extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Angiotensin II Type 1 Receptor-associated Protein Inhibits Angiotensin II-induced Insulin Resistance with Suppression of Oxidative Stress in Skeletal Muscle Tissue

Enhancement of AT1 receptor-associated protein (ATRAP) in adipose tissue improves high fat diet (HFD)-induced visceral obesity and insulin resistance, and suppresses adipose oxidative stress. However, HFD loading is not a direct stimulatory factor for AT1 receptor. In the present study, we investigated the effect of chronic, low-dose angiotensin II (Ang II) stimulation on glucose and lipid metabolism in mice and functional role of ATRAP. ATRAP expression was higher in adipose tissue (5–10-fold) and skeletal muscle tissue (approximately 1.6-fold) in ATRAP transgenic (TG) mice compared with wild-type (WT) mice. After Ang II infusion, insulin sensitivity was impaired in WT mice, but this response was suppressed in TG mice. Unexpectedly, Ang II infusion did not affect the adipose tissue profile in WT or TG mice. However, in skeletal muscle tissue, Ang II stimulus caused an increase in oxidative stress and activation of p38 MAPK, resulting in a decrease in glucose transporter type 4 expression in WT mice. These responses were suppressed in TG mice. Our study suggests that Ang II-induced insulin resistance is suppressed by increased ATRAP expression in skeletal muscle tissue. Hyperactivity of AT1 receptor could be related to formation of insulin resistance related to metabolic syndrome.

. ATRAP expression in adipose tissue and skeletal muscle tissue of WT and TG mice. (a) Relative ATRAP mRNA levels in WAT, BAT, and skeletal muscle tissue from WT and TG mice. Values are expressed as mean ± SE (n = 3 in each group). † P < 0.05, † † P < 0.01, vs WT mice. (b) Immunohistochemical analysis for ATRAP in skeletal muscle tissue of TG mice. The consecutive sections from the soleus muscle tissue of TG mice stained for ATRAP and myoglobin. Positive areas for ATRAP and myoglobin are shown as brown dots in each section. Original magnification, ×100 (upper panels), and ×400 (lower panels). Scale bar = 200 μm. WT, wildtype; TG, ATRAP transgenic mice; WAT, white adipose tissue; BAT, brown adipose tissue.
SCieNTifiC RepoRTs | (2018) 8:2846 | DOI: 10.1038/s41598-018-21270-8 induction analysis of mRNA levels in each tissue showed that ATRAP mRNA expression in skeletal muscle tissue of TG mice was slightly, but significantly, higher compared with WT mice (approximately 1.6-fold). To further investigate distribution of ATRAP in skeletal muscle tissue, we performed an immunohistochemical analysis using an anti-ATRAP antibody and a polyclonal antibody against myoglobin, which is specifically expressed in skeletal muscle cells. The results of consecutive sections stained with specific antibodies suggested that ATRAP was expressed in skeletal muscle cells (i.e., muscle fibres), but not other cells (e.g., macrophages and lymphocytes) in skeletal muscle tissue of TG mice (Fig. 1b).
Effects of Ang II infusion on body weight, blood pressure, heart rate, and cardiac hypertrophy in WT and TG mice. Age-matched WT and TG mice were divided into four groups: (1) vehicle-infused WT mice; (2) Ang II (100 ng/kg/min)-infused WT mice; (3) vehicle-infused TG mice; (4) Ang II (100 ng/kg/ min)-infused TG mice. Ang II infusion did not affect body weight gain, systolic blood pressure and heart rate in WT and TG mice. There were also no differences in these parameters between the genotypes of vehicle or Ang II groups (Fig. 2a-c). We further examined the heart weight/body weight ratio in WT and TG mice because cardiac hypertrophy is closely associated with an elevation in blood pressure 16 . We found that the heart weight/body weight ratio was comparable in the vehicle-and Ang II-infused groups in WT and TG mice (Fig. 2d). Therefore, in this study, a 100 ng/kg/min dose of Ang II corresponded to a subpressor dose of Ang II.
Effects of Ang II infusion on glucose and lipid metabolism in WT and TG mice. We next examined the parameters of glucose and lipid metabolism using blood samples obtained by cardiac puncture at the time mice were sacrificed. Total cholesterol, triglyceride, free fatty acid, and non-fasting blood glucose levels were comparable in the vehicle-and Ang II-infused mice in both genotypes (Table 1). Although Ang II infusion significantly increased plasma insulin levels in WT mice, this increase in plasma insulin levels in response to Ang II infusion was suppressed in TG mice (Table 1). To further examine effects of Ang II infusion on insulin sensitivity, we performed the glucose tolerance test (GTT) and insulin tolerance test (ITT), which reflect insulin secretion and resistance, respectively (Fig. 3). Chronic Ang II infusion caused insulin resistance as estimated by the ITT in WT mice, but this response was suppressed in TG mice (vehicle vs Ang II in WT mice; at 60 minutes after insulin injection, 54.6 ± 3.7 vs 84.5 ± 9.6 mg/dl, P = 0.006; at 120 minutes after insulin injection, 64.4 ± 3.0 vs 82.0 ± 7.2 mg/dl, P = 0.022; area under the curve, 8562 ± 425 vs 11045 ± 1068 min × mg/dl, P = 0.027) (Fig. 3a-d).
On the other hand, the results of GTT showed that Ang II infusion tended to cause impaired glucose tolerance in WT mice without statistical significance (vehicle vs Ang II; at 60 minutes after glucose injection, 165.1 ± 11.7 vs Figure 2. Ang II infusion does not affect body weight gain, blood pressure, heart rate, and the heart weight/ body weight ratio in WT and TG mice. (a) Effects of Ang II infusion on body weight. (b) Effects of Ang II infusion on systolic blood pressure. (c) Effects of Ang II infusion on heart rate. (d) Effects of Ang II infusion on the heart weight/body weight ratio. Values are expressed as mean ± SE (n = 5 in each group). WT, wild-type; TG, ATRAP transgenic mice; Ang II, angiotensin II. 196.0 ± 18.9 mg/dl, P = 0.095; at 120 minutes after glucose injection, 110.7 ± 6.1 vs 130.3 ± 11.6 mg/dl, P = 0.081) ( Fig. 3e-h). These results suggest that chronic Ang II infusion provokes insulin resistance only in WT mice, but not in TG mice.
Effects of angiotensin II infusion on adipose oxidative stress, adipokines and adipocyte differentiation in WT and TG mice. To investigate the mechanisms involved in the inhibition of insulin resistance in response to chronic Ang II infusion in TG mice, we examined nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in WAT from WT and TG mice. NADPH oxidase-derived reactive oxygen species (ROS) function as important intracellular second messengers to impair insulin signalling in adipose tissue 17,18 . There were no significant differences in NADPH oxidase components in WAT between the vehicle-and Ang II-infused groups in WT and TG mice (Fig. 4a). We next investigated the function of adipose tissue as an endocrine organ for releasing adipokines. The mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and adiponectin in WAT was comparable in the vehicle-and Ang II-infused groups in WT and TG mice (Fig. 4b). There was also no significant difference in mRNA expression of adipokines in WAT between WT and TG mice, regardless of the presence or absence of Ang II (Fig. 4b). Ang II reportedly impairs adipogenic differentiation of preadipose cells 19 . Therefore, we examined mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα), which are critical transcription factors 20 , in WAT from WT and TG mice. Ang II infusion did not affect mRNA PPARγ and C/EBPα expression in adipose in either WT or TG mice (Fig. 4c). These results indicate that inhibition of Ang II-induced insulin resistance in TG mice is not mediated by suppression of AT1R signalling in adipose tissue.

Effects of Ang II infusion on oxidative stress in skeletal muscle tissue in WT and TG mice.
Skeletal muscle tissue is important as a major target organ for insulin 21,22 . We next examined mRNA expression of the NADPH oxidase components (NOX2, p22 phox , p47 phox , and p40 phox ) in skeletal muscle tissue of WT and TG mice. mRNA expression levels of p47 phox and p40 phox in skeletal muscle tissue were significantly higher after Ang II infusion compared with vehicle infusion in WT mice (Fig. 5a). However, the Ang II-induced increase in p47 phox and p40 phox mRNA expression in skeletal muscle tissue was completely suppressed in TG mice (Fig. 5a). To further evaluate the changes in ROS status in skeletal muscle tissue of WT and TG mice, we examined immunostaining of 4-hydroxy-2-nonenal (4-HNE), which reflects tissue ROS generation in tissue 23,24 . Immunostaining of 4-HNE in skeletal muscle tissue was enhanced after Ang II infusion compared with vehicle infusion in WT mice (Fig. 5b). However, the Ang II-induced increase in 4-NHE immunostaining was not observed in skeletal muscle tissue of TG mice (Fig. 5b). These results indicate that the Ang II-induced increase in several NADPH oxidase components and ROS generation are inhibited in skeletal muscle tissue of TG mice.

Effects of Ang II infusion on Akt signalling and glucose transporter type 4 expression in skeletal muscle of WT and TG mice.
We further investigated the mechanism of insulin resistance caused by skeletal muscle tissue NADPH oxidase-derived ROS generation in Ang II-infused mice. We examined protein levels of phospho-Akt and glucose transporter type 4 (GLUT4) in skeletal muscle tissue. There was no significant difference in phospho-Akt in skeletal muscle tissue between the vehicle-and Ang II-infused groups in WT and TG mice (Fig. 6a). However, GLUT4 expression was significantly lower after Ang II infusion compared with vehicle infusion in skeletal muscle tissue of WT mice (Fig. 6b). This Ang II-induced downregulation of GLUT4 expression was inhibited in skeletal muscle tissue of TG mice.
Effects of Ang II infusion on skeletal muscle p38 mitogen-activated protein kinase activation in WT and TG mice. As the downstream signalling pathway of the AT1 receptor, we compared the activation of mitogen-activated protein kinase (MAPK) in response to Ang II infusion in skeletal muscle tissue of WT and TG mice. Skeletal muscle tissue p38 MAPK was significantly activated after 2 weeks of Ang II infusion in WT mice (Fig. 7). However, Ang II-induced activation of p38 MAPK was completely suppressed in skeletal muscle tissue of TG mice. These results indicate that suppression of over-activated AT1 receptor signalling by ATRAP enhancement in skeletal muscle tissue contributes to inhibition of Ang II-induced insulin resistance in TG mice.

