-FDG uptake. Additionally, we found that fasentin, a selective GLUT1 inhibitor, abolished the CRH-mediated increased glucose uptake, confirming the potential role of GLUT1 in glucose uptake in corticotropinomas. Given the dependence of adenomatous cells on glucose as a primary energy source, these results may lead to selective anti-tumor therapies by targeting glucose metabolism.

Conclusion
In this study, we demonstrate that CRH stimulation results in differentially increased glucose uptake from adenomatous but not in normal corticortrophs. A clinically relevant transient CRH exposure time-course study revealed that maximum uptake is 4 h post exposure. Corticotrophs do not normally utilize glucose at baseline as an energy source, however, glucose kinetic analyses suggest that a potential metabolic switch to aerobic glycolysis occurs in adenomatous cells. Based on these findings, we suggest a pathophysiologic basis for delayed 18 F-FDG PET imaging following  (Tumor 1 and 2) utilized the majority of their glycolytic capacity but barely used glucose for oxidation (B). OCR and ECAR were detected using Seahorse in human primary cells Tumor 1 and Tumor 2. The first stages of C and D and the first stages of E and F were generated using the same data. X-axis was extended to mimic continuous treatments. Data between two dash lines in each part were average of the first and third stages. Note that UK5099 caused minimal changes of OCR while oxamate led to significant alterations of ECAR in both samples. Abbreviations: ECAR e extracellular acidification rate, OCR e oxygen consumption rate.
CRH stimulation for detection of corticotropinomas. We are now confirming these findings with a clinical trial to detect corticotropinomas with 18F-FDG PET imaging 2e6 h following oCRH stimulation.

Disclosure statement
The authors declare no potential conflicts of interest.

Financial support
This research was supported by the Intramural Research Program of the National Institute of Neurological Diseases and Stroke, Bethesda, MD (NIH ZIA NS003150-01).


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Acid-degradable Dextran as an Image Guided siRNA Carrier for COX-2 Downregulation

Purpose: Effective in vivo delivery of siRNA to silence genes is a highly sought-after goal in the treatment of multiple diseases. Cyclooxygenase-2 (COX-2) is a major mediator of inflammation and its effective and specific downregulation has been of major interest to treat conditions ranging from auto-immune diseases to gastric inflammation and cancer. Here we developed a novel and efficient method to produce a multiple imaging reporter labeled cationic dextran nanopolymer with cleavable positive charge groups for COX-2 siRNA delivery. Methods: Small molecules containing amine groups were conjugated to the dextran scaffold through acetal bonds that were cleaved in weak acid conditions. With multiple imaging reporters located on different regions of the nanopolymer, cleavage of acetal bonds was visualized and quantified by imaging, for the first time, in cancer cells and tumors. Results: The biocompatibility of dextran and the rapid cleavage and release of amine groups minimized proinflammatory side effects and COX-2 induction observed with other siRNA carriers, to successfully achieve COX-2 downregulation in cancer cells and tumors. Imaging results confirmed that this nanoplex, consisting of the dextran nanopolymer with COX-2 siRNA, accumulated in tumors, and the amine functional groups were rapidly cleaved in cancer cells and tumors. Along with effective downregulation of COX-2, we also demonstrated, for the first time, effective downregulation of its major product prostaglandin E2 (PGE2). Conclusions: We successfully developed an efficient method to produce an acid-degradable dextran nanopolymer containing cleavable amine groups as the siRNA carrier. Because of its biocompatibility, this degradable dextran delivered COX-2 siRNA within tumors and efficiently downregulated COX-2 expression.