Discussion
ATRAP TG mice used in the present study were generated using a 5.4-kb adiponectin promoter, which has recently been reported to have high specific gene tractability for adipose tissue 14,[25][26][27] . Although the adipose tissue is the main tissue in which ATRAP is highly expressed, we found that there is also significantly higher ATRAP expression in skeletal muscle tissue in TG mice (approximately 1.6-fold higher expression than WT mice at the mRNA level). Adiponectin in the blood stream is mainly secreted by adipocytes, but several studies have indicated that endogenous adiponectin is also produced by skeletal muscle tissues. In addition, adiponectin production in skeletal muscle tissue is increased in response to metabolic stress, such as that induced by a high-fat diet [28][29][30] . The finding that there is a slight increase in ATRAP expression in skeletal muscle tissue of TG mice compared with that of WT mice is consistent with previous knowledge regarding ATRAP.
We assessed physiological parameters of WT mice that were affected by administration of a low dose of Ang II (100 ng/kg/min) over 2 weeks. We found that although there were no significant changes in body weight and blood pressure, there was an increase in non-fasting plasma insulin levels. In general, administration of Ang II Figure 3. Ang II infusion induces insulin resistance and tends to impair glucose tolerance only in WT mice. The insulin tolerance test in WT (a) and TG mice (b), and the glucose tolerance test in WT (e) and TG mice (f) after 2 weeks of vehicle or Ang II infusion. The AUCs of the insulin tolerance test in WT (c) and TG mice (d), and those of the glucose tolerance test in WT (g) and TG mice (h) after 2 weeks of vehicle or Ang II infusion. Values are expressed as mean ± SE (n = 7-10 in each group). *P < 0.05, **P < 0.01, vs vehicle. WT, wild-type; TG, ATRAP transgenic mice; Ang II, angiotensin II; AUC, area under the curve. results in elevated blood pressure, and causes muscle atrophy and cachexia, among other effects 31,32 . With respect to animal species differences in susceptibility to Ang II treatment, rats exhibit susceptibility to progressive hypertension in response to Ang II infusion, whereas mice are relatively resistant to Ang II-induced hypertension 7,33-35 . In the present study, 100 ng/kg/min doses of Ang II infusion into mice did not cause elevated blood pressure and weight loss, which is consistent with the previous reports 33,34 . However, administration of subpressor doses of Ang II promoted insulin resistance as estimated by the ITT. Additionally, Ang II stimulus tended to cause impaired glucose tolerance as estimated by the GTT, although this did not reach statistical significance.
There are several reports that chronic administration of low doses of Ang II promotes insulin resistance in mice and rats 7,9 . Studies on the mechanism of this promotion have reported that an increase in oxidative stress by activation of NADPH oxidase through skeletal muscle tissue AT1 receptor, disorders of GLUT4 expression and translocation in the insulin downstream pathway through activation of MAPK resulting from increased oxidative stress 36,37 , and suppression of Akt phosphorylation are involved 38,39 . In our study, Ang II infusion decreased GLUT4 expression, but did not affect Akt activation in skeletal muscle tissue of WT mice. This finding is partially   consistent with previous reports that chronic Ang II infusion induces insulin resistance without decreasing Akt activation in mice and rats 7,9 . Although administration of low doses of Ang II caused activation of the p47 phox / p40 phox -ROS-p38MAPK pathway and decreased GLUT4 expression in skeletal muscle tissue of WT mice, this change was completely suppressed in skeletal muscle tissue of TG mice. Collectively, these findings indicate that increased ATRAP expression in skeletal muscle tissue suppresses hyperactivation of AT1 receptor signalling 40,41 .
We recently conducted an investigation on the effect of a high-fat diet on the same type of ATRAP transgenic mice that were used in the present study 15,42 . We found that enhancement of adipose ATRAP expression suppressed oxidative stress and phosphorylation of p38 MAPK in response to the high-fat diet in adipose tissue. Additionally, enhancement of adipose ATRAP expression improved dysfunction of adipokine regulation and inhibited obesity-related metabolic disorders 15,42 . However, in the present study, enhancement of adipose ATRAP expression did not show a protective effect on the adipose tissue profile. This finding is probably related to the fact that administration of Ang II had no effect on oxidative stress and adipokine levels in adipose tissue of WT mice. Therefore, insulin resistance due to a direct effect of Ang II stimulation, unrelated to dietary obesity, might primarily occur in skeletal muscle tissue. As a result, adipose tissue has almost no involvement in formation of this pathological condition.
The following limitations of this study need to be considered. First, the TG mouse model is characterised by a high ATRAP expression level in adipose tissue. Therefore, there needs to be confirmation that the same results can be obtained when using a model in which a high ATRAP expression level is specific to skeletal muscle tissue 43 . If such transgenic mice can be generated, the possibility must be kept in mind that an increase in ATRAP expression levels only in skeletal muscle tissue may cause major deviations above physiological levels 44,45 . Our finding that increased ATRAP expression levels in skeletal muscle tissue just under 2-fold completely blocked insulin resistance caused by Ang II treatment suggests the powerful beneficial effect of ATRAP in skeletal muscle tissue. Second, ATRAP and AT1 receptor expression in skeletal muscle cells isolated from skeletal muscle tissue was not examined in the present study. A possibility that other non-skeletal muscle cells in skeletal muscle tissue or skeletal muscle blood flow might be related to formation of insulin resistance provoked by Ang II infusion cannot be excluded completely. Therefore, further studies will be needed to investigate expression and function of ATRAP and AT1 receptor in skeletal muscle cells isolated from skeletal muscle tissue between WT and TG mice.
In conclusion, chronic administration of low-dose Ang II in WT mice causes increased oxidative stress in skeletal muscle tissue and insulin resistance, but there is no effect on the adipose tissue profile. This Ang II-induced insulin resistance is suppressed by increased ATRAP expression levels in skeletal muscle tissue. Our findings suggest that even in patients who have not yet presented with obesity or hypertension, hyperactivity of AT1 receptor signalling is related to formation of pathological insulin resistance related to metabolic syndrome. In addition, ATRAP activation therapy in insulin target organs (e.g., adipose tissue and skeletal muscle tissue) may be widely applied for treating lifestyle diseases that are not accompanied by obesity or hypertension.

Methods
Animals. The mice were housed in a controlled environment with a 12-hour light-dark cycle at a temperature of 25 °C. The mice were allowed free access to food and water. Mice were fed a standard diet (0.3% NaCl, 3.6 kcal/g, and 13.3% energy as fat; Oriental MF, Oriental Yeast Co, Ltd.). This study was performed in accordance with the National Institute of Health guidelines for the use of experimental animals. All animal studies were reviewed and approved by the Animal Studies Committee of Yokohama City University.
Generation of TG mice overexpressing ATRAP in adipose tissue and skeletal muscle tissue. TG mice overexpressing ATRAP in adipose tissue and skeletal muscle tissue were generated on a C57BL/6 J background, as previously described 15 . Briefly, hemagglutinin-tagged mouse ATRAP (HA-ATRAP) cDNA was subcloned into a transgenic vector between the 5.4-kb fragment of the adiponectin promoter (kindly provided by Dr. Philipp E. Scherer, The University of Texas Southwestern Medical Center) and 3′ untranslated region (3′UTR). This construct was microinjected into the pronucleus of fertilised mouse embryos. TG mice were identified by PCR using forward (TGCTTGGGGCAACTTCACTATC) and reverse (ACGGTGCATGTGGTAGACGAG) primers. Mice were mated with C57BL/6 mice to obtain TG mice and their littermate WT mice for the experiments.
Ang II treatment. Ang II infusion was performed as previously described 46 . Briefly, Ang II (100 ng/kg/min) was infused subcutaneously in male WT and TG mice (9-11 weeks old) for 14 days using an osmotic minipump (Model 2002; ALZET, Palo Alto, CA, USA). Blood pressure measurement. Systolic blood pressure and heart rate were measured by the tail-cuff method (BP-Monitor MK-2000; Muromachi Kikai Co.). The MK-2000 monitor enabled measurement of blood pressure without preheating the animals and using anaesthesia. This procedure avoided this stressful condition, as described previously 15 . All measurements were performed between 10:00-14:00 hours. At least eight readings were taken for each measurement.
Biochemical assays. Blood samples were obtained by cardiac puncture when the mice were sacrificed in the fed state. Whole blood samples were centrifuged at 3000 rpm at 4 °C for 15 minutes to separate the plasma. Enzymatic assay kits were used for determining plasma glucose, free fatty acid, triglyceride, and total cholesterol levels (Wako Pure Chemical). Plasma insulin concentrations were measured with a commercially available ELISA kit (The Morinaga Institute of Biological Science, Inc.).
GTT and ITT. The GTT and ITT were performed as previously described 15 blood samples that were taken from the tail tip of overnight-fasted mice. Samples were taken at baseline and at 15, 30, 60, and 120 minutes after intraperitoneal injection of glucose (1 g/kg body weight). For the ITT, insulin (0.75 U/kg body weight in 0.1% BSA; Humulin R-Insulin; EliLilly & Co.) was administered via intraperitoneal injection after 4-hour fasting. Blood glucose concentrations were measured at baseline and at 15, 30, 60, and 120 minutes after the injection.
Real-time quantitative RT-PCR analysis. Total RNAs were extracted from WAT, interscapular BAT, and soleus muscle tissue with ISOGEN (Nippon Gene), and the cDNA was synthesized using the SuperScript III First-Strand System (Invitrogen). Real-time quantitative RT-PCR was performed with an ABI PRISM 7000 Sequence Detection System by incubating the reverse transcription product with TaqMan PCR Master Mix and a designed Taqman probe (Applied Biosystems), essentially as described previously 47 . The mRNA levels were normalized to those of the 18SrRNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control.
Immunoblot analysis. Immunoblot analysis was performed as described previously 15,46 . Briefly, total protein extract was prepared from tissues with sodium dodecyl sulfate-containing sample buffer. The protein concentration of each sample was measured with a Detergent Compatible Protein Assay Kit (Bio-Rad) using bovine serum albumin as the standard. Equal amounts of protein extract from tissue samples were fractionated on a 5-20% polyacrylamide gel (ATTO). The gel was then transferred to a polyvinylidene difluoride membrane using the iBlot Dry Blotting System (Invitrogen). Membranes were blocked for 1 hour at room temperature with phosphate-buffered saline containing 5% skim milk powder. Membranes were probed overnight at 4 °C with specific primary antibodies to GLUT4 (Abcam), Akt, phospho-Akt (Cell Signaling Technology), p38 MAPK (Santa Cruz Biotechnology), and phospho-p38 MAPK (Promega Corporation). Membranes were washed and further incubated with secondary antibodies for 15 minutes at room temperature. The sites of the antibody-antigen reaction were visualised by enhanced chemiluminescence substrate (GE Healthcare). Images were analysed quantitatively using a Fuji LAS-3000 image analyser (Fuji Film). For analysis of phospho-Akt, tissues were collected as described previously 15 . Briefly, after 4-hour fasting, mice were deeply anesthetized with an intraperitoneal injection of pentobarbital. Insulin (5 U/kg body weight in 0.1% BSA; Humulin R-Insulin; Eli Lilly & Co.) was injected through the inferior vena cava, and tissues were quickly collected 2 minutes after the injection.
Immunohistochemical analysis. Immunohistochemical analysis was performed as described previously 24,48 . Soleus muscle tissue from mice was fixed with 4% paraformaldehyde and subsequently embedded in paraffin. Sections of 4-μm thickness were dewaxed and rehydrated, and antigen retrieval was performed by microwave heating. The sections were blocked for endogenous biotin activity using peroxidase blocking reagent (DAKO) and treated for 60 minutes with 10% normal goat serum in phosphate-buffered saline. The sections were then incubated with anti-4-HNE antibody diluted to 1:100 (Abcam), anti-myoglobin antibody (Nichirei Biosciences), and anti-ATRAP antibody diluted to 1:100.
Statistical analysis. Data are expressed as mean ± SE. Differences were analysed as follows. 2-factor ANOVA with Bonferroni posttests was used to test for differences in Ang II infusion and genotype (Figs 2d, 4a and b, 5a, 6a and b and 7); Unpaired t-test was used to test for differences in WT vs TG mice (Fig. 1a) or differences in vehicle vs Ang II infusion within each genotype (Table and Fig . 3a-h); Repeated measures ANOVA was used to test for differences over time (Fig. 2a-c). A P value < 0.05 was considered statistically significant.


extfieldsofstudy: Biology Medicine

text:
An improved ternary vector system for Agrobacterium-mediated rapid maize transformation

Key message A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. Abstract The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86–103%) raw transformation frequency in an elite maize inbred. Electronic supplementary material The online version of this article (10.1007/s11103-018-0732-y) contains supplementary material, which is available to authorized users.

Introduction
Agrobacterium-mediated plant transformation is a preferred method for plant genetic engineering since it was known to reliably transfer large (30-150 kb) DNA fragments into plants to generate events with low transgene copy number (Gelvin 2003(Gelvin , 2009Komari et al. 2004). Early attempts to exploit Agrobacterium as a natural tool for transgenesis required introducing a target gene directly into the transfer DNA (T-DNA) region of the large tumorinducing (Ti) plasmid either through a single or doublehomologous recombination. This was challenging given the large size of the Ti plasmid (Fraley et al. 1985(Fraley et al. , 1983Zambryski et al. 1983). The complexity of plasmid construction was later solved through the development of the 'binary vector' system, where the T-DNA is separated on a smaller, independent episome from the virulence genes encoded by a disarmed Ti plasmid (Bevan 1984;Hoekema et al. 1983). This binary system is routinely used for plant genetic engineering.
Gene dosage effects have been observed with some of the virulence genes (Jin et al. 1987) which led to the development of a superbinary vector, pSB1. Plasmid pSB1 carries a DNA fragment (~ 14.8 kb) with vir genes (B, G, part of C and D) from the Ti plasmid, pTiBo542 (Komari 1990). Application of the pSB1 superbinary system broadened the host range of plants amenable to transformation with A. tumefaciens (Cheng et al. 1997;Hiei et al. 1994;Ishida et al. 1996;Komari et al. 1996Komari et al. , 2004Komari et al. , 2006Tingay et al. 1997) and is widely regarded as the primary choice of vector system for maize transformation Zhi et al. 2015). However, the large size of the plasmid pSB1 (~ 37 kb) and the co-integration step required for introduction of the T-DNA vector complicates vector construction and structural confirmation of the plasmid. Additionally, pSB1 contains a tetracycline resistance gene (tetAR) for selection and C58-based Agrobacterium strains are known to give rise to spontaneous tetracycline resistant mutants (Luo and Farrand 1999). The plasmid pSB1 was also found to have a defective set of virulence genes (specifically a frame-shift in virC1 gene and a truncated virD operon), repetitive and non-essential DNA sequences, and a large fragment containing the RK2 origin of replication (ORI), which we speculate negatively impacted plasmid function and plant transformation.
In this report, we describe the development of an improved ternary vector system for crop transformation using the newly described accessory "pVIR" plasmids. The pVIR plasmids have many desirable features, such as small size, enhanced vector stability, an improved bacterial selectable marker, and amended vir genes (operons virC, virD and virE) for improved T-DNA delivery (Anand et al. 2017). Introduction of these accessory plasmids in trans with the T-DNA binary vectors in the same Agrobacterium strain facilitated the generation of highly versatile ternary vectors ( Supplementary Fig. 1). Ternary vectors harboring a plasmid containing a constitutive virG mutant (virGN54D) have been described that efficiently transfer T-DNA in multiple dicot plant species (Kikuchi et al. 2005;Fits et al. 2000). The introduction of pVIR plasmids in the ternary vector design nearly doubled raw transformation rates and improved the number of transformation events of usable quality in difficult-to-transform maize genotypes. Incorporation of the ternary vector design enabled the development of a rapid maize transformation system (Lowe et al. 2018) that relies on expression of morphogenic genes Baby boom (Bbm) and Wuschel (Wus2) (Lowe et al. 2016), for rapid recovery of stable transgenic plants. We describe testing of different ORI in the accessory plasmid and T-DNA plasmid using the rapid transformation system. Through the testing, we identified unique ORI-by-ORI combinations with relatively high raw transformation frequencies: the percent of infected embryos producing stable events was 86-103%. This represents a six-to-seven-fold improvement over conventional random transformation (13-14%) using the plasmid pSB1 in an elite maize inbred.

Annotation of pSB1
We generated a complete annotated map of the pSB1 vector ( Fig. 1a) using the published sequence (GenBank: AB027255.2) and sequence assembled de novo. As shown in Fig. 1a, plasmid pSB1 is ~ 37 kb and includes a 14.8 kb KpnI fragment with vir genes from Ti plasmid pTiBo542 that enable transformation of a broad range of crops (Cheng et al. 1997;Hiei et al. 1994;Ishida et al. 1996;Tingay et al. 1997). pSB1 was found to have a 19.7 kb fragment containing the ORI and partitioning functions from the broad host range plasmid RK2 for replication in Agrobacterium (Knauf and Nester 1982;Schmidhauser and Helinski 1985). The assembled sequence identified several nonessential elements including a ~ 14 kb sequence between the trfA replication protein from its origin of replication target site that included some tra and trb genes encoding a non-functional conjugative transfer system. A 2.7 kb pBR322 fragment containing the ColE1 ORI and an inactive β-lactamase coding sequence with the embedded lambda phage COS site was also identified. Additionally, the ColE1 ORI (1.0 kb) was introduced with a 2.7 kb fragment from plasmid pBR322 with nonessential sequences. We also identified a 146 bp inverted repeat and a 32 bp palindrome of unknown origin in the vector backbone (Fig. 1a).

Overview of the pVIR accessory plasmids
A series of small and versatile accessory plasmids ( Fig. 1b-d) (Anand et al. 2017) were designed with the intent to simplify and optimize Agrobacterium-mediated crop transformation. These plasmids, designated pVIR referred to as pPHP70298, pPHP71539 and pPHP7976, contain sets of different vir genes (virG, virE, virA, virJ, virB, virC, and virD) from the hypervirulent pTiBo542 plasmid, as well as a stable high copy number pVS1 ORI (Zhi et al. 2015) and gentamicin bacterial selection marker. The respective sequences can be found under GenBank accession numbers MF788072, MF788073 and MF788074. The accessory plasmids were generated to support a ternary vector design for crop transformation ( Supplementary Fig. 1) and to simplify vector assembly.
Design of the pVIR plasmids was based on the open source superbinary vector pCAMBIA5105 (http://www. cambi a.org/daisy /cambi a/585). The large ORI (19.7 kb) containing the moderate copy plasmid replicon RK2 ORI (10-12 copies) and partitioning sequences in pSB1  was replaced with pVS1, a smaller (~ 2.6 kb), more stable (Stanisich et al. 1977) and high copy (~ 20 copies) ORI (Zhi et al. 2015). The choice of plasmid ORI is an important determinant of plasmid size, stability, copy number, host specificity and compatibility with other ORI when additional plasmids are present in the same bacterial cell. The original RK2 ORI is likely to have adversely affected plasmid performance due to its large size and the presence of intervening non-essential DNA sequence. In a previous study, we demonstrated the choice of ORI directly impacted maize transformation (Zhi et al. 2015).
We corrected several deficiencies in the virulence genes in plasmid pSB1. The single base pair frameshift mutation in the virC1 gene was replaced with a functional virC operon consisting of the virC1 and virC2 genes. The original Fig. 1 The maps of accessory plasmids described in this study, a complete annotation of the pSB1 vectors  following de novo assembly of the sequenced plasmid, b-d newly described pVIR plasmids, pPHP70298, pPHP71539 and pPHP79761 respectively, with complete annotation frame-shift in virC1 likely resulted in an inactive VirC protein. The VirC protein is known to bind to the cis-element "over-drive" sequences for processing of single stranded T-DNA and translocation of the T-DNA to the poles for enhanced T-DNA delivery (Close et al. 1987;Lu et al. 2009). The truncated virD2 gene in pSB1 was also replaced with a functional virD2 gene as in pPHP70298 (Fig. 1b). The VirD1 and VirD2 endonuclease complex is essential for T-DNA strand synthesis, processing and transfer (reviewed in Gelvin 2003;Tzfira and Citovsky 2006). With the addition of a more stable ORI and fully functional vir genes, the plasmid pPHP70298 is an improved version of plasmid pSB1, but only half the size (~ 19 kb).
Additional improvements incorporated in the pVIR plasmids included elimination of the 7.0 kb truncated tra and trb operons or flanking genes within the original 19.7 kb RK2 ORI, a 40 bp palindromic sequence and two copies of the 146 bp inverted repeat sequence on pSB1 (Fig. 1A). The 2.7 kb pBR322 fragment present in pSB1 that contains the ORI, an inactive β-lactamase coding sequence, and 18 bp poly-G flanked lambda COS sites were removed and replaced with the 1.2 kb ColE1 ORI. The tetracycline efflux transporter tetA gene, a potentially problematic marker in C58-based Agrobacterium strains (Luo and Farrand 1999), was replaced with a stringent selectable marker GmR (synthetic aacC1) conferring resistance to gentamicin. The plasmid containing this gentamicin selectable marker was highly stable in E. coli and multiple Agrobacterium strains AGL0, AGL1 and LBA4404; data from LBA4404 are presented below.