H2−6)
Compound 3: 40.0 mg compound 2 was dissolved in 2 ml HEPES buffer (pH 8.4), then 1 mg Cy5.5 NHS ester in 100 μL DMSO was added into the dextran solution. The mixture was stirred at room temperature for 2 h. After 2 h stirring, the pH value was adjusted to 7.0 by adding 10% HCl. Then 10 µl acetic acid was added into this solution, keeping the pH between 6.0-7.0. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 40 mg was added to form the amide bonds between the acetic acid and the unreacted amines of dextran. This reaction prevented the unreacted amines to be involved in further modifications of the dextran carriers. The Cy 5.5 labeled dextran was purified by centrifugal filter tubes (Amicon ultra-15, 10,000 MW cutoff) for 3 times. After lyophilization, 38.7 mg cyan solid was harvested. Colorimetric assay of Cy5.5 at 670 nm indicated that there were 1.1 Cy5.5 molecules in one dextran molecule. NMR could not characterize Cy5.5 on dextran due to the very low concentration of Cy5.5.
Compound 4: 30 mg of compound 3 was dissolved in 2 ml anhydrous DMSO containing 2 g 4Å active molecular sieves. Then p-toluenesulfonic acid monohydrate (270 mg, 4 eq of compound 1) was added into this mixture. This mixture was stirred at room temperature under nitrogen atmosphere for 4 h to remove water in the solution. Meanwhile 150 mg compound 1 (2 eq of glucose unit) was dissolved in anhydrous DMSO (1 mL) with 1 g 4Å active molecular sieves, and the solution was stirred at room temperature under nitrogen atmosphere for 4 h. Then compound 1 solution was transferred into compound 3 solution containing 4Å active molecular sieves, and this mixture was stirred at 65 ºC under nitrogen atmosphere for 16 h. This reaction was stopped by adding 100 μl triethylamine, and this DMSO solution was dropped into ethyl acetate to precipitate the cyan product. The suspension was centrifuged to obtain the cyan solid precipitate, which was purified by three times re-precipitation in ethyl acetate. After drying, 55.3 mg product was obtained. This product was dissolved in 2.0 ml HEPES buffer (pH 8.2), then 100 μl DMSO containing 0.5 mg rhodamine 6G NHS ester was added to this HEPES buffer. After stirring at room temperature for 2 h, this dextran product was purified by centrifugal filter tubes (Amicon ultra-15, 10,000 MW cutoff). After repeating purification 2 more times and lyophilization, 49.

N/P value dependent stability of siRNA/compound 4 nanoplex
The N/P ratio is crucial for determining the stability and delivery efficacy of the polymer/siRNA nanoplex. Electrophoretic gel mobility shift assay was used to evaluate the N/P ratio between compound 4 and COX-2 siRNA. Compound 4 and COX-2 siRNA were mixed in PBS (pH = 7.4) buffer with N/P values of 0, 1, 5, 10, and 15. After 30 min incubation at room temperature, the resulting nanoplexes were loaded on a 5% agarose gel (1.0 μg COX-2 siRNA per well). After electrophoresis, the nucleotide was stained by ethidium bromide and UV light images were obtained. Figure S2: Electrophoretic gel mobility shift assay of stability of dextran-siRNA nanoplex.

The Stability of Encapsulated siRNA in Fresh Mouse and Human Serum
A nuclease stability assay was conducted to determine the stability of siRNA encapsulated in siRNA/compound 4 nanoplex. The nanoplex was formulated in PBS, pH 7.4 with an N/P ratio of 15 for 20 min. The nanoplex was incubated for 4 and 8 h in 70% fresh serum at 37 °C. At the end of incubation, all encapsulated siRNA was displaced from the nanoplex by adding 2% SDS and loaded to the wells (1 μg/well siRNA) containing 4% agarose gel.

Degradation of compound 4
Compound 4 was incubated in pH 5.5 buffer for 8 h, after which the pH value was changed to pH 7.4 by adding NH3·H2O. After dialysis in pH 7.4 NH3·H2O solution, partially degraded compound 4 solid was collected by lyophilization. This solid was dissolved in D2O for 1 H NMR spectroscopy, or dissolved in PBS pH7.4 buffer for gel permeation chromatography (GPC).

Determination of Cytotoxicity of Compound 4 in vitro
The concentration dependent cytotoxicities of compound 4 in MDA-MB-231 human breast cancer cells and human mammary fibroblasts after 3 days incubation were measured using an MTT assay.
As demonstrated in Figure S6, compound 4 did not show toxicity up to 3 days incubation with concentrations up to 100 g/ml.