pVIR plasmids are stable
The stable maintenance and integrity of plasmids pPHP70298 and pPHP71539 in the different bacterial backgrounds (E. coli and Agrobacterium) were determined. Two bacterial strains, E. coli and an auxotrophic (thymidine mutant, THY-) strain of Agrobacterium LBA4404THYcontaining the pVIR plasmids, were grown as described in the Materials and Methods. The cultures after each passage (i.e., overnight culture) were serially diluted and plated on selective and non-selective media, followed by counting the numbers of colonies on each of the plates, respectively. A difference of less than 10% was observed in the colony numbers in the serial dilution plating, suggesting that both plasmids were highly stable in the two different bacterial strains. The integrity of the pVIR plasmids was inferred by restriction enzyme analysis of the pooled plasmid DNA extracted from two independent passages in LBA4404THY-and transformed into Top10 cells (Fig. 3). The DNA banding pattern across multiple enzyme digests (with PstI, BglII, NheI, BamHI and PvuII) was unchanged between the two independent E. coli passages (day 1-4) for both pPHP70928 and pPHP71539. A representative experiment showing the restriction digest fragments of pPHP70928 and pPHP71539 using PstI is presented in Fig. 3a, b. The same plasmid DNA was further analyzed by next-generation sequencing (Illumina), and the stability of the plasmids was confirmed at the base pair level by sequence alignment of the expected and observed bases ( Supplementary Fig. 2).

An improved ternary vector system for maize transformation
The utility of the accessory plasmids on maize transformation was analyzed by introducing the accessory plasmids pSB1 or pPHP70298 into A. tumefaciens strain LBA4404THY-. Subsequently, a binary plasmid pPHP45981, with repABC (pRi) replicon ORI and spectinomycin bacterial selectable marker harboring a simple T-DNA (7.8 kb) containing a reporter gene (ZMUbi1Pro-Intron:YFP:PINII TERM) and a selectable marker (ZMUbi1Pro-Intron:PMI:PINII TERM) between the borders (Fig. 4), was electroporated into the same Agrobacterium cell line (pSB1 or pPHP70298), to generate the ternary constructs, pSB1/pPHP45981 (control) and pPHP70298/pPHP45981. Immature embryo transformation was performed on difficult-to-transform maize inbreds HC69 and PH2RT Zhi et al. 2015). To visualize transient T-DNA transformation, the expression of the YFP (ZS-Yellow) reporter gene was observed after 3 and 5 days of co-cultivation. Representative experiments in the inbreds PH2RT and HC69 at 5 days post-infection are shown in Fig  Restriction mapping of the plasmid DNA in Agrobacterium tumefaciens to determine plasmid integrity. Agrobacterium strain LBA4404THY-harboring the plasmid pPHP70298 (a) or pPHP71539 (b) was isolated after four consecutive passage (day 1-4), and retransformed into E. coli. Pooled plasmid DNA from two independent E. coli passages (a and b colonies used for transforming Agrobacterium cells, day 1-4) was subjected to restriction enzyme digestion to determine plasmid integrity. The DNA banding pattern for PstI showed no detectable differences between the two colonies for all the time points. The plasmid DNA was further sequence confirmed by next generation sequencing (Illumina) Fig. 4 Schematic representation of the T-DNA elements on the binary plasmid pPHP45981 used in the ternary vector with the different accessory plasmids, pSB1 and pPHP70298 for transforming maize inbred lines HC69 and PH2RT. RB right border, Ubi1 Pro-intron maize ubiquitin 1 promoter-intron, ZS-yellow fluroscent protein (YFP), PMI phosphomannose isomerase, PINII TERM potato proteinase inhibitor II terminator, LB left border pPHP45981 alone (Fig. 5a). The above observation indicated that the accessory plasmid pPHP70298 improved transient T-DNA delivery when compared to pSB1. Importantly, pPHP70298 also increased the recovery of stable callus events compared to the control (Fig. 5d, e; Table 1). Taken together, these results demonstrate pPHP70298 improved both transient and stable T-DNA transformation in maize. We are not sure whether the above improvements resulted from the inclusion of functional vir genes (virC1, virC2, virD1 and virD2), or the high copy plasmid replicon pVS1, or the additional amendments made in plasmid pPHP70298.
Next, we assessed the effect of the accessory plasmids on stable transformation and stable transgene integration. The total number of T0 events was divided by the total number of embryos infected to compute stable transformation frequency. Stable T-DNA integration was confirmed by qPCR (for transgene copy number determination) and multiplex PCR (for detecting vector backbone integration)  using genomic DNA extracted from the putative T0 transgenic events as described in Zhi et al. 2015. Events that contained a single copy of the transgenes within the T-DNA and were free of vector backbone integration were identified as quality events (QE). The percentage of QEs was divided by the total number of events analyzed to calculate the QE frequency. The usable event (UE) frequency is a measure of the number of acceptable transgenic events per 100 embryos which was determined as the product of QE frequency and transformation frequency. Consistent with transient expression, T0 transformation frequency in the inbreds HC69 and PH2RT was significantly higher with pPHP70298/pPHP45981 compared to control pSB1/pPHP45981 (Table 1). In HC69, the raw T0 frequency almost doubled compared to the control (27 vs. 14%), while in PH2RT the increase was significant (61 vs. 45%). However, the QE frequency was found to be lower with pPHP70298/pPHP45981 compared to the control for inbreds HC69 (32 vs. 44%) and PH2RT (20 vs. 42%) ( Table 1). This suggests that the improved T0 transformation with accessory plasmid pPHP70298 may have contributed to a higher proportion of non-quality events (non-QE), i.e. events with backbone (BB) integration and/or multi-copy (MC) events. This is consistent with the observation in HC69 for pPHP70298/pPHP45981 that resulted in events with higher frequency of BB integration (22%) and MC (45%) compared to the control (14 and 35%), respectively. A similar pattern was observed for pPHP70298/pPHP45981 compared to the control in the second inbred, PH2RT (BB integration: 20 vs. 12%, MC events: 56 vs. 33%). Next, the UE frequency was calculated for each inbred. The UE frequency was higher than control in HC69 (9.9 vs. 6.5%) and lower than control in PH2RT (12.3 vs.18.6%) ( Table 1). Taken together, the use of ternary vectors carrying accessory plasmid pPHP70298 improved the transient and stable transformation frequency in the inbreds HC69 and PH2RT, but resulted in higher percentage of non-QE in the two genotypes. The higher proportion of non-QE with pPHP70298 likely resulted from the increased T-strand delivery (stronger transient gene expression) in maize cells producing complex extrachromosomal T-DNA structures as previously described (Singer et al. 2012) to generate a higher proportion of events with BB integration and multiple copies of the transgenes.

Plasmids pPHP70298 and pPHP71539 improved maize transformation
Since the observed UE frequency in PH2RT was lower with a ternary vector containing plasmid pPHP70298, we evaluated the accessory plasmid pPHP71539 (Fig. 1c) designed to have additional vir genes (VirD, VirE operons) to improve UE frequency on maize transformation. In this experiment, three different accessory plasmids (pSB1, pPHP70298 and pPHP71539) were introduced into A. tumefaciens strain LBA4404THY-harboring binary vectors with proprietary trait genes. Three different constructs were selected (A, B and C), each containing a different proprietary trait gene (variable DNA piece), the plant selectable marker phosphomannose-isomerase (PMI), and the herbicide resistance gene phosphinothricin acetyl transferase (moPAT). The size of the T-DNA in these three constructs was 11.5, 13.8 and 16.6 kb. Table 2 summarizes the three-way comparison of the different accessory plasmids and trait constructs A, B and C on transformation and event quality in the inbred PH2RT. Consistent with our previous data, ternary vectors containing accessory plasmids pPHP70298 and pPHP71539 resulted in significantly higher callus transformation frequency and putative T0 transformation frequency compared to pSB1 (Table 2). Plasmid pPHP71539 was the best performing accessory plasmid, averaging 1.6 and 2.3-fold higher callus transformation frequency and putative T0 transformation frequency compared to the plasmids pPHP70298 or pSB1, respectively.
A subset of the putative T0 events was subjected to molecular analysis. The QE frequency with plasmid pSB1 was higher, which is consistent with our earlier observation. Overall the QE frequency was lower with plasmid pPHP71539 (29.9%) when compared to control pSB1 (36.6%) ( Table 2). The low QE frequency as compared to control was a result of several factors including higher frequency of events with BB-insertion (19.8 vs. 11.7%), higher proportion of MC events (64 vs. 48%) and higher rates of null events or escapes (8.1 vs. 4.2%) (Supplementary Table 1). Compared to the control, plasmid pPHP70298 also resulted in lower QE frequency (29.1 vs. 36.6%; Table 2), with higher proportion of nulls (6.9%) and MC events (59%, Supplementary Table 1). A possible explanation for the higher frequency of non-QE with pPHP71539, could be due to the presence of extra VirE2 protein produced by the extra copy of the VirE operon in pPHP71539. The additional VirE2 protein would bind and protect extra copies of the T-strands (Christie et al. 1988;Citovsky et al. 1997) which, along with the synthesis of complex extrachromosomal T-DNA structures (Singer et al. 2012) potentially resulted in stable integration of additional copies of the T-strands into the plant chromosome (Supplementary Table 1). The increase in the number of null events could be explained by stable integration and high expression of multiple copies of the selectable marker (PMI). The presence of abundant phosphomannose isomerase protein may have helped protect adjoining non-transformed cells allowing them to escape selection, resulting in the higher number of null events. Interestingly, the observed data on transformation frequency and non-QE frequency is consistent with the previously described work using the hypervirulent strain of Agrobacterium Table 2 The effect of different ternary vectors harboring one of the accessory plasmids pSB1, pPHP70298 and pPHP71539 plus binary vector with proprietary trait genes (A, B and C) on callus transformation, transformation frequency and event quality in the inbred PH2RT Data from three independent transformers was used to determine the callus transformation frequency and T0 transformation frequency. The percentage of QEs was divided by the total number of events analyzed to calculate the QE frequency. The usable event (UE) frequency is a measure of the number of acceptable transgenic events per 100 embryos that was determined as the product of QE frequency and transformation frequency. AGL0 (Zhi et al. 2015). The hypervirulent strain AGL0 containing helper plasmid pTiBo542∆T-DNA significantly improved transformation frequency but produced a higher frequency of non-QE resulting in overall lower frequency of quality events. A similar effect on non-QE frequency from incorporation of hypervirulence genes from pTiBo542 in pVIR plasmids cannot be ruled out. Although QE frequency is reduced, without this technology it is difficult to get good number of events with proprietary trait gene constructs in the difficult -to -transform maize inbreds. Regardless, the overall UE frequency was almost doubled with plasmid pPHP71539 (9.3%) compared to control pSB1 (5.1%). The introduction of the accessory plasmid pPHP71539 enabled development of a superior ternary vector system for transforming recalcitrant maize inbreds.
An additional benefit to using the pVIR plasmids is the rapid callus growth seen in embryos transformed with plasmids pPHP70298 and pPHP71539, resulting in the events being advanced to maturation stage a week to 10 days earlier than with the control plasmid. We observed higher transient T-DNA delivery and a larger number of cells receptive to T-DNA, resulting in selective advantage for the transformed cells to proliferate on the selection medium compared to the control (pSB1). The putative T0 events were regenerated more rapidly, in total reducing the transformation process by 2-3 weeks. Therefore, the pVIR plasmids not only improved the transformation frequency and UE frequency in both inbreds tested but also shortened the transformation process. These improvements in speed and efficiency on maize transformation using the newly described ternary vector system are highly desirable for high-throughput transformation.

A rapid maize transformation system for evaluating the effect of plasmid origin of replication on maize transformation
A recently described rapid maize transformation method (Lowe et al. 2018) was used to assess the effect of 13 different ORI-by-ORI combinations (as listed in Table 3) on maize transformation. The accessory plasmid pPHP71539 was critical for developing rapid maize transformation technology with high transformation frequency in multiple difficult to transform maize inbreds (Lowe et al. 2018). Briefly, the rapid method of transformation relies on expression of morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2) (Lowe et al. 2016), allowing quicker recovery of transgenic maize plants at high frequency from immature embryo transformation. The method significantly reduces the number of steps involved in transformation, directly forming somatic embryos with no intermediate callus phase required. Transformed plants can be germinated rapidly, requiring a month Table 3 The effect of ternary vectors on transformation frequency containing different ORI-by-ORI combinations of the accessory plasmid and the T-DNA containing binary vector in the inbred HC69 A total of 13 different ORI-by-ORI combinations were tested using a rapid maize transformation protocol The effect of 13 ORI-by-ORI combinations on transformation was performed in four groups including the control. The transformation data was analyzed by mixed model using SAS (SAS Institute) and analysis conducted by group. Sample means with same letter were not significantly different at p < 0.05 from infection to sending plants to the greenhouse. This method results in much higher transformation frequencies compared to conventional Argobacterium-mediated transformation and is ideal for rapid gene-function analysis and developing swift crop transformation technologies. Using this rapid maize transformation method, four different bacterial ORI were tested (pVS1, pRi, pSa and RK2; Table 3). These ORI maintain different plasmid copy numbers, ranging from 2 to 3 copies (pRi, pSa), 10-12 copies (RK2) and ~ 20 copies (pVS1) per bacterial cell (Oltmanns et al. 2010;Zhi et al. 2015). Four binary vectors with different bacterial ORI were built, RV001750 (pRi), RV007067 (pVS1), RV007069 (pSa) and RV007070 (RK2), each harboring a 13.8 kb T-DNA containing a WUS2 cassette, a BBM cassette, a selectable marker gene (SB-ALS Pro: ZM-ALS: SB-PEPCI TERM) and a reporter gene (LTP2 Pro: YFP: PinII TERM). HRA is a modified acetolactate synthase gene (ALS) conferring a high level of resistance to imidazolinone and sulfonylurea herbicides (Green et al. 2009). Similarly, four different pVIR accessory plasmids based on pPHP79761 (Fig. 1d) with different ORI [namely RV002110 (pRi), RV004296 (pVS1), RV005072 (pSa) and RV005392 (RK2)] were built and mobilized into Agrobacterium strains harboring the binary vectors as detailed in Table 3. A schematic drawing of the different ORI-by-ORI (binary vector-by-accessory plasmid with different ORI) plasmid combination is presented in Fig. 6. The 12 ORIby-ORI (Fig. 6a) combinations were tested against the control, pPHP71539/RV001750 (Fig. 6b), in the inbred HC69 using ethametsulfuron (0.1 mg/L) herbicide selection to determine the effect of the two ORIs and their interaction on rapid maize transformation. The results from this study are summarized in Table 3. All 13 ORI-by-ORI combinations supported rapid maize transformation, producing T0 plants within a month following embryo infection. Transformation frequencies ranged from 65 to 103%, with the exception of the RK2/pVS1 combination that produced 35% (Table 3). Seven ORI-by-ORI combinations (pVS1/ pRi, pVS1/pSa, pVS1/RK2, pSa/pRi, pSa/RK2, RK2/pRi and pRi/pSa) resulted in significantly lower transformation frequency compared to the control (Table 3). We also identified four ORI-by-ORI combinations pSa/pVS1, RK2/pSa, pRi/RK2 and pRi/pVS1 (control, pPHP71539/RV001750) that resulted in very high raw transformation frequencies (86%-103%, Table 3). When compared to conventional transformation in HC69 with pSB1 (Table 1), this represents a six-to-seven-fold improvement. Based on these data, we concluded that pVS1 ORI on the pVIR plasmid and pRi ORI on the binary plasmid performed reasonably well with different ORI combination (except for the RK2 ORI-based binary vector). To the contrary, use of pSa and RK2 in either pVIR/ binary combination was identified to be a poorly performing Fig. 6 Schematic representation of the different ORI-by-ORI designs tested in the rapid maize transformation system. a The ternary vector design used for evaluating 12 different ORI-by-ORI designs containing the binary plasmid and accessory plasmid based on pPHP79761 differing in their replicons. b The ternary vector design with pRi (binary plasmid)/pVS1 (accessory plasmid pPHP71539) replicon used as the control in the experiments combination. In a previous study we showed direct correlation between binary plasmid copy number on transformation frequency and UE (Zhi et al. 2015). However, in the rapid transformation method an opposite trend was observed with pRi ORI (low copy) on the binary plasmid, resulting in higher transformation frequency. Improved transformation frequency was generally observed when the pVIR plasmid contained the high copy ORI (pVS1), with one exception (pVS1/RK2), suggesting that the choice of ORI is important for maize transformation. The work presented here and the recent work by Lowe et al. (2018) potentially opens the possibility for developing a genotype-independent transformation in maize by modulating the expression of the morphogenic genes along with the use of accessory plasmid pPHP71539. We recently reported the application of the accessory plasmid in a ternary vector for developing a highly efficient transformation system in sorghum (Che et al. 3) and are further testing it for improving transformation of multiple cereal crops including wheat and rice.

Construction of the pVIR plasmids
The pVIR plasmids, pPHP70298 (Fig. 1b), pPHP71539 (Fig. 1c) and pPHP79761 (Fig. 1d) were built in very similar manner. The construction of pPHP79761 is described here as an example of the methods used in the construction of pPHP70298 and pPHP71539. The virulence genes were identified and selected from the Ti plasmid, pTiBo542 (GenBank Accession No. NC_010929.1; Fig. 2). PCR primers were designed to amplify the virA, virJ, and virB1-virE3 coding and regulatory regions from a genomic DNA prep of A. tumefaciens strain AGL1. The 24 kb virB1-E3 sequence was amplified in 5 pieces to facilitate PCR. Fragments were designed with 40 bp overlapping ends to facilitate seamless cloning methods. Unique restriction enzyme sites were included between functional elements to facilitate their exchange with other functionally equivalent elements as shown in Fig. 1d. A gentamicin acetyltransferase gene (GenBank Accession No. DQ530421) conferring gentamicin resistance was synthesized with flanking restriction sites introduced upstream of the vir genes. The high copy ColE1 ORI of pBR322 (GenBank Accession No. J01749) was included for stable replication in E. coli and the pVS1 ORI was included for stable maintenance in Agrobacterium.
The pVIR plasmids were mobilized into an auxotrophic Agrobacterium strain LBA4404THY-by electroporation and the recombinant colonies were selected on minimal medium supplemented with 12.5 mg/L tetracycline (pSB1) or 25 mg/L gentamicin (pPHP70298, pPHP71539 and pPHP79761). For conventional maize transformation, only plasmids pSB1, pPHP70298 and pPHP71539 were tested with a research vector and three different trait gene vectors. In the research vector, the maize ubiquitin (Ubi-1) promoter with its intron1 was used to drive two different gene cassettes, a yellow fluorescent reporter gene (YFP), and phosphomannose isomerase (PMI) (Negrotto et al. 2000), fused to the PinII terminator (Fig. 4). These cassettes were generated as separate entry clones and introduced into a Gate-way™ binary vector with attR4 and attR3 sites, pRi ORI and spectinomycin as the bacterial selectable marker on the vector backbone. Additionally, three more constructs (A, B and C), each containing different proprietary trait gene (variable DNA piece), a maize ubiquitin (Ubi-1) promoter with its intron1 driving the phosphomannose-isomerase (PMI) fused to the PinII terminator and a rice action (Os-Act) promoter with its intron driving a maize codon-optimized phosphinothricin acetyltransferase (moPAT) (Jayne et al. 2000) fused to the CaMV35S terminator within the T-DNA borders were generated. The T-DNA containing binary vectors were mobilized into strain LBA4404 THY-harboring the plasmids (pSB1, pPHP70298 and pPHP71539) by electroporation and the resultant recombinant colonies were selected on appropriate antibiotic (rifampicin, 10 mg/L; spectinomycin,100 mg/L; tetracycline 12.5 mg/L or gentamicin 25 mg/L) supplemented media. All plasmids in Agrobacterium used plant transformation were sequenced verified by next gen sequencing (Illumina) prior to transformation. Some materials reported in this paper contained the selectable markers moPAT and PMI owned by third parties. Pioneer Hi-Bred International, Inc. (Pioneer) will provide materials to academic investigators for non-commercial research under an applicable material transfer agreement subject to proof of permission from any third-party owners of all or parts of the material and to governmental regulation considerations. Obtaining permission from third parties will be the responsibility of the requestor.
To generate various pVIR plasmids with different ORI, we replaced the pVS1 ORI in pPHP79761 with ORI RK2, pSa and pRi using seamless cloning strategy. Likewise, the binary vector with the pRi ORI harboring a T-DNA (13.8 kb, unpublished) containing a WUS2 cassette, a BBM cassette, a sorghum ALS promoter driving modified Zea mays acetolactate synthase gene (ZM-ALS) fused to a sorghum PEPCI terminator and a lipid transfer protein promoter driving a yellow fluorescent protein gene (YFP) fused to the PinII terminator was replaced with RK2 ORI, pSa ORI and pRi ORI using a seamless cloning strategy. The pVIR plasmids and binary vectors transformed into Agrobacterium strain LBA4404THY-were selected on appropriate antibiotic selection and sequence verified by next gen sequencing (Illumina).

Agrobacterium tumefaciens culture conditions
Agrobacterium tumefaciens strains were grown on solidified or liquid AB Sucrose or yeast peptone medium (YP) or Luria Broth (LB) medium supplemented with appropriate antibiotics (rifampicin, 10 mg/L; spectinomycin,100 mg/L; tetracycline 12.5 mg/L or gentamicin 25 mg/L) and thymidine (50 mg/L) at 28 °C. For plant transformation Agrobacterium strains were streaked from glycerol stocks on AB sucrose media supplemented with appropriate antibiotics; 1-3 colonies were picked 2-3 days post plating and streaked on freshly prepared YP media as previously described.

Analysis of plasmid stability
Two independent colonies of E. coli and Agrobacterium (LBA4404THY-) harboring plasmids pPHP70298 and pPHP71539 cultured on LB Agar and antibiotic selection, were used to inoculate 10 ml of LB with appropriate antibiotic, and incubated at 37 °C or 28 °C with shaking. Of this overnight culture 300 µl was used to re-inoculate 3 ml of LB in the presence/absence of antibiotic selection; at the same time a serial dilution of the bacteria grown under non-selective media was plated onto selective and nonselective media. The total viable count and antibiotic resistant bacterial count was used to calculate the mean colony-forming units per ml of culture, using the data from the dilution series. The inheritance of plasmid was monitored for four passages (overnight incubations). For plasmid integrity, the plasmid DNA from Agrobacterium cultures grown in the presence of antibiotic for four passages were retransformed into E. coli, followed by two passages and the pooled DNA subjected to restriction enzyme analysis. The banding pattern was used to infer the plasmid integrity after each growth passage. The same plasmid DNA was subjected to next gen sequencing for comparing the expected and observed plasmid DNA sequences.

Maize transformation and molecular analysis
Immature embryos derived from commercial elite inbred lines HC69 and PH2RT, were used for Agrobacterium-mediated transformation. Maize immature embryo transformation was performed using split ear transformation as described in Cho et al. (2014) using PMI with mannose selection. The T0 transformation frequency was determined as the number of events (plantlet with roots) produced to the total number of embryos infected. Only a single healthy looking event per embryo was used for determining the transformation frequency and for molecular analysis. For the ORI-by-ORI analysis in rapid maize transformation method transformation, the immature embryos were harvested from maize inbred (HC69), infected with Agrobacterium strain containing the ORI-by-ORI combinations using the transformation protocol described by Lowe et al. (2018) was used. Four independent transformation experiments were conducted for each ORI-by-ORI combination, and transformation frequencies were determined by analyzing all the events regenerated per construct.
Genomic DNA extracted from leaf slices derived from putative T0 events and wild-type non-transgenic PHR03 was used for determining the molecular event quality by quantitative PCR (qPCR) as previously described . We determined the copy number of T-DNA genes (PMI, YFP, moPAT and trait gene within the T-DNA borders) using qPCR and used a multiplex backbone PCR assay for detecting the presence/absence of the binary vector backbone . Events identified with a single copy (SC) of all the transgenes without vector backbone integration were defined as a quality event (QE). The usable event (UE) frequency was calculated as transformation frequency times QE frequency.

Statistical analysis
Data for the construct pPHP45981was collected following split ear transformation, with a minimum of 400 embryos, involving at least two independent researchers and three independent experiments. The T0 transformation frequency (stable events/embryos infected) was considered the response variable, with individual researchers as a fixed effect. The data on transformation frequency was collected for each inbred, mean and standard error values were determined. For trait gene (A, B and C) constructs involving three-way testing of the accessory plasmids, split ear transformation was performed on a minimum of four ears per construct involving three independent transformers. A minimum of 500 embryos/production construct was infected. For each individual construct callus transformation frequency and T0 transformation frequency was considered variable while the effect of different constructs/accessory plasmids on transformation rates was not compared. The mean callus and transformation frequency with standard error values was calculated and statistical significance determined by Tukey's test. In the ORI-by-ORI testing, a minimum of 30 embryos were infected by two independent transformers, replicated twice to determine the effect of the construct on putative T0 transformation frequency. The transformation data was analyzed by mixed model using SAS (SAS Institute) and analysis conducted by group. Both transformer and vectors were included as fixed effect in the model. LS-mean for each vector was generated from the model. DUNNETT was used for multiple comparison adjustments on the differences of LS-Means. The significant level was controlled as p < 0.05.


extfieldsofstudy: Medicine Biology

text:
Granulosa Cell Apoptosis in the Ovarian Follicle—A Changing View

Recent studies challenge the previous view that apoptosis within the granulosa cells of the maturing ovarian follicle is a reflection of aging and consequently a marker for poor quality of the contained oocyte. On the contrary, apoptosis within the granulosa cells is an integral part of normal development and has limited predictive capability regarding oocyte quality or the ensuing pregnancy rate in in vitro fertilization programs. This review article covers our revised understanding of the process of apoptosis within the ovarian follicle, its three phenotypes, the major signaling pathways underlying apoptosis as well as the associated mitochondrial pathways.

FiGURe 1 | Periluteal granulosa cells and cell morphology. Human granulosa cells were collected from a 15 mm follicle during an in vitro fertilization cycle at the time of oocyte collection. The granulosa cells are peri-luteal cells; however, the cytoplasm is still relatively compact compared to granulosa cells collected from ovulatory follicles (5). The granulosa cells have dense clustering of organelles around the large round nucleus (N). The cytoplasm appears granular during the late stages of follicular phase; large lipid droplets contain hormones. Cytoplasmic extrusions or blebbing, which indicates late apoptosis are shown (*); apoptotic bodies (a); organelles (o) clustered around the nucleus. Healthy granulosa cell (N) without blebbing is engulfing a neighboring apoptotic granulosa cell nucleus (N1) via phagocytosis (10). Bar 5 µm. typically have pyknotic, crescent-shaped or rounded dark bodies of dense DNA, fragmented into smaller sections. However, the majority of pyknotic cells in the middle layers of the membrana granulosa had been consumed by adjacent healthy cells. Necrosis and apoptosis both culminate in cell death of the granulosa cell, and nucleic dyes that stain DNA material are commonly used to indicate the vitality of the cell membranes (11). Uniquely, the apoptotic granulosa cell will continue to synthesize steroid hormones until the mitochondrial membranes are disrupted. Functioning apoptotic granulosa cells undergo reorganization of the cell cytoplasm, creating blebs of non-cytoplasmic organelles at the periphery; and mitochondria, Golgi apparatus, and endoplasmic reticulum, which are clustered around the nucleus; large fluid filled vacuoles containing steroids, lipids, and proteins also accumulate (12,13). The granulosa cell expands and reorganizes the contents of the cytoplasm, forming new organelles, particularly smooth endoplasmic reticulum (SER) for progesterone production (13). The SER and mitochondria are assembled in close association with the nucleus (8,13). The mitochondria and large, round, lipid droplets are closely associated with the SER, all of which have substantially increased in number, and cluster around the nucleus to increase efficiency of steroid synthesis (Figure 2) (8).
During luteinization, granulosa cells form irregular microvilli and tight junctions between the cells, whereas in an apoptotic granulosa cell, the cell membrane disintegrates and spaces form (12). The apoptotic granulosa cell continues to produce steroids in large antral follicles until complete mitochondrial breakdown occurs (16).

GRANULOSA CeLL APOPTOSiS: THRee PHeNOTYPeS, THeiR iNiTiATiON AND ReGULATiON
Atresia of ovarian follicles can be divided into three phenotypes, each with different mechanisms of initiation and regulation, but all involving granulosa cell apoptosis (17). The first, so-called "antral atresia", affects the middle prolific layers of granulosa cells, with apoptosis progressing toward the antrum. The second, "basal atresia", occurs in the granulosa cells closest to the basal lamina of very small antral follicles. The cells prematurely luteinize and begin to produce progesterone; however, they do not complete luteinization, with subsequent cell death (17). A third form of apoptosis in the preovulatory follicles is referred to as "terminal differentiation apoptosis" and is similar to epidermal skin cells sloughing off (10,18). The granulosa cells that are sloughed off from the antral surface form globules that aggregate and float into the antral fluid (10). The globules average 40 µm in diameter and stain positively for propidium iodide (PI), which indicates that their cell membranes are compromised. However, the DNA is not fragmented, being of high molecular weight, and has a negative DNA fragmentation laddering result for the TUNEL (terminal deoxy-UPT nick end-labeling) assay. The majority of cell death in the membrana granulosa is via "antral atresia. " Apoptotic bodies and cytoplasmic blebbing are typical of antral apoptosis, whereas apoptotic cells in the middle or basement section of the granulosa membrana are commonly engulfed by neighboring healthy granulosa cells and/or infiltrating macrophages. There are three main areas of apoptosis inducement; growth factors, death receptors and cell damage. Apoptosis can occur at any stage of the development of the follicle. In small follicles the granulosa cells are compact with large round nuclei. As the follicle matures the predominantly estrogen producing granulosa differentiates steroidogenically, increasing the volume of mitochondria and SER. Apoptosis at this stage of development would similarly result in condensation of the nucleic contents and clustering of the organelles around the nucleus. In the late stages of apoptosis, cell membranes are broken down and the contents compartmentalize into apoptotic bodies.

MAJOR APOPTOSiS SiGNALiNG PATHwAYS iN THe OvARiAN FOLLiCLe
There are three major signaling pathways for the development of apoptosis in granulosa cells (19). The first is growth factorinduced high levels of cAMP through granzyme B; the second affects mitochondrial function via the Bcl2 family member activation; and the third utilizes tumor necrosis factor alpha (TNFα) and Fas ligand (FasL)-Fas and other death receptors; all of which result in caspase-induced DNA fragmentation (Figure 3). In the ovary, granzyme B is stimulated by gonadotrophins and forskolin to bypass the mitochondria to preserve their function in the early stages of apoptosis, even though DNA fragmentation and nuclear collapse may have occurred (16).
Apoptosis can also be initiated by a number of extrinsic factors that may damage the cell such as DNA damage and oxidative FiGURe 2 | Schematic diagram of granulosa cell characterization from follicular to luteal phase. In a stage-dependent progression, the granulosa differentiates from a compact 8-15 µm cell with a large round nucleus and relatively small cytoplasm (12). The cytoplasm contains mitochondria, rough endoplasmic reticulum, Golgi apparatus, lipid droplets, and many other organelles (14). As the granulosa cell matures, the organelles proliferate and the cytoplasm expands to accommodate new steroidogenic capacity. A fully luteinized granulosa cell, 25-30 µm, contains a large volume of mitochondria, steroid filled lipid droplets, and smooth/rough endoplasmic reticulum, and has the capacity to produce progesterone directly (8). At any stage of follicular growth the granulosa cell can undergo apoptosis. Early apoptosis is characterized by collapse of the cell membranes and condensation of the chromatin, which often polarizes in the nucleus with the organelles clustered adjacent (15). Early apoptosis in a granulosa cell with an expanded cytoplasm is similarly changed with a greater volume of organelles clustered around a collapsing nucleus. Late stages of apoptosis end with compartmentalization of organelles into blebs and extrusion of apoptotic bodies that may contain nucleic matter. stress, which activate p53-specific signaling pathways to trigger apoptotic mechanisms (8,19,33).
Conversely, many growth factors [such as insulin-like growth factor (IGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF)] as well as gonadotrophins (FSH and LH) are antiapoptotic, which creates a microenvironment that ensures survival.
Estrogen and progesterone are the main antiapoptotic factors along with FSH, LH, EGF, IGF, FGF, prolactin, laminin, leptin, and glucocorticoids. The steroid producing capacity of a follicle is reflected in the hormone levels within serum and follicular fluid from the antral cavity of the follicle under the influence of steroidogenic acute regulatory protein (StAR) (34). The steroids produced by the theca and granulosa cells provide an antiapoptotic effect in a stage-specific manner during folliculogenesis.
After dominant follicle selection, the theca cell mitochondria convert cholesterol to pregnenolone by cytochrome P450 sidechain cleavage enzyme, mediated by StAR. The pregnenolone is transported out of the mitochondria and converted to FiGURe 3 | Apoptosis signaling pathways. Estrogen is a major driver of follicular growth and its effects results in inhibition of apoptosis. There are three main areas of apoptosis inducement: 1. growth factors, 2. death receptors, and 3. cell damage (20). Before antral cavity formation ovarian follicle androgens increase the receptor of follicle stimulating hormone (FSH) expression and high levels of BMPR1B activity, possibly via BMP6 and 7, suppress LHR expression (21). Antiapoptotic FSH induced cAMP-PKA promote ERK1/2 signaling, which increases Bcl-2 and promotes estrogen production in favor of progesterone synthesis (22)(23)(24). The bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 inhibit progesterone synthesis, which reduces caspase 3, 6, and 7 production during the follicular phase (25). Dysregulation of the BMPs induces granzyme B synthesis leading to increased DNA fragmentation (16). Fas ligand (FasL) and TNFR activity induces caspase 8 induced DNA fragmentation (26). BMP 7 inhibits caspase 3 activity. Estrogen via the estrogen receptor has also been shown to reduce FasL activity (27,28). Stress induced apoptosis via p53 causes mitochondrial breakdown via caspase 9 (29). Mitochondrial apoptosis is dependent on the ratio of pro and antiapoptotic factors of the Bcl-2 family (30). The Akt signaling pathway promotes antiapoptotic activity and is influenced by estrogen and growth factor-induced cAMP-PKA activity (31,32).
progesterone by 3β-hydroxysteroid dehydrogen. The progesterone is then converted to androgens, predominantly androstenedione, dehydroepiandrosterone, and testosterone, and transported from the theca cell to the granulosa cell where it used to synthesize estrogen.
During the estrogen-driven proliferative phase of folliculogenesis, granulosa cell apoptosis is inhibited by antiapoptotic signaling pathways such as protein kinase B (Akt/PKB), extracellular signal-regulated kinase (ERK), EGF, and the BMPs. The Akt signaling pathway promotes antiapoptotic activity and is influenced by estrogen and growth factor induced cAMP-PKA activity. Estrogen directly inhibits FasL-induced apoptosis via the estrogen receptor, which activates the Akt-dependent pathway (20). In addition, the BMP ligands have been shown to inhibit apoptosis signaling down-stream of these signaling pathways in a number of studies (25,(35)(36)(37)(38).
The LH surge-induced differentiation of the granulosa cell enables it to convert cholesterol to progesterone directly, and the levels rise substantially. At the time of ovulation the LH surge induces the granulosa cells to express progesterone receptors. The follicle undergoes progesterone-driven luteinization and enters the luteal phase, cell proliferation ceases, and high levels of progesterone inhibit apoptosis during corpus luteum formation (39).

MiTOCHONDRiAL PATHwAYS OF APOPTOSiS AND THe BMPs
Apoptosis is induced via a change in the balance of proapoptotic factors versus antiapoptotic factors. In most cells of the body, mitochondrial apoptosis is the primary pathway involved in programmed cell death. However, in the ovary, early apoptosis begins in the nucleus of granulosa cells, bypassing the mitochondria until the later stage of apoptosis. The B-cell lymphoma 2 gene (Bcl-2) family consists of proapoptotic and antiapoptotic factors (~40 known members) associated with the regulation of apoptosis via the mitochondria (27,40).
Bone morphogenetic proteins have an inter-relationship with the proapoptotic factor Bax (a Bcl-2 derived prosurvival protein), and its ratio to antiapoptotic factors (Figure 3). Bcl-2 opposes Bax and its destruction of the mitochondrial membranes. Bax is present from the resting primordial follicle onward; whereas Bcl-2 is only present after primary to secondary transition, which may influence primordial follicle loss (41). BMP2, when overexpressed in renal tumors, signals via Smad 1, 5, and 8 (Smads, initially identified in drosophila, are signal transducers for receptors of the TGF-B superfamily), and increases GATA-4 expression, which inhibits Bax activity in the mitochondria (42). (The GATA transcription factor family are key mediators of sp hemopoietic progenitors.) In the mouse ovary, GATA-4 inhibits Bax-induced mitochondrial membrane permeabilization (41). Conversely, BMP2 induces apoptosis in bone by increasing caspase 3, 6, 7, and 9 expression (35), indicating a tissue-specific involvement of BMP2.
In the ovary BMP2 increases granulosal FSHRs in small follicles, and FSHR-mediated signaling is antiapoptotic in nature (43). This action occurs independently of granulosa cell proliferation induced by BMP2, highlighting the dual action capability of BMPs to inhibit and to induce cellular activity, and to induce differential responses during different stages of folliculogenesis.
In vitro culture of caprine granulosa cells with BMP2, 4, 6, and 7 reduces the percentage of DNA fragmentation (38). In the same study, FSHR silencing increased apoptosis. BMP4 neutralization in rodents causes an increase in apoptosis of the granulosa cells and the oocyte (37). BMP7 activated caspase 9 in granulosa cells; however, BMP4 and 7 had no effect on the levels of mitochondrial apoptotic factors, Bax and Bcl-x1 mRNA (25). Furthermore, BMP7 induced survivin and X-linked inhibitor of apoptosis protein to stimulate caspase 3 and 9 expression. BMP4, however, reduced factors downstream of caspase 3 that culminated in the reduction of apoptosis (Figure 3) (25).
In the transition from pre-antral to antral follicle, high levels of BMPs promote FSHR expression within the granulosa cells. The high levels of BMP6, 4, 7 (25), and BMP15 (60) concurrently sp suppress LHR expression in granulosa cells. During the subsequent follicle section process, the FSHR and BMPR1B expression is reduced along with the BMP ligands 4, 6, 7, and 15, and this may facilitate up-regulation of granulosa cell LHR expression (61). The follicles with granulosa cells expressing LHRs are able to continue to produce estrogens, and are recruited into the dominant cohort. FSHR is protective against apoptosis; therefore, as the FSHR level falls, apoptosis increases (4).
During continued growth of the selected dominant follicle, the estrogen levels rise to a critical level and trigger luteinization. Luteinization down-regulates the BMPR1B and the FSHRs in the ovulatory follicle. The reduction in FSHR and the cessation of proliferation momentarily reduces estrogen synthesis and the balance between proapoptotic and antiapoptotic factors. In older women, the dysregulation of granulosal FSHR and the BMPR1Bs would alter this balance and would be expected to change the apoptosis levels. An altered profile of BMPR1B expression in granulosa cells was also observed in young compared to older sheep (47,62). Moreover, the BMPR1B mutation-induced reduction in apoptosis levels associated with the high ovulation rate in the Booroola sheep (47) was recently confirmed by the reduced GADD45A levels in the follicles of the Booroola sheep (62). (GADD45 alpha is a growth arrest and DNA-damage-inducible protein that is a gene marker for apoptosis.) The cumulus granulosa cells surrounding the oocyte have reduced apoptosis levels compared to the mural granulosa cells (2, 63). The reduced apoptosis level is associated with the BMP ligand concentration gradient emanating from the oocyte (36). At the time of ovulation, luteinization expands the oocyte-cumulus complex, which closes the communication gap junctions between the oocyte and the cumulus cells. The disruption of the concentration gradient of BMPs results in an increase in the apoptosis level and reduced expression of LHR (64). Once luteinization has occurred, progesterone becomes the main antiapoptotic hormone.

THe ReLATiONSHiP BeTweeN APOPTOSiS AND OOCYTe QUALiTY
In IVF programs it has long been recognized that fewer than 10% of oocytes collected become live births (65); hence, in the 1990s there was a concerted effort to find a marker for oocyte quality to better determine the chance of pregnancy. This led to investigations into the relationship between apoptosis and oocyte quality. Oocyte degeneration in the form of DNA fragmentation was linked to oocyte quality (66), and greater levels of DNA fragmentation were reported in the aged mouse oocyte. The level of granulosa cell apoptosis was increased in older IVF patients, similar to that observed in mice, and this was associated with a reduction in oocyte quality, fertilization, pregnancy, and live birth rate (1, 2, 67-69).

earlier Studies and Apoptosis Theory
The close proximity of the cumulus cells to the oocyte led to the investigation of cumulus cell apoptosis, and its relationship to fertility and oocyte quality. Lee et al. (70) found a strong association with fertility; however, conflicting results were also found (71). The level of cumulus cell apoptosis before the LH surge is reported to be less than 3% (2, 72). After the acquisition of LHR (73) and expansion of the cumulus away from the oocyte, apoptosis levels increase in both the oocyte and in the cumulus cells (71).
However, the techniques initially employed for evaluating apoptosis were based on light microscopy assessment of pyknotic cell count or index. What was clear from these early studies was the variance in development of the non-ovulatory smaller follicles compared to the larger preovulatory follicles (7). Nakahara et al.
(2) went on to examine the effect of the subject's age, and in contrast to other reports, they observed a reduced rate of granulosa cell apoptosis in the 40+ years age group. This finding was overshadowed by emphasis on the greater level of granulosa cell apoptosis in patients with a "poor ovarian responsiveness" to gonadotrophin stimulation. For example, when ovarian reserve was indirectly equated to the number of oocytes collected >12 mm in diameter, "independent of age, " the granulosa cell apoptosis rate was significantly greater. This implies that women with low ovarian reserve, or more correctly poor "ovarian responsiveness" to IVF stimulation, had greater levels of granulosa cell apoptosis.
Apoptosis levels within granulosa cells were also greater in the women who did not become pregnant, and was associated with patients with a high FSH level at the start of a cycle (implying poor ovarian reserve). Interestingly, when adjusted for the confounders of age and number of oocytes retrieved, granulosa cell apoptosis was related to pregnancy outcome and not the ovarian reserve (1). The results were significantly confounded by many uncontrolled variables and the methodology used to evaluate apoptosis had limitations.

Recent Studies and Revised Apoptosis Theory
With the newer technique of flow cytometry, more granulosa cells could be examined (~5,000 per follicle) and the white blood cell fraction could be selectively removed using CD45 magnetic beads for subtraction gating to improve the accuracy of identifying granulosa cells. However, the common practice of pooling the granulosa cells collected from a range of follicle sizes from each patient precluded the identification of follicle size-related differences. Other errors were introduced by selectively discarding follicles because they were contaminated with blood and by the spectral overlap between fluorescein isothiocyanate and PI that was not compensated for during flow cytometry (1, 68).
In the current era of research, it is possible to individually analyze the granulosa cells collected from a single follicle of a known size determined using ultrasonography (5). The granulosa cell population can be selectively gated to remove contaminating white blood cells using a CD45 monoclonal antibody. Common leukocyte antigen; 7AAD (far red DNA stain) can be used in place of PI to minimize spectral overlap or flow cytometric compensation can be performed.
The recent study of Regan et al. (4,5) examined the relationship between ovarian reserve, granulosa cell receptor density and apoptosis during healthy human follicle development. The effect of the age of women undergoing the IVF protocol on granulosa cell apoptosis rate related to cell surface gonadotropin receptor and BMPRIB receptor expression. The study showed that the apoptotic rate of granulosa cells was higher in follicles during the two critical stages of dominant follicle selection and the preovulatory maturation stage of folliculogenesis in young women with better ovarian reserve, as determined by ultrasound-defined antral follicle counts (AFCs), compared to older women with a decreased AFC. The reduced apoptosis was associated with low levels of BMPR1B at the time of dominant follicle selection, whereas the lack of down-regulation of the BMPR1B, FSHR, and LHRs was associated with reduced granulosal apoptosis at the time of pre-ovulatory maturation (3, 4). The authors suggested that this result is a reflection of a poor mitogenic turnover rate of granulosa cells in healthy follicles in the older patients.
Notably, administration of gonadotropin-releasing hormone (GnRH) was shown to increase apoptosis of both cumulus and mural granulosa cells. However, in clinical practice, the predictability of the LH surge by its suppression with GnRH has outlived the importance of the finding (74); however, only Hoechst staining was performed for apoptosis determination in that study. This change may have been helped by the finding from another study that the apoptosis level in granulosa cells was not correlated with oocyte quality or fertilization rate, which was determined by using a new technique of identifying very early apoptosis via Annexin V and PI and flow cytometry (75). In another study using PI and Bcl2 fluorescent staining of granulosa cells in patients with the same ovarian reserve and age, the fertilization rate was the same but the patients (women) who achieved a successful pregnancy had a reduced apoptotic index (76). However, variation was very wide in the non-pregnant group (13.61 ± 9.26). Internalization of the phospholipid membrane (Annexin V assay) occurs after the caspase proteolytic cascade, but before DNA condensation and fragmentation (11). Unfortunately, unintentionally induced apoptosis by centrifuging cells at >300 g would limit the value of some studies (67,74). In the study of Lee et al. (70), cumulus cell apoptosis was found to be greater in the 40+ years age group, corresponding to a poorer fertilization rate; however, only 200 cumulus cells were counted per cumulus-oocyte complex, and only four patients were in the older age groups.
As new techniques were developed, and their sensitivity increased, the granulosa caspase activity was also analyzed (71,(76)(77)(78)(79). Yuan et al. (71) found no difference in TUNEL staining and caspase activity until the LH surge, whereas caspase activity for the combined caspases 1-9 was present before the LH surge but then disappeared after the LH surge (71). In a recent study, the same technique of counting pyknotic cells found that the level of apoptosis was reduced in patients who became pregnant (80). The finding was supported by qPCR data of a greater level of caspase 3 in the non-pregnant group. Although the results appear robust, an undisclosed number of follicles were sampled (between 1 and 4 per patient), and were not necessarily the corresponding follicle that produced the pregnancy. To confound the finding further, two embryos were transferred per patient; therefore, the outcome of the fate for each follicle, pregnant or not, was unclear.
At this time, the mechanisms of apoptosis were being investigated to determine whether various factors were proapoptotic or antiapoptotic (81). Whole ovary investigations also took place and revealed the level of DNA fragmentation (TUNEL assay) and caspase 3 activity to be undetectable in adult primordial, primary, and secondary follicles (59,78,82), whereas high levels of caspase-dependent apoptosis were recorded in antral follicles. However, in pre-ovulatory follicles, apoptosis was again rarely found (59). This change in apoptosis corresponds to the two critical times of FSHR down-regulation and LHR acquisition during dominant follicle selection; and later during pre-ovulatory down-regulation of LHR, FSHR, and BMPR1B (3, 5).
Early research by Sadraie et al. (67), reported that young patients produced more oocytes that were mature compared to the older age groups, which is consistent with the recently reported lack of down-regulation of BMPR1B, FSHR, and LHR, and final maturation of the follicle in older women (3-5). However, Jancar et al. (83), found no effect of granulosa cell apoptosis on fertilization rate or blastocyst development. Even though CD45-coated beads were used to purify the granulosa cells, they were pooled from multiple follicles. Moreover, the cells were centrifuged at 400 g, which is known to induce apoptosis. These factors may limit the significance of this study as well as a similar study examining granulosa cells from pooled follicles, where cells were also centrifuged at 400 g (84).
In older patients, the follicular levels of oxidative stress were increased, which can be associated with reduced oocyte quality (29). However, both the embryo and the oocyte produce reactive oxygen species (ROS) as a normal part of metabolism, and the production of the ROS may reflect other changes to the ovarian environment that are associated with aging and not necessarily a result of changed apoptosis levels (29,70,(85)(86)(87)(88).
Recently, in human fetal whole ovaries, granulosa cells from primordial follicles were reported to be positive for caspase 3 and DNA fragmentation (89). This limited semi-quantitative study also reported high levels of BAX in primordial follicles and Bcl2 in secondary follicles. However, the level of caspase dependent apoptosis was not commensurate with the expected loss of primordial follicles before birth or postnatally (89,90). The inconsistent low levels of apoptosis indicate that other forms of caspase-independent cell death are in operation.
Several new proposals have surfaced in relation to the mechanisms of programed cell death that do not have features typically associated with apoptosis. These include autophagy (91), self-sacrifice to provide nutrients for neighboring follicles (92) and neuronal-endocrine induced apoptosis via the endoplasmic reticulum (93). Clearly, there are still many unanswered questions associated with the mechanism of cell death in the follicle.

CONCLUSiON
The complex control of folliculogenesis, ovulation, and the luteal phase of follicle development relies on the balance of proapoptotic and antiapoptotic factors to regulate cell survival. Apoptosis in the granulosa cell is predominantly via caspase-dependent signaling pathways, and the majority of apoptosis results in terminal differentiation of the granulosa cell at the antral surface of the follicle. The type of apoptosis and the signaling pathway depends on the stage of development of the follicle and the origin of the intrinsic or extrinsic trigger. A large volume of granulosa cell apoptosis within the granulosa membrana leads to death of the follicle.
Optimal receptor expression during folliculogenesis ensures maximum estrogen synthesis, which is essential for follicle survival. Cessation of estrogen driven-proliferation leads to ovulation, which imposes a new regulatory paradigm. The progesterone dominated luteal phase is also regulated by receptor expression to promote cell survival and corpora lutea function.
Using the techniques available at this time, it is apparent that apoptosis levels of the granulosa cells are reflective of the proliferative stage of the follicle rather than a predictor of oocyte health. Apoptosis is not used as a marker in the clinical setting because of its poor predictive capability regarding oocyte quality and ensuing pregnancy rate. Recent studies challenge the longheld view that increased granulosa cell apoptosis is typical of older patients and is related to oocyte quality. Rather, granulosa cell apoptosis appears to be an integral part of normal follicle development and reflects the mitogenic growth of the follicle that varies in a stage-dependant manner.